WO2020239822A1

WO2020239822A1 - Nucleic acid construct binding to influenza polymerase pb1 rna synthesis active site

Info

Publication number: WO2020239822A1
Application number: PCT/EP2020/064674
Authority: WO
Inventors: Stephen Cusack; Petra DRNCOVA; Tomas KOUBA
Original assignee: The European Molecular Biology Laboratory
Priority date: 2019-05-27
Filing date: 2020-05-27
Publication date: 2020-12-03

Abstract

The present invention relates to a new nucleic acid construct capable of binding to the PB1 RNA synthesis active site of influenza polymerase. The nucleic acid construct allows capturing the structure of the transcription initiation state of influenza polymerase. The present invention further pertains to methods for obtaining images or crystallography data on an influenza polymerase in a functional or active state, methods for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, and to such compounds per se. Further provided are pharmaceutical compositions comprising such compounds and the compounds or pharmaceutical compositions for use in treating, ameliorating, or preventing a disease caused by viral infections with negative-sense single stranded RNA viruses.

Description

NUCLEIC ACID CONSTRUCT BINDING TO INFLUENZA POLYMERASE PB 1 RNA

SYNTHESIS ACTIVE SITE

TECHNICAL FIELD OF INVENTION

The present invention relates to a new nucleic acid construct binding to the PB1 RNA synthesis active site of influenza polymerase. The nucleic acid construct allows capturing the structure of the transcription initiation state of influenza polymerase. The present invention further pertains to methods for obtaining images or crystallography data on an influenza polymerase in a functional or active state, methods for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, and to such compounds per se. Further provided are pharmaceutical compositions comprising such compounds and the compounds or pharmaceutical compositions for use in treating, ameliorating, or preventing a disease caused by viral infections with negative-sense single stranded RNA viruses.

BACKGROUND OF THE INVENTION

Influenza is responsible for much morbidity and mortality in the world and is considered by many as belonging to the most significant viral threats to humans. Annual influenza epidemics swipe the globe and occasional new virulent strains cause pandemics of great destructive power. At present the primary means of controlling Influenza virus epidemics is vaccination. However, mutant influenza viruses are rapidly generated which escape the effects of vaccination. In light of the fact that it takes approximately 6 months to generate a new influenza vaccine, alternative therapeutic means, i.e., antiviral medication, are required especially as the first line of defense against a rapidly spreading pandemic.

An excellent starting point for the development of antiviral medication is structural data of essential viral proteins. The crystal structure determination of the influenza virus surface antigen neuraminidase (von Itzstein et al., 1993) led directly to the development of neuraminidase inhibitors with anti-viral activity preventing the release of virus from the cells, however, not the virus production. These neuraminidase inhibitors and their derivatives have subsequently developed into the anti-influenza drugs, Zanamivir (Glaxo) and Oseltamivir (Roche), which are currently being stockpiled by many countries as a first line of defense against an eventual pandemic. However, these medicaments provide only a reduction in the duration of the clinical disease. Alternatively, other anti-influenza compounds such as amantadine and rimantadine target an ion channel protein, i.e., the M2 protein, in the viral membrane interfering with the uncoating of the virus inside the cell. However, they have not been extensively used due to their side effects and the rapid development of resistant virus mutants (Magden et al., 2005). In addition, more unspecific viral drugs, such as ribavirin, have been shown to work for treatment of influenza infections (Eriksson et al., 1977). However, ribavirin is only approved in a few countries, probably due to severe side effects (Furuta et al., 2005). Clearly, new antiviral compounds are needed, preferably directed against different targets.

Influenza virus as well as Thogoto virus belong to the family of Orthomyxoviridae which, as well as the family of the Bunyaviridae, including the Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus, are negative stranded RNA viruses. Their genome is segmented and comes in ribonucleoprotein particles that include the RNA dependent RNA polymerase which carries out (i) the initial copying of the single-stranded virion RNA (vRNA) into viral mRNAs and (ii) the vRNA replication.

The heterotrimeric influenza polymerase, which comprises subunits PA, PB1 and PB2, binds to the conserved 5' and 3' termini (the 'promoter') of each of the eight negative sense single- stranded viral RNA (vRNA) genome segments. It both transcribes and replicates the vRNA in the infected cell nucleus (Eisfeld et al., 2015; Pflug et al., 2017; Te Velthuis et al., 2016) and is the major target for novel anti-influenza drugs (Pflug et al., 2017; Zhang et al., 2018; Hayden et al., 2018). While PB 1 harbors the endonuclease and polymerase activities, PB2 contains the RNA cap binding domain. In transcription, the cap-binding domain of PB2 binds to the 5' cap structures of host pre-mRNAs (Guilligay et al., 2008). The cap, together with about 10-13 nucleotides downstream of the cap, remain bound to PB2, while the reminder of the RNA is cleaved off by the N-terminal endonuclease domain of PA (Yuan et al., 2009; Dias et al., 2009). Subsequently, the generated 5 '-capped RNA fragment bound to PB2 serves as the primer to synthesize viral mRNA by the PB 1 subunit.

WO 2009/046983 to some of the present inventors discloses high resolution structural data of the RNA cap binding pocket within PB2 by X-ray crystallography within an independently folded domain. To obtain the high resolution data, the inventors recombinantly produced soluble PB2 polypeptide fragments comprising a functional RNA cap binding pocket, which allowed performing in vitro high-throughput screening for inhibitors of a functional site on Influenza virus polymerase using easily obtainable material from a straightforward expression system.

Attempts to capture the structure of the transcription initiation state, in which a short primer- template duplex is expected to be positioned in the active site to allow further nucleotide incorporation, have so far failed (Pflug et al., 2018; Reich et al., 2017). There are several likely reasons for the difficulty in trapping this complex, whose formation is the rate-limiting step in RNA synthesis and whose instability can result in abortive product formation (Klumpp et al., 1998). Firstly, the 3' end of the template is flexible as shown by single-molecule FRET (Robb et al., 2016) and consistent with this, crystal structures either locate it on the surface of the polymerase or threaded through the template entrance channel into the active site cavity (Reich et al., 2014 and 2017; Pflug et al., 2014). Secondly, an element of PB 1 known as the priming loop, which is thought to promote unprimed RNA synthesis (Te Velthuis et al., 2016), normally obstructs the active site cavity and would clash with the presumed position of the 3' extremity of the primer (Reich et al., 2017). How the priming loop is displaced to allow initiation and processive RNA synthesis to proceed was so far unknown. Thirdly, template translocation requires disruption of the highly stable distal 5 '-3' base paired region of the promoter. This is energetically unfavorable without the formation of compensating primer/product base pairs in the active site (Reich et al., 2017; Tickle et al., 2018).

In order to better understand the mechanisms underlying viral transcription and for identifying specific inhibitors of influenza RNA polymerase and in particular its active site, it would be highly desirable to structurally characterize the different steps of actively transcribing influenza polymerase.

SUMMARY OF THE INVENTION

The present inventors identified a new nucleic acid construct that binds to the PB1 RNA synthesis active site of influenza polymerase, allowing capturing the structure of the transcription initiation state, in which the primer-template pair is positioned in the active site to allow further nucleotide incorporation, giving rise to new crystal structures showing the influenza polymerase in a functional/active state. Further, using the nucleic acid construct of the present invention, the active site dynamics of influenza polymerase during the nucleotide addition cycle can be characterized. The new crystal structures aid in revealing the functions of influenza polymerase and in identifying new compounds as antiviral medication.

To circumvent the reported problems and enable visualization of the initiation and early elongation steps of transcription by influenza polymerase, the present invention provides a new nucleic acid construct. Further provided are methods for obtaining structures of influenza polymerase, methods for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, and compounds obtained by such methods.

According to a first aspect, the present invention provides a nucleic acid construct comprising a primer strand and a template strand. The primer strand has the sequence 5'-Xi-X_m-3' and the template strand has the sequence 3'-Y₁-Y_n-5', wherein X and Y are independently nucleotides, and wherein m is an integer of from 12 to 16 and n is an integer of from 13 to 50. Between five and six consecutive nucleotides of the primer strand and of the template strand are complementary to each other such that either (i) nucleotides X_m-4, X_m-3, X_m-2, X_m-₁, and X_m of the primer strand are complementary to nucleotides Y₁, Y₂, Y3, Y4 and Y₅ of the template strand, or (ii) nucleotides X_m-5, X_m-4, X_m-3, X_m-2, X_m-₁, and X_m of the primer strand are complementary to nucleotides Y₁, Y₂, Y₃, Y₄, Y₅ and Y₆ of the template strand.

According to one embodiment, at least 50% of the nucleotides of the primer strand and of the template strand are ribonucleotides.

According to an embodiment, the primer strand has a m⁷Gppp-cap connected to the 5' end, wherein m⁷Gppp is 7-methyl-guanosine linked to a triphosphate bridge.

According to one embodiment, m is an integer of from 14 to 15 and n is an integer of from 16 to 30.

According to a preferred embodiment, the nucleic acid construct further comprises an activator strand having the sequence 5'-Z_rZ₀-3', wherein Z is independently selected from the group consisting of adenine, thymine, uracil, guanine and cytosine, and wherein o is an integer of from 12 to 18. In case of (i) four complementary consecutive nucleotides between the primer strand and the template strand, at least two consecutive nucleotides of the activator strand beginning with Z₁₁ are complementary to at least two consecutive nucleotides of the template strand beginning with Y₁₂. In case of (ii) five complementary consecutive nucleotides between the primer strand and the template strand, at least two consecutive nucleotides of the activator strand beginning with Z ₁₁ are complementary to at least two consecutive nucleotides of the template strand beginning with Y₁₃.

According to one embodiment, o is an integer of from 13 to 17, preferably of from 14 to 16.

According to a further embodiment, at least four consecutive nucleotides of the activator strand beginning with Z ₁₁ are complementary to at least four consecutive nucleotides of the template strand beginning with Y₁₃.

According to one embodiment, nucleotides Z_\ to Z_w of the activator strand together form a stem-loop structure.

According to a further embodiment, the activator strand has a triphosphate bridge connected to its 5' end.

According to a further embodiment, the 3' end of the activator strand is connected to the 5' end of the template strand.

According to a further embodiment, the primer strand has the sequence as denoted in SEQ ID NO: l or 3 or a sequence at least 80% identical to SEQ ID NO: l or 3, and the template strand has the sequence as denoted in SEQ ID NO:2 or a sequence at least 80% identical to SEQ ID NO:2. Optionally, the nucleic acid construct further comprises an activator strand having the sequence as denoted in SEQ ID NO:4 or a sequence at least 80% identical to SEQ ID NO:4.

According to a further aspect, the present invention provides a polynucleotide encoding the nucleic acid construct of the present invention.

According to a further aspect, the present invention provides a recombinant vector comprising the polynucleotide of the present invention.

According to a further aspect, the present invention provides a recombinant host cell comprising the polynucleotide or the recombinant vector of the present invention. According to a further aspect, the present invention provides a construct comprising an influenza PB1 RNA synthesis active site and the nucleic acid construct of the present invention bound thereto.

According to a further aspect, the present invention provides a construct comprising an influenza PB2 cap binding site and the nucleic acid construct of the present invention bound thereto.

According to a further aspect, the present invention provides a construct comprising an influenza polymerase and the nucleic acid construct of any one of the present invention bound to the PB1 RNA synthesis active site of the influenza polymerase. Preferably, the nucleic acid construct is also bound to the PB2 cap binding site of the influenza polymerase.

According to a further aspect, the present invention provides the nucleic acid construct of the present invention bound to an influenza PB1 RNA synthesis active site.

According to a further aspect, the present invention provides the nucleic acid construct of the present invention bound to an influenza PB2 cap binding site.

According to a further aspect, the present invention provides an influenza polymerase comprising the nucleic acid construct of the present invention bound to the PB 1 RNA synthesis active site.

According to a further aspect, the present invention provides an influenza polymerase comprising the nucleic acid construct of the present invention bound to the PB2 cap binding site.

According to a further aspect, the present invention provides a method for crystallizing an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site, comprising the steps of:

(i) contacting the nucleic acid construct of the present invention with the influenza polymerase or variant or fragment thereof and allowing binding of the nucleic acid construct to the PB1 RNA synthesis active site, and (ii) crystallizing the influenza polymerase or variant or fragment thereof with the bound nucleic acid construct.

According to one embodiment, the method further comprises obtaining crystal diffraction data.

According to one embodiment, the nucleic acid construct is further allowed to bind to the PB2 cap binding site.

According to a further aspect, the present invention provides a method for cryogenic electron microscopy of an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site, comprising the steps of:

(i) contacting the nucleic acid construct of the present invention with the influenza polymerase or variant or fragment thereof to allow binding of the nucleic acid construct to the PB 1 RNA synthesis active site, and

(ii) freezing the influenza polymerase or variant or fragment thereof with the bound nucleic acid construct.

According to a further aspect, the present invention provides a method for in vitro testing of a function of influenza polymerase. The method comprises the steps of

(i) providing an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site;

(ii) adding to the influenza polymerase or variant or fragment thereof the nucleic acid construct of any one of claims 1 to 9 and allowing binding of the nucleic acid construct to the PB 1 RNA synthesis active site;

(iii) adding nucleosides or nucleoside analogues;

(iv) evaluating the polymerase activity of the influenza polymerase.

According to a further aspect, the present invention provides a method for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, comprising the steps of: (i) contacting an influenza polymerase or variant or fragment thereof comprising at least the PB 1 RNA synthesis active site with (a) the nucleic acid construct of the present invention and (b) a test compound, and

(ii) analyzing the ability of said test compound to inhibit influenza RNA polymerase.

According to one embodiment, contacting the influenza polymerase or variant or fragment thereof with the nucleic acid construct comprises contacting the influenza polymerase or variant or fragment thereof consecutively with different nucleic acid constructs having different sequences and/or lengths of the primer strand, the template strand, and/or the activator strand.

According to a further embodiment, the strands of the nucleic acid construct are contacted with the influenza polymerase or variant or fragment thereof prior to, concomitantly with, or after addition of said test compound.

According to a further aspect, the present invention provides a method for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, comprising the steps of:

(a) constructing a computer model of the influenza polymerase with the nucleic acid construct bound to the PB 1 RNA synthesis active site based on the crystal diffraction data obtained with the method according to the present invention;

(b) selecting a potential compound by a method selected from the group consisting of:

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound;

(c) employing computational means to perform a fitting program operation between computer models of the said compound and the PB 1 RNA synthesis active site with the bound nucleic acid construct; and

(d) evaluating the results of said fitting operation to quantify the association between the said compound and the influenza polymerase PB1 RNA synthesis active site.

According to one embodiment, the methods further comprise the step of synthesizing said compound and optionally formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s). According to one embodiment, the compound is selected from the group consisting of a small molecule, a peptide or a protein. Preferably, the compound is an NTP analogue.

According to a further aspect, the present invention provides a compound identified or obtained by the methods of the present invention.

According to one embodiment, the compound of the present invention is for use in medicine.

According to a further aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention. The pharmaceutical composition preferably or optionally further comprises a pharmaceutically acceptable carrier.

According to a further aspect, the present invention provides the compound, the compound for use or the pharmaceutical composition of the present invention for use in treating, ameliorating, or preventing a disease caused by viral infections with negative-sense ssRNA viruses.

According to one embodiment, the disease is caused by viral infections of the Mononegavirales order comprising the Bornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae families, preferably wherein said disease condition is caused by the Orthomyxoviridae, Arenaviridae, or Bunyaviridae families. Preferably, the disease is caused by a virus selected from the group consisting of Borna disease virus, Marburg virus, Ebola virus, Sendai virus, Mumps virus, Measles virus, Human respiratory syncytial virus, Turkey rhinotracheitis virus, Vesicular stomatitis Indiana virus, Nipah virus, Henda virus, Rabies virus, Bovine ephemeral fever virus, Infectious hematopoietic necrosis virus, Thogoto virus, Influenza A virus, Influenza B virus, Influenza C virus, Hantaan virus, Crimean-congo hemorrhagic fever virus, Rift Valley fever virus, and La Crosse virus.

According to one embodiment the disease is selected from the group consisting of hemorrhagic fever, rabies, influenza, mumps or measles.

Further aspects and embodiments of the present invention are disclosed in the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the transitions between the (a) and (b) pre-initiation, (c) initiation, and (d) elongation states for transcribing influenza polymerase. In the promoter-bound pre- initiation state the template 3’ end is either (a) on the polymerase surface or (b) takes a sinous route into the active site (A in red sphere). Eventually stochastic displacement of the priming loop tip allows primer-template hybridization and incorporation of the first few nucleotides (c). Template translocation could initially occur by straightening of the template entrance pathway but eventually promoter melting has to occur. The associated collapse of the b-ribbon and closure of the initial template entry tunnel disfavors backtracking, re-formation of the promoter and abortive product formation. NTP incorporation driven translocation provides a counter force to the priming loop, which back extrudes in steps through the active site cavity and out of the template exit channel. Thumb rotation and active cavity opening allow growth of the template -product duplex to the full nine base pairs characteristic of the elongation state before abutting against the PB2 helical lid which forces strand separation (d).

FIG. 2 schematically shows RNA-protein interactions in the active site cavity of (A) the pre- translocation initiation and (B) the post-translocation elongation states. Protein residues are colored according to polymerase subunits (PBEcyan, 653 PB2:ruby). The template (yellow) is numbered with respect to the +1 site, the primer/product (blue) with respect to the capped 5' end. Interaction type is color coded as indicated.

FIG. 3 shows RNA-protein interactions in the active site cavity. (A) View into the PB1 catalytic site showing the conserved RNA-dependent RNA polymerase functional motifs with color code motif A (green), B (light grey), C (magenta), D (green-cyan), E (orange) and F (orange-yellow). Template (yellow), primer (blue), metal B (magenta sphere) and pyrophosphate (orange) are shown as observed in the pre-translocation initiation state. Metal A (magenta sphere), as observed in the post-translocation elongation state, is superimposed. The G+l template base stacks on Ile241 and makes a polar interaction with Lys229, and its 2' hydroxyl hydrogen bonds to the Ala242 carbonyl oxygen (all motif F residues). The opposite base in the primer (Cl 5) stacks on Met410 (motif B) and its ribose interacts with Asn310 (motif A). (B) Interactions of the capped RNA primer (m7GpppGAAUG... ) with specific residues (yellow sticks) of the cap- binding (orange), PB2-midlink (violet) and PB2-N (ruby) domains as observed in the initiation state structure. Nucleotides 2-AAU-4 are stacked on each other and whereas G5 is in a separate pocket. (C) Conformation and interactions of the flexible, methionine-rich motif B loop (PB 1/407-413) as observed in occupied (light grey) and vacant (dark grey) active site structures. When incoming CTP (orange) is present, the motif B loop remodels to stabilize the base by stacking with Met410 and the backbone of Gly411. The specific conformation of the motif B loop is further secured by a hydrogen bond interaction of Gly411 carbonyl with the 2' hydroxyl of the template at the -1 position. The +1 NTP position 2' and 3' hydroxyls are sensed by hydrogen bonds to the side-chain of Asn310. When the +1 NTP absent, Met410 flips into a specific hydrophobic pocket (arrow).

FIG. 4 shows conformational dynamics of the methionine-rich motif B loop. (A) Superposition of the methionine-rich motif B loop conformations as observed in the pretranslocate state (light grey), post-translocated state (dark grey) and intermediate positions as observed in two different pre-initiation state structures (this study and PDB entry 5MSG). PB 1/Met410 gradually flips (gradient of greys) from the position where it contacts (orange dashed lines) the product base at the +1 position (marked by blue C15) towards a disengaged position when there is no base. Notably, the pathway would clash (red dashed line) with the +1 template position (marked by yellow +1G) position in both pre and post translocated states. (B) and (C) Details of the methionine-rich motif B loop conformations when the incoming NTP site vacant or occupied, respectively, and their superposition (D) showing that PB1/Phe344 and Phe413 both reorient to accommodate Met410 in a distinct hydrophobic pocket when the incoming NTP site is vacant. (E) and (F) Fit of the methionine-rich motif B loop residues into the Fo - Fc omit map electron density of the vacant (capped 14-mer X-ray structure) and occupied (capped 15-mer X-ray structure) states of the active site at 3s.

FIG. 5 shows structural snapshots of the initiation to elongation transition of influenza polymerase. (A) Pre-initiation, (B) initiation and (C) elongation state complexes. In the top row sequence and secondary structure of nucleic acid moieties in the complex are shown. The vRNA 5' and 3' ends are respectively pink and yellow (with the template +3 nucleotide extension in grey) and the capped primer/product is blue/black. Nucleotides not visible in the structure are in italics. Nucleotides are numbered from the 5' end for the capped RNA and the vRNA 5' moiety. Numbers for the vRNA 3' moiety (template) are with respect to the transcription start site as +1 (unbracketed numbers) or from the 3' end of the unextended template (bracketed numbers). The middle row shows the structure of the RNA moieties, represented as spheres. The bottom row shows ribbon diagrams of the pre-initiation (cryoEM), initiation (crystal) and elongation (cryoEM) state complexes colored according to domain structure 14. In the initiation state structure, the PB1 b-ribbon position is distorted by crystal contacts.

FIG. 6 shows that the priming loop extrudes out of the active site in stages during the progression from pre-initiation to elongation state. (A) Pre-initiation state 1 (cryoEM). The priming loop (grey) is fully ordered in its b-ribbon configuration when neither the template (yellow) nor the primer (blue) are in the active site cavity. Nucleotide numbering as in Fig. 5. (B) Pre-initiation state 2 (PDB entry 5MSG 11). The tip of the priming loop disorders when the template 3' end enters the active site cavity. (C) Initiation state (crystal). The partially extruded priming loop accommodates five template/primer base pairs in the active site cavity. (D) Elongation state (cryoEM). The priming loop is fully extruded allowing the active site cavity to accommodate an RNA duplex of nine template/product base pairs in the post-translocation state (positions -1 to -9) and ten base pairs in the pre-translocation state (positions +1 to -9).

FIG. 7 shows an elongation intermediate stability and on pathway assay. Pre-incubated complexes formed with either 18 (left) or 18+3 (right) 3’ vRNA templates together with 15- mer primer and ATP and GTP are able to continue the transcription reaction upon CTP addition even in the presence of heparin competitor, whereas addition of heparin at the beginning of the reaction completely inhibits RNA synthesis (last two columns of each panel). Read-through and endonuclease cleavage products are indicated. Radiolabelled capped RNA markers, of size indicated, are marked by letter M.

FIG. 8 shows strand separation and template exit channel opening. The figure shows superposition 635 of the PB2 helical lid domain in the pre-initiation (light grey) and elongation state (red). The lid is held in place by PB2 N2 domain residues 112-119 (ruby), PB1 C-terminal residues 656-659 and 699-705 (cyan), the PB1 priming loop (dark grey) and the PA endonuclease residues 89-97 (green). Lid helix al2 faces the product (blue)-template (yellow) duplex and Tyr207 (black) stacks on the template base of the last base pair, preventing duplex continuation beyond position -9.

FIG. 9 also shows strand separation and template exit channel opening. (A) and (B) are cartoon and surface representations of the initiation state showing that the partially extruded priming loop blocks the template exit channel and the five base pair primer/template duplex remains buried in the active site cavity. (C) and (D) are cartoon and surface views along the open template exit channel after priming loop extrusion in the elongation state. The template 3' end emerges from the active site cavity upon strand separation. The open channel is lined by parts of the priming loop (dark grey), the following linker PB 1/661-670 (cyan), PB 1 helix a22 (cyan), PB 1/514-517 (cyan), the PB2 lid helix a22 (red) and PB2 residues 34-37 (green).

FIG. 10 shows the electron density for the RNA template-primer pair in the crystal structure of the initiation state.

FIG. 11 shows promoter disruption and remodeling of the template entry channel. (A) The promoter 3' (yellow) and 5' (pink) base pairing and sinuous pathway into the active site in the pre-initiation state is stabilized by interactions with PA residues Met473 and His506 (green), the short PB 1/670-677 hΐq helix (cyan), the PB 1 b-ribbon (light orange) and PB2/37-44 (salmon). Nucleotide numbering as in Fig. 5. (B) Upon promoter melting, as observed in the elongation state, the PB 1 b-ribbon (orange) collapses onto the 5' end vRNA and forms a new three- stranded sheet together with the remodeled hΐq helix residues (grey). PB2/37-44 move to block the positions in the initial template channel previously occupied by 3' nucleotides 7-8. (C) Details of the promoter configuration in the pre-initiation state. The 3' G9 position is stabilized by stacking to PA/Met473 (green) and pseudo-base pairing with PA/His506. 3Ά11 packs against PA/Met473. The PB 1/670-677 hΐq helix stabilizes the tight turn between nucleotides 8 and 9 of the template.

FIG. 12 also shows promoter disruption and remodeling of the template entry channel. (A) Details of melted promoter state. 5' A11-G12 are sandwiched between PA/His506 and PB 1/Leu200 of the collapsed b-ribbon. The paths of the incoming template and outgoing 5' end (arrows) are not defined by the structure. (B) Superposition of Fig. 11A and B showing concerted conformational changes associated with promoter disruption, priming loop extrusion and primer-product/template duplex growth in the active site chamber.

FIG. 13 shows a high resolution bat FluA crystal structure. Left: Ribbon representation with PA green except endonuclease (forest green), PB 1 (cyan), PB2 (red) except cap-binding domain (orange). The promoter 3' strand is yellow and the 5' strand is violet. Right: Surface representation in the same orientation coloured according to B-factors (blue-white-red, 20-120 A2). The core and active site region is highly ordered, whilst the cap-binding and endonuclease domains appear less ordered. FIG. 14 shows a view on the active site of the native crystal structure. There is no incoming NTP and therefore the motif B loop 407-MMMGM-411 (green) is in the open position. The priming loop (magenta) appears fully ordered.

FIG. 15 shows a crystals soaked with GTP and CTP. Left: schematic showing positioning of the soaked GTP and CTP with respect to the template RNA. Right: Unbiased difference electron density for the soaked GTP and CTP. Crosses represent water molecules.

FIG. 16 shows a view of the active site of the GTP+CTP soaked crystal structure. There is incoming CTP at the +1 position and therefore the motif B loop 407-MMMGM-41 l(cyan) is in the closed position. The priming loop (magenta) is fully ordered. There are direct and water- mediated contacts between PA/658-RK and PB1/H649 and the priming GTP triphosphate. The three active site aspartates and two magnesium ions are in the catalytic configuration.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger et aI. 1995.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The open term “comprise” however also explicitly encompasses the meaning“consists of’, yet, it is not limited thereto. For example, if a substance or method is described as comprising compounds or steps A and B, this also means that according to a preferred embodiment, the substance or method consists of the compounds or steps A and B.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

A large number of natural PB2 variants are known and have been described in scientific and patent literature. In this respect it is exemplarily referred to Fechter et al., 2003, as well as to WO 2009/046983, which discloses natural PB2 and respective variants thereof. All these PB2 variants can be used in the context of the present invention. The term "RNA-dependent RNA polymerase subunit PB2" preferably refers to the PB2 of Influenza A, Influenza B and/or Influenza C virus.

The PB1 RNA synthesis active site is well known and described in the art, for example in Pflug et al., 2014, and Reich et al., 2014. According to e.g. Pflug et al., 2014, the catalytic centre responsible for template-directed nucleotide addition is located in the PB lintemal cavity and formed mainly by the highly conserved RdRp motifs pre-A/F and A-E. Motif pre-A/F is partly contained in the fingertips, a loop (residues 222-246) that extends from the fingers towards the thumb domain and the tip of which is stabilized by contacts with PA helix. Whereas HepC and Norwalk virus polymerases have two fingertip loops (one corresponding to motif F and the other closer to the polymerase N terminus), influenza polymerase PBl-Nteris analogous to the second loop with residues 24-38 crossing from thumb to fingers in intimate association with the fingertips. Several conserved basic residues from motif pre-A/F are likely to be involved in template binding, and NTP channel ligand binding. Motif A contains the conserved active site Asp 305, which, together with Asp 445 and Asp 446 on motif C, coordinate two divalent metal ions and promote catalysis. These residues have been shown to be essential for PB1 activity. Motif B has a characteristic methionine-rich loop in PB 1 (406-GMMMGMF), and is probably involved in stabilizing the base-pair between the incoming NTP and the template. Motif D contains conserved Lys 480 and Lys 481 residues (involved in NTP binding) and is stabilized by contacts with PA helix (656-663) and the PA peptide 671-684. Motif E forms another beta- hairpin containing conserved residues thought to stabilize the position of the substrate/priming NTP. As in other polymerases, a narrow tunnel, lined with positively charged residues, connects the internal cavity to the outside and this is presumed to attract and channel NTPs into the active site electrostatically. In PB 1, this putative NTP tunnel directly leads to the tip of the putative priming loop and involves highly conserved PB1 basic residues Arg 45, Lys 235, Lys 237 and Arg 239 (motif F3), Lys 308 (motif A), and Lys 480 and Lys 481 (motif D). A second tunnel constitutes the putative template entrance channel that is lined by conserved residues from all three subunits. The PB1 RNA synthesis active site preferably contains an amino acid sequence as denoted in SEQ ID NO:8 SEQ ID NO:8 shows amino acid residues within 12 A of the catalytic center of influenza B/Memphis polymerase. The residues within the catalytic center among influenza A and B strains are highly conserved, hence, when referring to the PB 1 RNA synthesis active site to have an amino acid as set forth in SEQ ID NO:8, also a sequence at least about 80% identical to SEQ ID NO:8 is encompassed. At least about 80% identical includes at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to SEQ ID NO: 8.

The terms“RNA cap”,“capped primer” and“capped” refers to a cap structure found on the 5' end of an RNA molecule and may consist of a guanine nucleotide connected to the RNA via an unusual 5' to 5' triphosphate linkage. This guanosine is preferably methylated on the 7 position. Further modifications include the possible methylation of the 2' hydroxy-groups of the first 3 ribose sugars of the 5' end of the RNA.

As used herein, the terms“primer”,“primer strand”,“RNA primer strand” and the like all denote a strand of ribonucleotides of a specific sequence and may be interchangeably used. Likewise, the terms“template”,“template strand”,“RNA template strand” and the like all denote a further strand of ribonucleotides of a specific sequence and may be interchangeably used. Likewise, the terms“activator”,“activator strand”,“RNA activator strand” and the like all denote a further strand of ribonucleotides of a specific sequence and may be interchangeably used. If the activator strand is connected via hydrogen bonds between complementary nucleotides with the template strand, the resulting construct is referred to as“promoter”.

The term "nucleotide" as used herein refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the G position, including 2'-deoxy and 2'- hydroxyl forms, e.g., as described in Komberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) and further include, but are not limited to, synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, N.Y., 1980). Preferred nucleotides according to the present invention are ribonucleotides. The term“type of nucleotides” refers to the backbone of the nucleotides, i.e. to ribose or deoxyribose or an artificial or modified backbone of the nucleotides. Non limiting examples of modified nucleotides as in the meaning of the present invention include 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2’-0-methylcytidine, 5- carboxymethylaminomethyl-2-thiouridine, queuosine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2’-0-methylpseudouridine, beta D-galactosylqueuosine, 2’-0- methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1- methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2- methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6- methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-

2-thiouridine, beta D-mannosylqueuosine, 5-methoxycarbonylmethyl-2-thiouridine, 5- methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine, N-((9-beta-D- ribofuranosylpurine-6-yl)N-methylcarbamoyl)threonine, wybutoxosine, uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid, pseudouridine, 2-thiocytidine, 5-methyl-2- thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine, N-((9-beta-D-ribofuranosylpurine-6- yl)-carbamoyl)threonine, 2’-0-methyl-5-methyluridine, 2’-0-methyluridine, wybutosine, and

3 -(3 -amino-3 -carboxy-propyl)uri dine.

The RNA cap binding domain of PB2 can be described also as PB2 cap binding domain and RNA cap binding pocket. The term“PB2 cap binding site” refers to the minimal polypeptide fragment of PB2 that comprises the RNA binding pocket in its native three-dimensional structure.

As used herein, the term“influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site” means that all of the influenza polymerase, the variant and the fragment thereof comprise at least the PB1 RNA synthesis active site and optionally further structures or subunits of influenza polymerase such as but not limited to PA and PB2. According to the present disclosure, variants and fragments of the influenza polymerase are capable of binding the nucleic acid construct of the present invention. As used herein, the term“nucleic acid construct” refers to at least two nucleic acid strands that have between four and five consecutive complementary nucleotides which allow a base to base pairing via hydrogen bonds. It is, however, to be understood that the individual strands of the nucleic acid construct may be provided without any hydrogen bonds formed therebetween. Upon use of the nucleic acid construct in e.g. one of the methods of the present invention, the hydrogen bonds will form between complementary nucleotides. It is therefore possible to consecutively provide a first nucleic acid strand, then add the second nucleic acid strand, and optionally add the third nucleic acid strand. The order in which the nucleic acid strands are added is not important. Thus, the nucleic acid strands can be added to an influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site consecutively essentially in any order or simultaneously. It is also possible to allow base to base pairing via hydrogen bonds of two nucleic acid strands such as the template and the activator strand, and then adding the third strand, in this non-limiting example the primer strand, which will then form base to base pairing via hydrogen bonds with complementary nucleotides in the template strand (Figs. 5 A to C). It is emphasized that also a primer-template strand duplex can be added first, to which optionally the activator strand is added, which will then form base to base pairing via hydrogen bonds with complementary nucleotides in the template strand. Thus, the order of providing the nucleic acid strands of the nucleic acid construct of the present invention either alone or in already bound form is of no particular relevance and may be adapted to individual needs. It is however preferred to provide a combination of the template strand and the activator strand with hydrogen bonds between their consecutive complementary nucleotides, and to subsequently add the primer strand, which will form hydrogen bonds with consecutive complementary nucleotides of the template strand. Thus, according to the principles of the present invention, the nucleic acid construct can comprise the primer strand and the template strand. The activator strand can be optionally added to the construct or to a reaction mixture comprising the influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site, and the nucleic acid construct. Alternatively, a nucleic acid construct according to the principles of the present invention may comprise the template strand and the activator strand connected to each other via base to base pairings between consecutive complementary nucleotides. The primer strand may then be added to this pre-construct or to a reaction mixture comprising the influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site, and the pre-construct. The term "recombinant vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

"Recombinant host cell", as used herein, refers to a host cell that comprises a polynucleotide that codes for the construct of interest, i.e., the nucleic acid construct according to the invention. This polynucleotide may be found inside the host cell (i) freely dispersed as such, (ii) incorporated in a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. The recombinant cell can be used for expression of a polynucleotide of interest or for amplification of the polynucleotide or the recombinant vector of the invention. The term "recombinant host cell" includes the progeny of the original cell which has been transformed, transfected, or infected with the polynucleotide or the recombinant vector of the invention. A recombinant host cell may be a bacterial cell such as an E. coli cell, a yeast cell such as Saccharomyces cerevisiae or Pichia pastoris, a plant cell, an insect cell such as SF9 or Hi5 cells, or a mammalian cell. Preferred examples of mammalian cells are Chinese hamster ovary (CHO) cells, green African monkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HELA cells, and the like.

As used herein, the term "crystal" or "crystalline" means a structure (such as a three- dimensional solid aggregate) in which the plane faces intersect at definite angles and in which there is a regular structure (such as internal structure) of the constituent chemical species. The term "crystal" can include any one of: a solid physical crystal form such as an experimentally prepared crystal, a crystal structure derivable from the crystal (including secondary and/or tertiary and/or quaternary structural elements), a 2D and/or 3D model based on the crystal structure, a representation thereof such as a schematic representation thereof or a diagrammatic representation thereof, or a data set thereof for a computer. In one aspect, the crystal is usable in X-ray crystallography techniques. Here, the crystals used can withstand exposure to X-ray beams and are used to produce diffraction pattern data necessary to solve the X-ray crystallographic structure. A crystal may be characterized as being capable of diffracting X- rays in a pattern defined by one of the crystal forms depicted in Blundell and Johnson, 1976.

As used herein, the term "constructing a computer model" includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models. The term "modeling" includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry, and other structure-based constraint models.

The term "fitting program operation" refers to an operation that utilizes the structure coordinates of a chemical entity, binding pocket, molecule or molecular complex, or portion thereof, to associate the chemical entity with the binding pocket, molecule or molecular complex, or portion thereof. This may be achieved by positioning, rotating or translating the chemical entity in the binding pocket to match the shape and electrostatic complementarity of the binding pocket. Covalent interactions, non-covalent interactions such as hydrogen bond, electrostatic, hydrophobic, van der Waals interactions, and non-complementary electrostatic interactions such as repulsive charge-charge, dipole-dipole and charge-dipole interactions may be optimized. Alternatively, one may minimize the deformation energy of binding of the chemical entity to the binding pocket.

As used herein, the term "test compound" refers to an agent comprising a compound, molecule, or complex that is being tested for its ability to bind to influenza polymerase or variants or fragments thereof comprising the PB 1 RNA synthesis active site. Test compounds can be any agents including, but not restricted to, peptides, peptoids, polypeptides, proteins (including antibodies), lipids, metals, nucleotides, nucleotide analogs, nucleosides, nucleic acids, small organic or inorganic molecules, chemical compounds, elements, saccharides, isotopes, carbohydrates, imaging agents, lipoproteins, glycoproteins, enzymes, analytical probes, polyamines, and combinations and derivatives thereof. The term“small molecules” refers to molecules that have a molecular weight between 50 and about 2,500 Daltons, preferably in the range of 200-800 Daltons. In addition, a test compound according to the present invention may optionally comprise a detectable label. Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds. Well known methods may be used for attaching such a detectable label to a test compound. The test compound of the invention may also comprise complex mixtures of substances, such as extracts containing natural products, or the products of mixed combinatorial syntheses. These can also be tested and the component that binds to the target polypeptide fragment can be purified from the mixture in a subsequent step. Test compounds can be derived or selected from libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, CA), or Aldrich (Milwaukee, WI). A natural compound library is, for example, available from TimTec LLC (Newark, DE). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal cell and tissue extracts can be used. Additionally, test compounds can be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures. A collection of compounds made using combinatorial chemistry is referred to herein as a combinatorial library.

The term “antibody” refers to both monoclonal and polyclonal antibodies, i.e., any immunoglobulin protein or portion thereof which is capable of recognizing an antigen or hapten, i.e., the influenza polymerase or variant or fragment thereof comprising the PB1 RNA active synthesis site, preferably recognizing an antigen or hapten of the PB 1 RNA active synthesis site. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')₂, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies such as humanized antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide (Harlow et al., 1990).

As described herein, a compound“inhibiting influenza RNA polymerase” refers to a compound that is capable of binding to influenza RNA polymerase, where it has an effect on the active site so that transcription is reduced, hampered or inhibited. For example, upon binding of the compound to influenza RNA polymerase, the three dimensional conformation of the active site can be altered. Alternatively, a compound inhibiting influenza RNA polymerase may refer to a compound that targets the influenza polymerase PB 1 RNA synthesis active site, for example by being incorporated into viral RNA and stopping further elongation or disturbing the RNA chain. The active site of RNA polymerase is well known in the art and described in literature (e.g. Li, 2001; Yuan et al., 2009; Nudler, 2010; Boivin et al., 2010; Te Velthuis & Fodor, 2016).

The molecular biology methods applied in this application are generally known to the person skilled in the art (for standard molecular biology methods see Sambrook et al., 1989, which is incorporated herein by reference). For example, RNA can be isolated from Influenza virus infected cells and cDNA generated applying reverse transcription polymerase chain reaction (RT-PCR) using either random primers (e.g., random hexamers of decamers) or primers specific for the generation of the fragments of interest. The fragments of interest can then be amplified by standard PCR using fragment specific primers.

The present inventors identified the problem with visualizing the actual transcription initiation state of influenza polymerase to be the short complementary overlap between ribonucleotides of the primer and the template strand. Native vRNA sequences provide an overlap between complementary ribonucleotides and thus binding between these ribonucleotides over a stretch of only three ribonucleotides. Using an artificially extended template strand, the nucleic acid construct of the present invention provides a more stable binding between template strand and primer strand, which allows the desired crystal structures to be obtained. For the first time, the present invention provides the basis for identifying, selecting, or designing potential compounds which target the influenza RNA polymerase in its active state and interfere with transcription.

According to a first aspect, the present invention provides a nucleic acid construct comprising a primer strand and a template strand, the primer strand having the sequence 5'-Xi-X_m-3' and the template strand having the sequence 3'-Y₁-Y_n-5', wherein X and Y are independently nucleotides, wherein m is an integer of from 12 to 16 and n is an integer of from 13 to 50, wherein between five and six consecutive nucleotides of the primer strand and of the template strand are complementary to each other such that

(i) nucleotides X_m-4, X_m-3, X_m-2, X_m-i, and X_m of the primer strand are complementary to nucleotides Yi, Y₂, Y₃, Y₄ and Y₅ of the template strand, or

(ii) nucleotides X_m-5, X_m-4, X_m-3, X_m-2, X_m-i, and X_m of the primer strand are complementary to nucleotides Yi, Y₂, Y₃, Y4, Y5 and Y₆ of the template strand. The nucleotides are preferably selected among deoxyribonucleotides and ribonucleotides. Nucleotides can be selected among the group consisting of but not limited to adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, synthetic nucleosides having modified base moieties and/or modified sugar moieties. According to a preferred embodiment, at least 50% of the nucleotides of the primer strand and of the template strand are ribonucleotides. Preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the nucleotides of a strand are ribonucleotides selected from the group consisting of adenine, guanine, cytosine, and uracil. The strands may also consist of a mixture of different species of nucleotides. For example, a strand may consist of a first type of nucleotides such as deoxyribonucleotides and a second type of nucleotides such as ribonucleotides. Strands may also consist of different combinations of nucleotides such as ribonucleotides and synthetic or modified nucleotides. It is also possible to combine a first strand comprising one type of nucleotides such as ribonucleotides, and a second strand comprising a different type of nucleotides. It is also possible to combine strands having mixtures of different types of nucleotides and to combine such strands with strands comprising one type of nucleotides only. This applies for all nucleic acid strands of the nucleic acid construct of the present invention, i.e. for the primer strand, the template strand, and the activator strand and their combinations.

The consecutive complementary nucleotides allow building of hydrogen bonds between the primer strand and the template strand over a length of between four and five nucleotides, giving rise to a nucleic acid construct in which primer and template are bound to each other. Although at least four and preferably five consecutive nucleotides of the primer strand are complementary to a respective number of nucleotides of the template strand, it is emphasized that the present invention can also be put into practice with more than six consecutive nucleotides of the primer strand being complementary to a respective number of nucleotides of the template strand. For example, there can be seven, eight, nine, or ten consecutive nucleotides of the primer strand being complementary to a respective number of nucleotides of the template strand.

The number of nucleotides of the primer strand is between 12 and 16, accordingly m is indicated to be an integer of from 12 to 16. This range includes all subranges, meaning that m can be any integer of 12, 13, 14, 15 and 16, and any subrange thereof, i.e. 13 to 14, 13 to 15, 13 to 16, 14 to 16, 15 to 16, 14 to 15, and 15 to 16. All intercombinations within the ranges disclosed herein are explicitly disclosed. The number of nucleotides of the template strand is between 13 and 50, accordingly n is indicated to be an integer of from 14 to 50. This range includes all subranges, meaning that n can be any integer of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, and any subrange thereof, such as 14 to 25, 15 to 20, 16 to 19, 17 to 18 and not limited thereto. Preferably, n is 19 to 23, 20 to 22 and most preferably 21. All intercombinations within the ranges disclosed herein are explicitly disclosed.

With the disclosed subranges for the length of the primer strand and the template strand, the nucleic acid construct of the present invention may have according to a preferred embodiment a template strand length of 18 nucleotides and a primer strand length of 13 to 15 nucleotides. In a further preferred embodiment the template strand has a length of 18 nucleotides and the primer strand has a length of 14 nucleotides. In a further preferred embodiment the template strand has a length of 18 nucleotides and the primer strand has a length of 15 nucleotides. According to alternative embodiments, the template strand may have a length of 20, 21 or more nucleotides and the primer strand may have a length of 13, 14, 15 or 16 nucleotides. It is noted in this respect that any length of the primer strand can be combined with any length of the template strand as long as the indicated minimum number of nucleotides is taken into account.

In an exemplary embodiment, which is applicable to all lengths of the primer and template strands as noted herein above, the primer strand has the sequence of m⁷GpppXi-X_m.5AAUAG- 3', and the template strand has the sequence 3'-UUAUCY₆-Y_n-5'. According to a further exemplary embodiment, which is applicable to all lengths of the primer and template strands as noted herein above, the primer strand has the sequence of m⁷GpppXi-X_m.6AAUAGC-3', and the template strand has the sequence 3'-UUAUCY₆-Y_n-5'. According to a further exemplary embodiment, the primer has the sequence of m⁷Gppp-GAAUGCUAUAAUAG-3' (m⁷Gppp- SEQ ID NO: l) and the template has the sequence of 3 '-UUAUCGUCUUCGUCUCC AUAU- 5' (SEQ ID NO:2). According to an alternative embodiment, the template has the sequence of 3'-UUAUCUUCUUCGUCUCCAUAU-5' (SEQ ID NO: 10). According to a further exemplary embodiment, the primer has the sequence of m⁷Gppp-GAAUGCUAUAAUAGC-3' (m⁷Gppp- SEQ ID NO:3) and the template has the sequence of 3 '-UUAUCGUCUUCGUCUCC AUAU- 5' (SEQ ID NO:2) or 3'-UUAUCUUCUUCGUCUCCAUAU-5' (SEQ ID NO: 10). In accordance with the present invention, the sequences for template and primer may provide a complementary coupling between the primer and the template as schematically and exemplarily illustrated in the following:

Template strand

Primer strand

It will be understood that the coupling via complementary nucleotides as indicated above can be achieved with any complementary nucleotides and that the present invention is not limited to the specific nucleotides and sequences indicated above, which merely represent an exemplary embodiment of the present invention.

For providing a more natural binding to the influenza polymerase, the nucleic acid construct of the present invention further comprises an activator strand having the sequence 5'-Z_rZ₀-3', wherein Z is independently selected from the group consisting of adenine, uracil, guanine and cytosine, wherein o is an integer of from 12 to 18. The activator strand has at least two consecutive nucleotides being complementary to the same number of consecutive nucleotides in the template strand. The at least two consecutive complementary nucleotides start in the activator strand with nucleotide Z₁₁. Depending on the length of complementary nucleotides between the primer strand and the template strand, the respective at least two consecutive nucleotides of the template strand complementary to the activator strand start with nucleotide Y i2 in case of alternative (i), i.e. five consecutive nucleotides are complementary between primer and template strand, and with nucleotide Y₁₃ in case of alternative (ii), i.e. six consecutive nucleotides are complementary between primer and template strand. This structure will leave a bridge of six or seven nucleotides on the template strand between the base to base pairings of the template-primer strands and template-activator strands. A non-limiting exemplary embodiment of a respective nucleic acid construct with build hydrogen bonds between respective complementary nucleotides of different strands is illustrated in the following: Template strand

The activator strand may have a length of at least 12 nucleotides and it is preferred that the length is between 13 and 17, even more preferred of from 14 to 16 nucleotides. The length of the activator thus can be 12, 13, 14, 15, 16 nucleotides or longer, such as 17, 18, 19, 20 and even more such as 30, 40 or 50 nucleotides. All intercombinations within the ranges disclosed herein are explicitly disclosed. According to a preferred embodiment of the present invention, the activator strand has a length of 14 nucleotides, preferably with the sequence of 5'- AGUAGUAAC AAGAG-3 ' (SEQ ID NO:4). According to a further preferred embodiment of the present invention, the activator strand has a length of 16 nucleotides, preferably with the sequence of 5’-AGUAGUAACAAGAGGG-3' (SEQ ID NO: 8).

It is noted that any length of the activator strand can be combined with any length of the template strand and any length of the primer strand as long as the indicated minimum number of nucleotides is taken into account. A preferred embodiment of the present invention comprises a primer strand having a length of 14 nucleotides, a template strand having a length of 21 nucleotides and an activator strand having a length of 14 nucleotides. A further preferred embodiment of the present invention comprises a primer strand having a length of 15 nucleotides, a template strand having a length of 21 nucleotides and an activator strand having a length of 14 nucleotides. A third preferred embodiment of the present invention comprises a primer strand having a length of 15 nucleotides, a template strand having a length of 21 nucleotides and an activator strand having a length of 16 nucleotides.

It is further emphasized that the number of consecutive complementary nucleotides between the activator strand and the template strand can be larger than 2, for example 3, 4, 5, 6 or more such as 7, 8, 9 and 10. According to a preferred embodiment, at least three or more, preferably at least four or at least five consecutive nucleotides of the activator strand are complementary to at least four consecutive nucleotides of the template strand. Nucleotides Z₁ to Z₁₀ of the activator strand may form a stem-loop structure such that this loop structure precedes the consecutive nucleotides Z₁₁ to Z_x with x being an integer of from 12 to 50 (in the example below Z₁₁ to Z₁₄ representing AGAG) complementary to the template strand, as exemplary indicated in the following:

The activator strand may further have a triphosphate bridge connected to its 5' end.

According to a very preferred embodiment, the 3' end of the activator strand is connected to the 5' end of the template strand.

According to a preferred embodiment, the nucleic acid construct of the present invention comprises a primer strand having the sequence as denoted in SEQ ID NO: 1 or 3, and a template strand having the sequence as denoted in SEQ ID NO:2. Optionally, the nucleic acid construct may further comprise an activator strand having the sequence as denoted in SEQ ID NO:4. According to a further embodiment, the sequence of the primer strand and/or the template strand and/or the optional activator strand have a sequence at least 80% identical to the sequences identified above. At least 80% identical includes at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, and at least 99% identical to any of the sequences as denoted in SEQ ID NO: 1 to 4.

In some of the methods of the present invention it may be desirable to use different nucleic acid constructs having varying lengths and sequences of the primer, the template and/or the activator strand in order to evaluate the capabilities of compounds for targeting and/or inhibiting influenza polymerase.

The nucleic acid construct of the present invention is particularly suitable in methods for obtaining structural or visual data of influenza RNA polymerase. Thus, according to one aspect, the present invention provides a method for crystallizing an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site. The method comprises the steps of contacting the nucleic acid construct of the present invention with the influenza polymerase or variant or fragment thereof and allowing binding of the nucleic acid construct to the PB1 RNA synthesis active site, and crystallizing the influenza polymerase or variant or fragment thereof with the nucleic acid construct bound thereto. The crystallized complex of the influenza polymerase or variant or fragment thereof to which the nucleic acid construct of the present invention is bound allows obtaining crystal diffraction data of the polymerase or variant or fragment thereof during the nucleotide addition cycle and thus in its active state. The data can be obtained for example by standard X-ray crystallography as is well known in the art (e.g. Drenth, 1999).

According to a preferred embodiment, the nucleic acid construct of the present invention is further allowed to bind to the influenza polymerase PB2 cap binding site. Preferably, the nucleic acid construct of the present invention binds with all or parts of the complementary consecutive nucleotides of the primer and the template strand, between which hydrogen bonds have been formed, to the influenza polymerase PB 1 RNA synthesis active site and with the 5’ end of the primer strand to the influenza polymerase PB2 cap binding site. Such a binding to the PB 1 RNA synthesis active site and the PB2 cap binding site allows even better simulation of the natural processes taking place during influenza polymerase activity. Such simulation can even be improved by the addition of the activator strand to the construct of the present invention, which will bind to a pocket (Fig. 5 and further described in Pflug et aI. 2014) of the influenza polymerase.

Crystals can be grown by any method known to the person skilled in the art including, but not limited to, hanging and sitting drop techniques, sandwich-drop, dialysis, and microbatch or microtube batch devices. It would be readily apparent to one of skill in the art to vary the crystallization conditions disclosed above to identify other crystallization conditions that would produce crystals of influenza with the influenza polymerase PB 1 RNA synthesis active site or variants thereof alone or in complex with a compound. Such variations include, but are not limited to, adjusting pH, protein concentration and/or crystallization temperature, changing the identity or concentration of salt and/or precipitant used, using a different method for crystallization, or introducing additives such as detergents (e.g., TWEEN 20 (monolaurate), LDOA, Brij 30 (4 lauryl ether)), sugars (e.g., glucose, maltose), organic compounds (e.g., dioxane, dimethylformamide), lanthanide ions, or poly-ionic compounds that aid in crystallizations. High throughput crystallization assays may also be used to assist in finding or optimizing the crystallization condition.

Microseeding may be used to increase the size and quality of crystals. In brief, micro-crystals are crushed to yield a stock seed solution. The stock seed solution is diluted in series. Using a needle, glass rod or strand of hair, a small sample from each diluted solution is added to a set of equilibrated drops containing a protein concentration equal to or less than a concentration needed to create crystals without the presence of seeds. The aim is to end up with a single seed crystal that will act to nucleate crystal growth in the drop.

The present invention also provides a method for cryogenic electron microscopy of an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site. The method comprises the steps of:

(ii) freezing the influenza polymerase or variant or fragment thereof with the bound nucleic acid construct. Cryogenic electron microscopy can be performed as is well known in the art and includes techniques such as transmission electron cryomicroscopy.

As with the method for crystallizing an influenza polymerase, more natural results are achieved if the nucleic acid construct is further allowed to bind to the influenza polymerase PB2 cap binding site. Preferably, the nucleic acid construct of the present invention binds with all or parts of the complementary consecutive nucleotides of the primer and the template strand, between which hydrogen bonds have been formed, to the influenza polymerase PB1 RNA synthesis active site and with the 5’ end of the primer strand to the influenza polymerase PB2 cap binding site. Such a binding to the PB1 RNA synthesis active site and the PB2 cap binding site allows even better simulation of the natural processes taking place during influenza polymerase activity. Such simulation can even be improved by the addition of the activator strand to the construct of the present invention, which will bind to a pocket (Fig. 5 and further described in Pflug et al., 2014) of the influenza polymerase. The nucleic acid construct of the present invention is further particularly suitable in methods for testing a function of influenza polymerase and in methods for identifying, selecting or designing a compound which targets the influenza polymerase RNA synthesis active site. Thus, according to one aspect, the present invention provides a method for in vitro testing of a function of influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site. The method comprises the step of providing an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site; adding to the influenza polymerase or variant or fragment thereof the nucleic acid construct of the present invention and allowing binding of the nucleic acid construct to the PB1 RNA synthesis active site; and adding nucleosides or nucleoside analogues. Under conditions allowing binding of the nucleic acid construct to the PB1 RNA synthesis active site, elongation of the primer can be observed. By choosing respective nucleotides or nucleotide analogues, elongation can be interrupted. For example, if only ATP and GTP are provided, elongation of the primer will stop when reaching a guanine or uracil in the template sequence. Alternatively, nucleotide analogues can be used which comprise a blocking group that interrupts further elongation. Such protection groups are well known in the art, such as 3’ -OH protecting groups, for example a 3'-0-NH₂ protecting group or a 3'-0-CH₂N₃ protecting group. Further nucleotides comprising protecting groups are exemplified for example in US 2018/023108.

According to a further aspect, the present invention provides a method for identifying, selecting or designing a compound which inhibits influenza RNA polymerase. The method comprises the steps of:

(i) contacting a PB2 polypeptide or variant or fragment thereof comprising the PB2 cap binding site with the nucleic acid constructof the present invention and with a test compound, and

(ii) analyzing the ability of said test compound to target the influenza polymerase RNA synthesis active site.

In the above described methods of the invention, the nucleic acid construct can be contacted with the PB1 RNA synthesis active site prior to, concomitantly with, or after addition of said test compound. It is again emphasized that for enabling the methods of the present invention, the nucleic acid construct of the present invention must not exhibit base to base bindings via hydrogen bonds upon being provided to the influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site. That is the primer strand, the template strand and optionally the activator strand can be added to the polymerase or variant or fragment thereof concomitantly in any order or simultaneously or with hydrogen bonds already build between the template strand and the primer strand or between the template strand and the activator strand. The reaction conditions allowing binding of the nucleic acid construct also enable formation of hydrogen bonds between complementary nucleotides, allowing the formation of an nucleic acid construct in which the consecutive complementary nucleotides form hydrogen bonds to create base to base pairings.

In the above described methods of the invention, the influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site can be contacted with varying primer, template and/or activator sequences and lengths. This enables analysis of the polymerase active site in different settings and comparing the data for evaluating potential structural differences also under taking into consideration different test compounds.

Compounds that inhibit influenza RNA polymerase can also be identified, selected or designed by using a computer model. Thus, according to one aspect, the present invention provides a method for identifying, selecting or designing a compound which inhibits influenza RNA polymerase. The method comprises the steps of:

(a) constructing a computer model of the influenza polymerase with the nucleic acid construct bound to the PB1 RNA synthesis active site based on the crystal diffraction data obtained with the methods of the invention;

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound;

(d) evaluating the results of said fitting operation to quantify the association between the said compound and the influenza polymerase PB 1 RNA synthesis active site.

The methods for identifying, selecting or designing a compound which inhibits influenza RNA polymerase may further comprise the step of synthesizing the compound. Optionally, the methods further comprise formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s).

The compound to be tested, also referred to as“test compound”, can be any agent including, but not restricted to, peptides, peptoids, polypeptides, proteins (including antibodies), lipids, metals, nucleotides, nucleosides, nucleic acids, small organic or inorganic molecules, chemical compounds, elements, saccharides, isotopes, carbohydrates, imaging agents, lipoproteins, glycoproteins, enzymes, analytical probes, polyamines, and combinations and derivatives thereof. The term“small molecules” refers to molecules that have a molecular weight between 50 and about 2,500 Daltons, preferably in the range of 200-800 Daltons. In addition, a test compound according to the present invention may optionally comprise a detectable label. Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds. According to a preferred embodiment, the compound is selected from the group consisting of a small molecule, a peptide or a protein. A preferred compound is a chain perturbing compound. Preferred chain perturbing compounds include nucleotide analogues that potently inhibit viral replication by being incorporated into viral RNA in competition with one or more of ATP, GTP, CTP and UTP. Preferably, the nucleotide analogue is an NTP analogue. The incorporation of such analogue will lead to lethal mutagenesis of the viral genome. Nucleotide analogues are well known in the field. Individual examples of such compounds are disclosed in Baranovich et al., 2013; Jin et al., 2013; and in Yoon et al., 2018.

Once suitable compounds have been selected, they can be designed or assembled into a single compound or complex. This manual model building is performed using software such as Quanta or Sybyl. Useful programs aiding the skilled person in connecting individual compounds or fragments include, for example, (i) CAVEAT (Bartlett et al., 1989; Lauri and Bartlett, 1994); CAVEAT is available from the University of California, Berkley, CA), (ii) 3D Database systems such as ISIS (MDL Information Systems, San Leandro, CA; reviewed in Martin, 1992), and (iii) HOOK (Eisen et al., 1994); HOOK is available from Molecular Simulations Incorporated, San Diego, CA).

Another approach enabled by this invention, is the computational screening of small molecule databases for compounds that inhibit influenza RNA polymerase. In this screening, the quality of fit of such compounds may be judged either by shape complementarity or by estimated interaction energy (Meng et al., 1992).

Alternatively, a potential binding partner inhibiting influenza RNA polymerase may be designed de novo on the basis of the 3D structure of PB2 in its active state. There are various de novo ligand design methods available to the person skilled in the art. Such methods include (i) LUDI (Bohm, 1992); LUDI is available from Molecular Simulations Incorporated, San Diego, CA), (ii) LEGEND (Nishibata et al., 1991); LEGEND is available from Molecular Simulations Incorporated, San Diego, CA), (iii) LeapFrog (available from Tripos Associates, St. Louis, MO), (iv) SPROUT (Gillet et al., 1993); SPROUT is available from the University of Leeds, UK), (v) GROUPBUILD (Rotstein et al., 1993), and (vi) GROW (Moon et al., 1991).

In addition, several molecular modeling techniques that may support the person skilled in the art in de novo design and modeling of potential compounds targeting the influenza polymerase RNA synthesis active site have been described and include, for example, Cohen et al., 1990; Navia et al, 1992; L. M. Balbes et al.,“A Perspective in Modem Methods in Computer-Aided Drug Design”, Reviews in Computational Chemistry, Vol. 5, K. B. Lipkowitz and D.B. Boyd, Eds., VCH, New York, pp. 37-380 (1994); Guida, 1994).

A molecule designed or selected as inhibiting influenza RNA polymerase may be further computationally optimized so that in its bound state it preferably lacks repulsive electrostatic interaction with the target region. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the binding compound and the binding pocket in a bound state, preferably make a neutral or favorable contribution to the enthalpy of binding. Specific computer programs that can evaluate a compound deformation energy and electrostatic interaction are available in the art. Examples of suitable programs include (i) Gaussian 92, revision C (M. J. Frisch, Gaussian, Incorporated, Pittsburgh, PA), (ii) AMBER, version 4.0 (P.A. Kollman, University of California, San Francisco, CA), (iii) QUANTA/CHARMM (Molecular Simulations Incorporated, San Diego, CA), (iv) OPLS-AA (Jorgensen, 1998), and (v) Insight II/Discover (Biosysm Technologies Incorporated, San Diego, CA). These programs may be implemented, for instance, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages are known to those skilled in the art. Once a molecule of interest has been selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will approximate the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for their efficiency for inhibiting influenza RNA polymerase by the same computer methods described in detail above.

If computer modeling according to the methods described hereinabove indicates inhibition of influenza RNA polymerase, said compound may be synthesized and optionally the ability of said compound to inhibit influenza RNA polymerase can be tested in vitro or in vivo comprising the step of synthesizing said compound, and optionally contacting said compound with influenza polymerase or variant or fragment thereof comprising at least the PB 1 RNA synthesis active site to determine the ability of said compound to inhibit influenza polymerase activity. Methods for synthesizing said compounds are well known to the person skilled in the art or such compounds may be commercially available. Examples for methods for determining said inhibitory effect of the identified compounds are described hereinafter.

An interaction between a PB1 RNA synthesis active site polypeptide and a test compound may be analyzed in form of a pull down assay. For example, the PB 1 RNA synthesis active site polypeptide may be purified and may be immobilized on beads. For example, the PB1 RNA synthesis active site polypeptide can be immobilized on beads and may be contacted, for example, with (i) another purified protein, polypeptide fragment, or peptide, (ii) a mixture of proteins, polypeptide fragments, or peptides, or (iii) a cell or tissue extract, and binding of proteins, polypeptide fragments, or peptides may be verified by polyacrylamide gel electrophoresis in combination with Coomassie staining or Western blotting. Unknown binding partners may be identified by mass spectrometric analysis.

Alternatively, the interaction between the PB1 RNA synthesis active site polypeptide and a test compound may be analyzed in form of an enzyme-linked immunosorbent assay (ELISA)-based experiment. The PB1 RNA synthesis active site polypeptide may be immobilized on the surface of an ELISA plate and contacted first with the nucleic acid construct of the invention and then with the test compound. Binding of the test compound may be verified, for example, for proteins, polypeptides, peptides, and epitope-tagged compounds by antibodies specific for the test compound or the epitope-tag. These antibodies might be directly coupled to an enzyme or detected with a secondary antibody coupled to said enzyme that - in combination with the appropriate substrates - carries out chemiluminescent reactions (e.g., horseradish peroxidase) or colorimetric reactions (e.g., alkaline phosphatase). Binding of compounds that cannot be detected by antibodies might be verified by labels directly coupled to the test compounds. Such labels may include enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds. The test compounds might be immobilized on the ELISA plate and contacted with the soluble PB 1 RNA synthesis active site polypeptide. Binding of said polypeptide may be verified by a PB 1 RNA synthesis active site polypeptide specific antibody and chemiluminescence or colorimetric reactions as described above.

Accordingly, purified soluble PB1 RNA synthesis active site polypeptide may be incubated with the nucleic acid construct of the invention and then with a peptide array. Binding of the PB 1 RNA synthesis active site polypeptide to specific peptide spots corresponding to a specific peptide sequence may be analyzed, for example, by PB1 RNA synthesis active site polypeptide specific antibodies, antibodies that are directed against an epitope-tag fused to the PB1 RNA synthesis active site polypeptide, or by a fluorescence signal emitted by a fluorescent tag coupled to the PB1 RNA synthesis active site polypeptide.

The ability of the test compound to inhibit influenza RNA polymerase can be tested e.g. by testing binding of said compound to influenza polymerase in the presence of the nucleic acid construct of the invention or the ability of said test compound to inhibit binding of said nucleic acid construct is analyzed. A compound is considered to inhibit nucleic acid construct binding if binding is reduced by the compound at the same molar concentration as the nucleic acid construct by more than 20%, by more than 30%, by more than 40%, by more than 50%, preferably by more than 60%, preferably by more than 70%, preferably by more than 80%, preferably by more than 90%. In preferred embodiments, the above-described pull down, ELISA, peptide array, FRET, and co-immunoprecipitation experiments may be carried out in presence of the nucleic acid construct of the present invention, in presence or in absence of a test compound. In one embodiment, the nucleic acid construct is added prior to addition of said test compound. In a further embodiment, the nucleic acid construct is added concomitantly with addition of said test compound. In yet another embodiment, the nucleic acid construct is added after addition of said test compound. It is not necessary that a test compound directly binds to the PB1 RNA synthesis active site. Inhibition of polymerase activity can also be achieved if a test compound binds to any other site on influenza polymerase and having an effect on polymerase activity.

In a preferred embodiment, the ability of the identifiable test compound to interfere with the interaction of PB1 RNA synthesis active site and the nucleic acid construct of the present invention may be tested by incubating influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site with 7-methyl-GTP Sepharose 4B resin (GE Healthcare) in presence or absence of said compound and comparing, preferably quantifying, the amount of bound PB1 RNA synthesis active site polypeptide with and without said compound, e.g., on a coomassie stained SDS PAGE gel or using Western blot analysis.

The ability of the nucleic acid construct of the present invention to associate with the PB 1 RNA synthesis active site in presence or absence of the test compound is evaluated. In one embodiment, this may be achieved by providing a fluorescein-label to the nucleic acid construct as described e.g. in Natarajan et al. (2004). In a further embodiment, radioactively labeled nucleic acid constructs may be incubated with the influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site in presence or absence of a test compound, wherein the nucleic acid construct may be added prior to, concomitantly with, or after addition of the test compound. In another embodiment, a PB 1 RNA synthesis active site polypeptide may be immobilized on a microtiter plate, incubated with a labeled nucleic acid construct of the present invention in presence or absence of a test compound, wherein the nucleic acid construct may be added prior to, concomitantly with, or after addition of the test compound, and binding is analyzed by verifying the presence of the label after thorough washing of the plate. Signal intensities of the nucleic acid construct label in wells with test compound to signal intensities of said label without test compound are compared and a compound is considered to inhibit nucleic acid construct binding to PB1 RNA synthesis active site if binding is reduced by more than 50%, preferably by more than 60%, preferably by more than 70%, preferably by more than 80%, preferably by more than 90% as described above.

In a preferred embodiment, the above-described method for identifying compounds is performed in a high-throughput setting. In a preferred embodiment, said method is carried out in a multi -well microtiter plate as described above using immobilized PB 1 RNA synthesis active site polypeptide and labeled nucleic acid constructs of the present invention. In a preferred embodiment, the test compounds are derived from libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, CA), or Aldrich (Milwaukee, WI). A natural compound library is, for example, available from TimTec LLC (Newark, DE). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be used. Additionally, test compounds can be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures. The nucleic acid construct may be added prior to, concomitantly with, or after the addition of the library compound. The ability of the compound to inhibit binding of the nucleic acid construct of the present invention may be assessed as described above.

In another embodiment, the inhibitory effect of the identified compound on the Influenza virus life cycle may be tested in an in vivo setting. A cell line that is susceptible for Influenza virus infection such as 293T human embryonic kidney cells, Madin-Darby canine kidney cells, or chicken embryo fibroblasts may be infected with Influenza virus in presence or absence of the identified compound. In a preferred embodiment, the identified compound may be added to the culture medium of the cells in various concentrations. Viral plaque formation may be used as read out for the infectious capacity of the Influenza virus and may be compared between cells that have been treated with the identified compound and cells that have not been treated.

The test compound applied in any of the above described methods can be a small molecule. The small molecule can be derived from a library, e.g., a small molecule inhibitor library. Alternatively, said test compound can be a peptide or protein. Said peptide or protein can be derived from a peptide or protein library.

In another embodiment of the above-described methods for computational as well as in vitro identification of compounds that inhibit influenza RNA polymerase and/or inhibit binding of the nucleic acid construct of the present invention to the PB1 RNA synthesis active site, said methods further comprise the step of formulating the identifiable compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s). In another aspect the present invention provides a pharmaceutical composition producible according to the afore-mentioned method. A compound according to the present invention can be administered alone but, in human therapy, will generally be administered in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (see hereinafter).

In the aspect of computational modeling or screening of compounds that inhibit influenza RNA polymerase and/or inhibit binding of the nucleic acid construct of the present invention to the PB 1 RNA synthesis active site, it may be possible to introduce into the molecule of interest chemical moieties that may be beneficial for a molecule that is to be administered as a pharmaceutical. For example, it may be possible to introduce into or omit from the molecule of interest, chemical moieties that may not directly affect binding of the molecule to the target area but which contribute, for example, to the overall solubility of the molecule in a pharmaceutically acceptable carrier, the bioavailability of the molecule and/or the toxicity of the molecule. Considerations and methods for optimizing the pharmacology of the molecules of interest can be found, for example, in "Goodman and Gilman's The Pharmacological Basis of Therapeutics" (Goodmanet al., 1985); W. L. Jorgensen & E. M. Duffy, Bioorg. Med. Chem. Lett., 10, pp. 1155-1158 (2000). Furthermore, the computer program "Qik Prop" can be used to provide rapid predictions for physically significant descriptions and pharmaceutically-relevant properties of an organic molecule of interest. A 'Rule of Five' probability scheme can be used to estimate oral absorption of the newly synthesized compounds (Lipinski et al., 1997). Programs suitable for pharmacophore selection and design include (i) DISCO (Abbot Laboratories, Abbot Park, IL), (ii) Catalyst (Bio-CAD Corp., Mountain View, CA), and (iii) Chem DBS-3D (Chemical Design Ltd., Oxford, UK).

For the purpose of the methods of the present invention, the nucleotides of the strands of the nucleic acid construct are preferably at least 50% ribonucleotides, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the nucleotides.

The compounds identified by the present invention are particularly suitable for use in medicine. Thus, the present invention provides a compound identified, selected, designed or obtained by any of the methods of the present invention, and a pharmaceutical composition comprising the compound of the present invention. The pharmaceutical composition preferably or optionally further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition contemplated by the present invention may be formulated in various ways well known to one of skill in the art. For example, the pharmaceutical composition of the present invention may be in solid form such as in the form of tablets, pills, capsules (including soft gel capsules), cachets, lozenges, ovules, powder, granules, or suppositories, or in liquid form such as in the form of elixirs, solutions, emulsions, or suspensions.

Solid administration forms may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, and starch (preferably com, potato, or tapioca starch), disintegrants such as sodium starch glycolate, croscarmellose sodium, and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions, solutions, elixirs, and emulsions suitable for oral administration the compound may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol, and glycerin, and combinations thereof.

The pharmaceutical composition of the invention may contain release rate modifiers including, for example, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, camauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer, and mixtures thereof.

The pharmaceutical composition of the present invention may be in the form of fast dispersing or dissolving dosage formulations (FDDFs) and may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavoring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol. For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

The pharmaceutical composition of the present invention suitable for parenteral administration is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.

The pharmaceutical composition suitable for intranasal administration and administration by inhalation is best delivered in the form of a dry powder inhaler or an aerosol spray from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoro- alkane such as 1, 1,1,2-tetrafluoroethane (HFA 134A.TM.) or 1,1, 1,2,3,3,3-heptafluoropropane (HFA 227EA.TM.), carbon dioxide, or another suitable gas. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate.

According to a further aspect, the present invention provides the compound or the pharmaceutical composition of the present invention for use in treating, ameliorating, or preventing a disease caused by viral infections with negative-sense single stranded RNA viruses. Preferably, the disease is caused by viral infections of the Mononegavirales order comprising the Bornaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae families, more preferably wherein said disease condition is caused by the Orthomyxoviridae, Arenaviridae, or Bunyaviridae families. Preferably, the disease is caused by a virus selected from the group consisting of Borna disease virus, Marburg virus, Ebola virus, Sendai virus, Mumps virus, Measles virus, Human respiratory syncytial virus, Turkey rhinotracheitis virus, Vesicular stomatitis Indiana virus, Nipah virus, Henda virus, Rabies virus, Bovine ephemeral fever virus, Infectious hematopoietic necrosis virus, Thogoto virus, Influenza A virus, Influenza B virus, Influenza C virus, Hantaan virus, Crimean-congo hemorrhagic fever virus, Rift Valley fever virus, and La Crosse virus, most preferably Influenza A virus. The compound or the pharmaceutical composition of the present invention is thus particularly suitable for use in the treatment of diseases such as but not limited to hemorrhagic fever, rabies, influenza, mumps or measles.

The compound or pharmaceutical composition for use in treating, ameliorating, or preventing said disease conditions can be administered to an animal patient, preferably a mammalian patient, preferably a human patient, orally, buccally, sublingually, intranasally, via pulmonary routes such as by inhalation, via rectal routes, or parenterally, for example, intracavernosally, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally intrastemally, intracranially, intramuscularly, or subcutaneously, they may be administered by infusion or needleless injection techniques.

Also disclosed are methods of treatment comprising administering a person in need thereof a pharmaceutical composition comprising the compound identified by the methods of the invention. Optionally, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

The methods disclosed herein encompass methods of treating, ameliorating, or preventing a disease caused by viral infections with negative-sense ssRNA viruses.

According to the methods disclosed herein, the disease is caused by viral infections of the Mononegavirales order comprising the Bomaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae families, preferably wherein said disease condition is caused by the Orthomyxoviridae, Arenaviridae, or Bunyaviridae families.

According to the methods disclosed herein, the disease is caused by a virus selected from the group consisting of Boma disease virus, Marburg virus, Ebola virus, Sendai virus, Mumps virus, Measles virus, Human respiratory syncytial virus, Turkey rhinotracheitis virus, Vesicular stomatitis Indiana virus, Nipah virus, Henda virus, Rabies virus, Bovine ephemeral fever virus, Infectious hematopoietic necrosis virus, Thogoto virus, Influenza A virus, Influenza B virus, Influenza C virus, Hantaan virus, Crimean-congo hemorrhagic fever virus, Rift Valley fever virus, and La Crosse virus. According to the methods disclosed herein, the disease is selected from the group consisting of hemorrhagic fever, rabies, influenza, mumps and measles.

In order to allow production of the nucleic acid construct of the present invention, the present invention also provides a polynucleotide encoding for the nucleic acid construct of the present invention, a recombinant vector comprising the polynucleotide of the present invention, and a recombinant host cell comprising the polynucleotide or the recombinant vector of the present invention.

Further provided and according to another aspect is a construct comprising an influenza PB1 RNA synthesis active site and the nucleic acid construct of the present invention bound thereto. Also provided is a construct comprising an influenza PB2 cap binding site and the nucleic acid construct of the present invention bound thereto.

According to one preferred embodiment, the present invention provides a construct comprising an influenza polymerase and the nucleic acid construct of the present invention bound to the PB 1 RNA synthesis active site of the influenza polymerase. Preferably, the nucleic acid construct is also bound to the PB2 cap binding site of the influenza polymerase.

The conserved catalytic core of viral RNA-dependent RNA polymerases share six characteristic structural elements (motifs A to F), which are responsible, together with two divalent cations (denoted A and B), for controlling the nucleotide addition cycle (Te Velthuis et al., 2017). With the present invention it is now possible to see how these motifs, and other parts of the influenza polymerase core interact with the nucleic acid construct of the present invention and with incoming nucleotides during transcription initiation and elongation. The present disclosure provides the first characterization of the active site dynamics of influenza polymerase during the nucleotide addition cycle.

The results obtained with the nucleic acid construct and the methods of the present invention are summarized in Figure 1, schematically showing the transitions between the (A) pre- initiation, (B) initiation, and (C) elongation states for transcribing influenza polymerase.

The present invention further provides a first structure-based, mechanistic understanding of the cap-dependent transcription cycle of influenza polymerase from initiation to termination and any features, such as priming loop extrusion and strand separation are also likely to occur in the same way during unprimed replication. By varying the template and primer sequence and/or length in combination with various NTPs or nucleoside analogues, the methods presented herein can be used to capture multiple RNA synthesis intermediates. Structural analysis of such states, by the complementary techniques of X-ray crystallography and/or cryoEM, not only gives further insight into the mechanisms of RNA synthesis by influenza polymerase, but is also valuable for anti-influenza drug development. In this respect, knowledge of the different configurations of the motif B loop is of particular help in optimizing specific inhibitors, such as nucleoside analogues, inhibiting influenza RNA polymerase.

As shown in Figures 2 and 3, in the promoter bound pre-initiation state the template strand’s 3' end takes a sinuous route into the active site. Eventually stochastic displacement of the priming loop tip allows primer-template hybridization and incorporation of the first few nucleotides. Template translocation can initially occur by straightening of the template entrance pathway but eventually primer melting has to occur. The associated collapse of the b-ribbon and closure of the initial template entry tunnel disfavors backtracking, re-formation of the promoter and abortive product formation. NTP incorporation driven translocation provides a counter force to the priming loop, which back extrudes in steps through the active site cavity and out of the template exit channel. Thumb rotation and active cavity opening allow growth of the template- product duplex to the full nine base pairs characteristic of the elongation state before abutting against the PB2 helical lid which forces strand separation.

Hence, during the early stages of transcription by influenza polymerase, the initiation to elongation transition involves progressive priming loop extrusion coupled to active cavity opening. This accommodates growth of the product-template duplex to a steady state of nine base pairs in the post-translocation state. Subsequently, the PB2 helical lid enforces strand separation, directing the template into the newly opened template exit channel, whereas the increasingly bulged capped mRNA transcript eventually forces the release from the cap-binding domain. Concomitantly, template translocation causes promoter disruption resulting in collapse of the PB1 b-ribbon onto the vRNA 5' end and template entry channel remodeling. The steric hindrance of the priming loop to RNA duplex growth is consistent with priming loop deletions increasing transcription and may also explain its role in the process of template backtracking and realignment during early transcription (Te Velthuis et aI. 2016 and 2017; Oymans et aI. 2018). The intermediate position of the priming loop that was observed in the initiation state structure correlates well with a previously proposal, based on the anomalously high Km for ATP incorporation at template position 5, that a significant structural transition had to occur to permit elongation at this stage (Klumpp et al., 1998). Hepatitis C (HepC) virus polymerase has a b-hairpin priming loop similar to that of influenza polymerase and an analogous extrusion mechanism has been implicated in the transition to elongation, although a significantly larger rotation (about 20°) of the thumb domain is observed (Appleby et al., 2015; Mosley et al., 2012). However to obtain the HepC elongation structure, deletion of the priming loop was required (Appleby et al., 2015; Mosley et al., 2012). Thus the influenza polymerase structures reported here are the first to show successive configurations of an intact priming loop during the transition to elongation.

In the elongation state, the nucleic acid construct comprising a primer-template-strand duplex fills the active site cavity and residues lining it make numerous van der Waals and polar interactions to the backbone of both strands (Fig. 2B). Of particular note is the flexible methionine rich motif B loop, which is observed in several conformations during the pre- to post translocation transition (Fig. 3C, Fig. 4A-F). In one positon, PB1/Met410 would clash with the base in the +1 template position. It is assumed that the motif B loop pushing on the template strand might be part of the mechanism promoting product/template translocation after nucleotide incorporation. At the other end of the template-product duplex, flipping of Tyr207 could act as a shutter during translocation, allowing individual template bases to sequentially enter the exit tunnel.

EXAMPLES

To circumvent the problems and enable visualization of the initiation and early elongation steps of transcription by influenza polymerase, a nucleic acid construct comprising a primer strand and a template strand having at least five consecutive nucleotides at its 3’ end that are complementary to at least five consecutive nucleotides at the primer strand 3’ end according to the present invention is provided.

In the specific examples that follow, two different variants of the nucleic acid construct of the present invention have been used:

1) a construct comprising: a 15-mer primer having the sequence m⁷GpppGAAUGCUAUAAUAGC-3' (m⁷Gppp-SEQ ID NO:3); a 21-mer template having the sequence 3'-UUAUCGUCUUCGUCUCCAUAU-5' (SEQ ID NO:2); and a 14-mer activator strand having the sequence 5'-AGUAGUAACAAGAG-3' (SEQ ID NO:4).

2) a construct comprising: a 14-mer primer having the sequence m⁷Gppp- GAAUGCUAUAAUAG-3' (m⁷Gppp-SEQ ID NO: l); a 21-mer template having the sequence 3'-UUAUCGUCUUCGUCUCCAUAU-5' (SEQ ID NO:2); and a 14-mer activator strand having the sequence 5'-AGUAGUAACAAGAG-3' (SEQ ID NO:4).

3) a construct comprising: a 15-mer primer having the sequence m⁷GpppGAAUGCUAUAAUAGC-3' (m⁷Gppp-SEQ ID NO:3); a 21-mer template having the sequence 3'-UUAUCGUCUUCGUCUCCAUAU-5' (SEQ ID NO:2); and a 16-mer activator strand having the sequence 5'-AGUAGUAACAAGAGGG-3' (SEQ ID NO:8).

Example 1

Influenza B (FluB) virus /Memphis polymerase (SEQ ID NO: 9) was co-cry stallized with nucleic acid constructs identified under 1) and 2) above. Anisotropic diffraction data were integrated using an ellipsoidal mask to a maximum resolution of 2.9 to 3.1 A, as shown in table

1

Table 1 : X-ray data collection and refinement statistics

Structure solution revealed clear electron density for the complete primer-template RNA construct. Whereas the promoter structure was unchanged from that previously observed (Reich et al., 2017 and 2014), a new feature was the five base pair primer-template duplex in the active site cavity, mimicking an initiation complex (Fig. 5B). Using a cap-dependent transcription assay, it was confirmed that this complex was functional for RNA synthesis and yielded the expected elongation products (Fig. 7). In particular, addition of only ATP and GTP to the initiation complex extended the primer by five nucleotides, before lack of CTP caused polymerase stalling (Fig. 7). After four hours incubation such a stalled elongation reaction sample was plunge frozen on cryo-electron microscope (cryoEM) grids and data permitting 3D single particle reconstruction were collected.

One structure, at an overall resolution of 3.16 A, shows a pre-initiation state with unperturbed promoter base pairing, the template 3' end located on the surface of the protein and the capped RNA primer partially ordered (Fig. 5A) (see table 2).

Table 2: CryoEM data collection, refinement and validation statistics

This conformation is similar to previous crystal structures of promoter bound influenza B polymerase, but with the position of the PB 1 b-ribbon now being undistorted by crystal contacts (compare Figs. 5 A and 5B). A further cryoEM structure, at an overall resolution of 3.20 A, shows an early elongation state the template having translocated by five nucleotides, resulting -- - §§markable nine base pair template-product duplex in the active site cavity (Fig. 5C). The cryoEM map is of sufficient resolution to confirm that the duplex sequence is as expected. The transition from initiation to elongation state involves a 4.5° outward rotation of the thumb domain (PB 1/509-670) together with the PB2/N1-N2 domains (PB2/54-153). The structures (Figs. 5 A-C) reveal the successive transitions that transcribing influenza polymerase undergoes in progressing from pre-initiation to early elongation.

Progressive extrusion of the priming loop allows growth of the product-template duplex

A priming loop inserted into the active site cavity has been shown to promote initiation of un primed RNA synthesis for several viral polymerases (Butcher et al., 2001; Appleby et al., 2015). For influenza polymerase, the priming loop plays a mechanistic role in vRNA to cRNA replication (Te Velthuis et al., 2016) and in early stages of transcription (Te Velthuis et al., 2017; Oymans et al., 2018). In a cryoEM structure of the pre-initiation state (Fig. 5A), the complete priming loop (PB 1/631-660) is visible in a characteristic b-hairpin conformation (Fig. 6 A), preventing the 3' end of the primer approaching the active site. The tip of the priming loop (648-AHGP) forms a platform above the active site, leaving just enough space to accommodate and align the two nucleoside triphosphates at the +1 (GTP) and -1 (ATP) positions consistent with its importance for de novo initiation of vRNA to cRNA replication (Te Velthuis et al., 2016). In this structure, the expected base pairing between the primer and the template does not occur due to the presence of the priming loop. Instead, the 3' end of the template turns away from the template entry tunnel and binds to the PB 1 b-ribbon (Fig. 6A), as previously observed (Reich et al., 2014). Similarly, the cap proximal part of the primer binds to the PB2 cap binding and midlink domains as previously described (Pflug et al., 2018). The primer 3' end descends into the active site cavity but is not visible beyond the fifth nucleotide (Fig. 6A). In the pre - initiation state, the mobile template 3' end can also flip into the active site adjacent to the tip of the priming loop, which then becomes slightly disordered (Reich et al., 2017) (Fig. 6B).

In the new crystal structures of the initiation complex provided by the present invention (Fig. 1 c), the primer forms five base pairs (position +1 to -4) with the template and this is only possible due to the partial retraction of the priming loop (Fig. 6C). The last template base U21 (-5 position) is however not paired with A10 of the primer, instead, these two bases as well as primer base A8 form a close packed arrangement that butts against residues PB 1/652-655 of the repositioned priming loop with conserved PB1/Asp655 forming a salt-bridge with PB2/Arg218. The rest of the priming loop (residues 632-637 and 643-656) refolds into and still blocks the template exit channel with residues 638-642 forming a partially disordered loop projecting into the solvent, near the N-terminus of PB2. The observed position of the priming loop likely does not correspond to that in the true initiation state but to a later post-initiation state, in line with the mixed nature of the structure.

Following addition of ATP and GTP, five nucleotides were incorporated into the product and a nine base pair duplex (positions -1 to -9) was established in the active site cavity of the elongation state (Fig. 5C and 6D). This early elongation complex is in the post-translocation state, with the incoming nucleotide position at +1 being vacant (i.e. similar to the crystal structure with the 14-mer capped RNA primer). Nucleotide addition would thus result in a ten base pair duplex (position +1 to -9) before translocation. To accommodate the extended RNA duplex, the priming loop has to be fully displaced from the active site cavity with 17 residues being extruded into the solvent in a disordered loop that projects towards the PB1-PB2 interface helical bundle (Fig. 6D). These results show for the first time how the priming loop is successively displaced during early transcription.

The mechanism of product-template strand separation and template exit channel opening

Product-template strand separation is an important necessity for transcribing polymerases. The elongation state structure clearly identifies the PB2 helical lid domain (PB2/153-212) as responsible for product-template strand separation in influenza polymerase (Fig. 8), confirming a previous suggestion (Reich et aI. 2014). The helical lid lies on top of the active site chamber with helix al2 facing the growing product-template duplex. Tyr207 (conserved in all influenza A and B strains, histidine in influenza C) changes rotamer to stack on the template base of the last base pair, preventing duplex continuation beyond position -9 (Fig. 8). The stable, nine base pair long product-template duplex in the active site chamber disfavors product dissociation. This likely explains the high processivity of elongating influenza polymerase, compared to the initiation phase where abortive products frequently occur due to the instability of a too short product-template duplex (Klumpp et aI. 1998). In the initiation state, the partially extruded priming loop still blocks the template exit channel (Fig. 9A-D). Only in the elongation state, when the priming loop is fully extruded, is there an unobstructed exit channel for the template after strand separation (Fig. 9B, C). There is a sharp kink at the -9/- 10 junction as the template exits and the -10 nucleotide is accommodated in the exit channel without specific interactions (Fig. 8). Conformation and protein-RNA interactions of the template and primer/product

The conserved catalytic core of viral RNA-dependent RNA polymerases share six characteristic structural elements (motifs A to F), which are responsible, together with two divalent cations (denoted A and B), for controlling the nucleotide addition cycle (Te Velthuis et al., 2017). With the present invention it is now possible to see how these motifs, and other parts of the influenza polymerase core, interact with the primer, template and incoming nucleotide during transcription initiation and elongation (Figs. 2, 3). In the elongation state, the product-template duplex fills the active site cavity and residues lining it make numerous van der Waals and polar interactions to the backbone of both strands (Fig. 2B). The proximal part of the template strand (+1 to -4 position) binds to the fingers (residues 127-136), fingertips (residues 227-229 and 241-249 of motif F) and palm (residues 271-274 and motif B 412-415) domains of PB1. These interactions are conserved in the initiation complex (Fig. 2A). The distal part of the template (positions -5 to -9), only present in the elongation complex, crosses over to the thumb domain to be contacted by PB1 residues 527-531 as well as conserved PB2 basic residues Arg211, Arg216 and Arg218 which interact with the phosphates at positions -8, -7 and -6 respectively. The template enters the active site by undergoing the characteristic kink at the +1/+2 junction (Shu et al., 2016) with the +1 guanine base stacking on PB 1/Ile241 and making a polar interaction with PB1/Lys229, both conserved motif F residues (Figs. 2A, B; 3 A). In the initiation complex the template then follows a sinuous path back to the double stranded region of the promoter (Reich et al., 2017; Pflug et al., 2014), whereas the entire promoter region is remodelled in the elongation complex.

The complete 15-mer capped primer is observed in the initiation complex (Fig. 10). Both the cap and the first base of the primer bind in specific pockets formed by the PB2 cap binding and midlink domain as previously described (Pflug et al., 2018; Reich et al., 2017). The following three bases (2-AAU-4) are stacked on each other and sandwiched between PB2/Tyr434 of the cap-binding domain and a salt-bridge between PB2/Arg217 and Glul55 of the PB2 N2 domain (Fig. 2A, 3B). Between nucleotides five to ten, the capped primer has an irregular backbone structure that probably varies according to its exact length and sequence. There is a distinct pocket for the splayed out base of G5, which is stacked between of PB2/Argl46 and Arg425 and makes base contacts with PB 1/Glu227. Poorly ordered bases C6 and U7 are turned outwards, whereas A8 stacks on A10 at the top of the primer-template duplex region and U9 is bulged out. Primer bases 11-15 form an A form duplex with the template (+1 to -4). The distal part of the primer backbone interacts with PB1 Tyr24, Arg233, Ser493 and the peptide PB 1/506-509 whereas the proximal part interacts with residues PB 1/443-445 from motif C and PB 1/309-310 from motif A (Fig. 2A).

In the elongation complex, the interactions with the cap proximal region of the primer/product (m7GpppGAAU) are maintained, with virtually no change in the position of the cap-binding domain, but the next seven nucleotides are disordered, presumably due to bulging out of the product, before connecting with the end of the duplex after strand separation (Fig. 8). In the nine base pair duplex region, the backbone of nucleotides 16-20 of the product interact as the equivalently placed nucleotides 11-15 in the initial position of primer (Figs. 2A, B), whereas the backbone of primer nucleotides 12-15, due to their translocation, now interact with PB2/35- 45 and PB 1/124-126 and Lys706 (Fig. 2B).

The active site methionine-rich motif B loop reconfigures to stabilize the incoming nucleotide

The initiation complex crystal structure is in the pre-translocation state with the primer template base pair, C15-G(+l) at the active site position (Figs. 2A, 3A). By contrast, the elongation complex cryoEM structure is in the post-translocation state, with the incoming NTP position opposite template base G(+l) vacant, consistent with the lack of CTP in the elongation reaction (Fig. 4B). In neither case is the conserved triad of aspartates (PB1/Asp305, Asp444, Asp445) in an active configuration together with two divalent cations A and B, required for catalysis. In the initiation structure, there is a pyrophosphate at the expected position of the triphosphate of an incoming NTP, thus the structure mimics a reaction product complex. There is a presumed magnesium at the metal B site, octahedrally coordinated by motif A PB1/Asp305, the carbonyl oxygen of motif A PB 1/Gly306, motif C PB1/Asp444, the phosphate of the terminal primer nucleotide Cl 5 and the pyrophosphate (Figs. 2A, 3 A). In the elongation structure, a presumed Mg²⁺ ion, coordinated by motif C PB1/Asp445, motif E PB 1/Glu490 and the carbonyl oxygen of motif A PB1/Gly304 is observed in a position typical for metal A in the inactive 'open' state (Shu et aI. 2016) (Figs. 2B, 3 A). Based on the architectural similarity of viral RNA polymerase active sites, local conformational changes of motifs A and D resulting in reorientation of the active site triad of aspartates and co-ordination of both metals A and B together with the incoming NTP in a catalytically active configuration (Shu et aI. 2016). In an attempt to visualize the pre-catalytic, active state of influenza polymerase, the initiation complex was crystallized with a capped 14-mer, (lacking C15 compared to the capped 15-mer) and determined structures with and without soaking crystals with CTP as incoming nucleotide opposite G(+l) of the template (Table 1). The soaked structure shows clear electron density in the expected place for the incoming CTP with magnesium ion B in place (Fig. 3C), but not the functional configuration of the triphosphate with metal A, perhaps due to the low pH and high phosphate content of the crystallization medium.

Comparing the post-translocation (NTP site vacant), pre-catalytic (NTP site occupied) and pre- translocation/product state structures highlights the role of the flexible, methionine rich motif B loop (PB1/407-GMMMGMF-413, highly conserved in all orthomyxoviruses) in adapting to the presence of a base at the incoming nucleotide position +1 (Fig. 4E). In the pretranslocation and pre-catalytic states (i.e. with CTP), this base stacks on the side-chain of Met410 and also contacts the backbone of Gly411. The carbonyl oxygens of Gly411 and Asp242 hydrogen bond to the 2' hydroxyls of the template nucleotides at respectively the -1 and +1 positions and the side-chain of Asn310 (Motif A) interacts with both hydroxyls of the primer nucleotide at position +1. In the post-translocation state, when the incoming nucleotide site is vacant, the motif B loop reconfigures, with Met410 being buried in a hydrophobic pocket, necessitating adjustments of the side-chains Phe344 and Phe413 as well (Figs. 3C, Fig. 4).

Progression to elongation requires promoter melting and remodeling of the template entrance

The intact vRNA promoter, as observed in the pre-initiation and initiation states, comprises both conserved extremities of the vRNA (Fig. 11 A). The 5' nucleotides 1-10 form a compact stem-loop (hook) structure that is tightly bound in a pocket formed by PA and PB 1 subunits and is required to activate polymerase functions (Pflug et al., 2014; Thierry et aI. 2016). The 5' nucleotides 11-14 base pair with 3' nucleotides 10-13 and project away from the polymerase (Fig. 11 A). The intact promoter configuration is stabilized by a network of specific interactions, including stacking and pseudo base pairing of PA/His506 with 5' A10 and 3' G9 respectively, and stacking of PA/Met473 on 3 ' G9 (Fig. 5 A,C). Of particular importance is the short PB 1/670- 677 h-helix, which stabilizes the pronounced turn of the 3' backbone between nucleotides 7-11 (Fig. 11 A). Finally, the tip of the PB 1 b-ribbon loosely engages with end of the promoter duplex from the solvent side in the region of 5' G14-3' C13 (Figs. 11 A, C).

A major reorganization of the promoter-binding region occurs during the initiation elongation transition, a consequence of the promoter melting necessitated by template translocation (Fig. 1 IB). The entire incoming template strand from nucleotide 7 into the duplex region is displaced from its initial position, allowing the b-ribbon to bend towards the 5' promoter strand (Figs. 11B, C; 12 A, B). The peptide PB 1/667-681 also reconfigures and the h-helix is replaced by a short b-strand PB 1/675-677 that establishes a three- stranded b-sheet with the b-ribbon, stabilising its new position (Figs. 1 IB; 12A). The triple stack of PA/His505 with 5' bases A11 and G12 remains in place, but with b-ribbon residue PB1/Leu200 now directly packing against 5' G12 and forcing 5' A13 into a new position (Fig. 12A). An additional consequence of the displacement of template nucleotides 7 to 8 is that peptide PB2/37-44 repositions into the space vacated by these nucleotides (Figs. 11B; 12B). This movement widens the active site cavity, allowing the extended product-template helix to grow, with residues from PB2/37-44 interacting with the product strand (Fig. 2B) rather than sterically clashing with it. These observations show that after the transition to elongation, the incoming template can no longer follow the entry pathway into the active site as observed in the pre-initiation and initiation states. However, the quality of the density in the elongation state cryoEM map does not allow a clear definition of the trajectories of the outgoing 5' end beyond nucleotide A13 or the incoming template prior to the +3 position (Fig. 12A), although uninterpretable density exists in the vicinity. It thus remains to be elucidated whether there is some mechanism to prevent interference between the translocating incoming template and the tethered 5' end, which are in close proximity. Interestingly, the remodeled PB 1/667-681 region immediately follows the priming loop (PB 1/631-660), which itself is being extruded during the initiation to elongation transition (Fig. 12B). This suggests that these conformational changes might be coupled. However a previous co-crystal structure of FluB polymerase with bound only to 5' vRNA nucleotides 1-12 (Thierry et aI, 2016) (PDB entry 5EPI) is very similar to the elongation state structure in showing the triple-stranded configuration of the collapsed b-ribbon and the shift in PB2/37-44 but without exhibiting priming loop extrusion. Thus removal of the 3' template from the promoter is sufficient to induce b-ribbon collapse whereas priming loop extrusion seems rather to be correlated with product-template helix growth and thumb domain opening as a second step after promoter melting.

Materials & Methods

Expression and purification

The influenza B/Memphis/13/03 (FluB) polymerase self-cleaving polyprotein heterotrimer construct was expressed in High Five insect cells as described (Reich et aI. 2014). Frozen cell pellets were re-suspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 8) containing protease inhibitors (Roche, complete mini, EDTA-free). Following lysis by sonication and centrifugation at 20,000 r.p.m. (JA20/Beckman Coulter) for 45 min at 10°C, the supernatant was precipitated by ammonium sulphate (0.5 g ml^-1) and centrifuged at 45,000 r.p.m. for 45 min at 10°C (45Ti/Beckman Coulter). The pellet was re-dissolved in lysis buffer, and finally re-centrifuged at the same settings. Cleared supernatant was incubated with nickel resin (His60 NiNTA, Clontech) for one hour at 10°C. Protein was eluted with lysis buffer supplemented with 500 mM imidazole and loaded on a Strep-Tactin matrix (Superflow, IB A). Elution was performed with 2.5 mM d-desthiobiotin in low salt buffer (50 mM Tris pH 8, 250 mM NaCl, 10 % glycerol). Pooled FluB polymerase fractions were filtered with 0.22 pm filter and loaded on a heparin column (HiTrap Heparin HP, GE Healthcare). Elution was performed with a gradient using buffers A and B (2 mM TCEP, 50 mM HEPES, pH 7.5, 150 mM (A) or 1 M NaCl (B), 5% glycerol). Homogeneous monomeric polymerase was pooled and dialysed overnight with 6-8 kDa molecular weight cut-off membrane tubing (Spectra/Por, Spectrum Labs) into 50mM HEPES, 500 mM NaCl, 5% glycerol at pH7. Finally, the protein was concentrated with Amicon® Ultra- 15 (50 KDa cutoff), flash-frozen and stored at -80°C.

Cap-dependent polymerase transcription assays

Separated synthetic 18-mer or 18+3-mer 3' and 14-mer 5' ends (IB A) were used as vRNA and synthetic 15-mer capped RNA (TriLink Biotechnologies) as a primer (Table 3, Fig. 5 A).

Table 3: RNA sequences used for biochemical assay, cryoEMand crystallization with influenza B polymerase.

For the cap-dependent transcription assay, 0.6 mM FluB polymerase, 0.75 mM vRNA, 0.75 pM capped RNA primer, 50 pM GTP, ATP and 2.5 pM a-32P-ATP were mixed 358 and incubated in reaction buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM MgCl₂ and 2 mM TCEP) at 30°C for 0.5, 1, 2, 3, 4, 5 and 6 hours. Samples were separated on a 7 M urea, 20% acrylamide gel in TBE buffer, exposed on a storage phosphor screen and read with a Typhoon scanner. 20-mer, 23-mer and 28-mer RNAs, 2-0'-methylated at first 5' ribose adjacent to the cap and radio-labelled at the 3' end were used as markers. For quantitative analysis, transcript bands were analyzed in Quantity One software (BIO-RAD). The elongation intermediate stability was determined by a transcription assay in the presence of heparin as a competitor similarly to as described in Gourse, 1988 and in Borukhov, 1993. The elongation intermediate complexes were reconstituted by mixing 0.2 mM FluB polymerase with 0.25 mM of either synthetic 18-mer or 18+3-mer 3', 14-mer 5' end, synthetic 15-mer capped RNA as a primer, and 100 pM GTP, ATP and 2.5 pM a-32P-ATP in reaction buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM MgC12 and 2 mM TCEP). After four hours pre-incubation at 28 °C, either 100 pM CTP or CTP and heparin at 50 pg/ml were added to the reaction and incubated for an additional 15 minutes. Positive control samples were incubated with either only GTP and ATP or GTP, ATP and CTP for 4 hours and 15 minutes. Samples were separated on an 8 M urea, 20% acrylamide gel in TBE buffer, exposed on a storage phosphor screen and read with a Typhoon scanner. Competitor test experiments were also conducted, demonstrating that the concentration of heparin (50 pg/ml) used was sufficient to completely abolish transcription if present in the reaction before the addition of influenza polymerase (Fig. 7).

Crystallization data collection and structure determination

FluB polymerase at 9 mg ml^-1 in dialysis buffer was mixed with 40 pM of the vRNA 5' end 14-mer, 40 pM of the vRNA 3 ' end 21-mer and 80 pM 14-mer or 15-mer capped RNA. Hanging drops for crystallization were set up at 4 °C. Marquise shaped crystals growing up to 200 pm in size appeared in two to three weeks in mother liquor containing 200 mM di-ammonium phosphate and 100 mM sodium acetate between pH 4.0 and 4.4. CTP was soaked for 18 hours at a final concentration of 5 mM in the drop. Crystals were cryoprotected with 30 % glycerol in mother liquor and flash-frozen in liquid nitrogen. Diffraction data were collected on the MASSIF beamline, ID30A-1, at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). Anisotropic data were integrated with AUTOPROC/STARANISO using an ellipsoidal mask with resolution cut-off criteria local (I/sI) = 1.2¹⁷ (Table 1). Crystal structures were solved by molecular replacement with PHASER (McCoy et aI, 2007), using the FluB polymerase structure (PDB 5MSG) (Reich et aI, 2017), rebuilt with COOT (Emsley et aI. 2004), refined using REFMAC5 (Murshudov, 1997) with TLS parameters and validated with Molprobity (Chen et aI. 2010) (Table 1). For the capped 15-mer structure 92.1 (0.4) % of residues were in the Ramachandran favoured (outlier) regions and the Molprobity score and clash score were respectively 1.72 and 1.47. For the capped 14-mer structure 94.4 (0.2) % of residues were in the Ramachandran favoured (outlier) regions and the Molprobity score and clash score were respectively 1.73 and 1.80. For the capped 14-mer+CTP structure 92.2 (0.7) % of residues were in the Ramachandran favoured (outlier) regions and the Molprobity score and clash score were respectively 1.80 and 1.99.

CryoEM of cap-dependent transcribing complexes

To assemble transcribing complexes, 0.8 mM FluB polymerase, 1 mM 5' and 3' vRNA, 1 pM of capped RNA and 50 pM ATP and GTP were incubated for 5 hours at 30 °C in cryoEM buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM MgC12 and 2 mM TCEP). The transcription reaction was then transferred to 8 °C and incubated with 50 pM 383 non-hydrolysable cytidine- 5'-[(a,P)-methyleno]tri phosphate (CMPCPP) for 10 minutes. Aliquots of 3 pi were applied to glow-discharged Quantifoil Rl .2/1.3 Au 300 mesh grids, immediately blotted for 2s and plunged into liquid ethane using an FEI Vitrobot IV (4 °C, 100% humidity). The grids were loaded into a 300 keV Tecnai Krios (FEI) electron microscope (beamline CMOl at the ESRF) equipped with a K2 Summit direct electron-counting camera positioned after a GIF Quantum energy filter (Gatan). CryoEM data were acquired using EPU software (FEI) at a nominal magnification of xl65000, with a pixel size of 0.86 A per pixel. Movies were acquired for 6 seconds at a flux of 8.3 electrons per Å 2 per second, giving a total exposure of - 50 electrons per A2. Each movie was fractionated into 40 frames of 150 ms. 4151 movies were acquired at a defocus range from -0.7 to -3.3 pm (Table 2).

CryoEM image processing

All movie frames were aligned using MotionCor2 (Zheng et al., 2017) and then used for contrast transfer function parameter calculation with Gctf (Zhang, K., 2016). Initially, particles were selected without a template by Gautomatch (provided by Dr. Kai Zhang, http://www.mrc- lmb.cam.ac.uk/kzhang) from a small portion of the data set (about 200 movies). The relatively high dose enabled sufficient contrast to allow particle picking even in low defocused movies. This initial small dataset was subjected to reference free 2D-classification using RELION 2.1 (Scheres, 2012). Eight representative classes of different views were selected from the two- dimensional averages and used as reference for automatic particle picking for the whole data set by Gautomatch. The resulting about 1,615,000 particles were iteratively subjected to two rounds of 2D-classification at a pixel size of 5.16 A and 2.58 A per pixel, respectively, and particles in classes with poor structural features were removed. The remaining particles (about 1,055,000) were then subjected to three dimensional classifications with image alignment. The first round of 3D-classification was restricted to eight classes and performed using bat FluA polymerase with bound vRNA promoter (PDB entry 4WSB) as a 60 A low-pass filtered initial model. Classification was done during three rounds of 25 iterations each, using regularization parameter T = 4. During the second and third round, local angular searches were performed at 3.5 ° and 1.8 ° to clearly separate structural species. The most abundant 3D-classes were pooled together (about 615,000 particles), re-extracted at the pixel size of 0.86 A per pixel, and a second round of 3D classification was performed again restricted to eight classes. Four major classes of the full complex of FluB polymerase with all periphery domains intact were identified. The final accuracy of the rotational alignment was about 1° and the translational alignment accuracy was about 0.5 pixels. In the next step, selected movie frames were aligned using a dose-weighting scheme of MotionCor2 (Zheng et al., 2017). The first frame (of total dose of approximately 1.2 e-/ A2) and the last 21 frames (of total dose of about 26 e-/ A2) were discarded to reduce the total exposure to about 22.5 e-/A2, thus limiting the radiation damage in the used frames. Particles from the four 3D-classes of the FluB complex were re-extracted from the newly movies and 3D auto-refined using respective masks in RELION. The results of the 3D auto-refinement were used for further 3D-classification with restricted angular searches into eight classes. The two most well defined 3D-classes, corresponding to the pre-initiation and elongation complexes, were separately 3D auto-refined. The final cryoEM density maps were generated by the post-processing feature in RELION and sharpened or blurred into MTZ format using CCP427 EM (Burnley et al, 2017). The resolution of the cryoEM density maps was estimated using the 0.143 gold standard Fourier Shell Correlation (FSC) cut off. The local resolution was calculated using RELION and reference-based local amplitude scaling was performed with LocScale (Jakobi et al., 2017). The angular distribution of particles was calculated using the cryoEF software package (Nay denova et al., 2017).

CryoEM model building and refinement

The known structure of promoter-bound FluB polymerase (PDB entry 5MSG) was first rigid body fitted into the cryoEM density by Molrep (Vagin et al., 2010). Individual protein sub- domain positions were optimised using the Jigglefit tool (Brown et al., 2015) in COOT (Emsley et al., 2004). The vRNA promoter and capped RNA primer atomic models were then manually adapted using COOT. The cryoEM atomic models were iteratively improved by manual building in COOT and refinement with Phenix real-space refinement (Afonine et al., 2018). Validation was performed using the Phenix validation tool and model resolution was estimated at the 0.5 FSC cut off (Table 2). Data Availability

Structure 1. Capl5R21

B /Memphis polymerase chains A (PA), B (PB 1), C (PB2) +3' vRNA 1-21 chain R

+5' vRNA 1-14 chain V

+ 15-mer capped RNA primer chain M

Complete structure Capl5R21

Active site only Cap 15R21 sphereAsnB31 OOD 1

(30 A sphere around Asn B310 atom OD1)

Structure 2. Capl4R21

B /Memphis polymerase chains A (PA), B (PB 1), C (PB2) +3' vRNA 1-21 chain R

+5' vRNA 1-14 chain V

+ 14-mer capped RNA primer chain M

Complete structure Capl4R21

Active site only Cap 14R21 sphereAsnB31 OOD 1

(30 A sphere around Asn B310 atom OD1)

Structure 3. Capl4R21CTP

B /Memphis polymerase chains A (PA), B (PB 1), C (PB2) +3' vRNA 1-21 chain R

+5' vRNA 1-14 chain V

+ 14-mer capped RNA primer chain M

+CTP chain N

Complete structure Capl4R21CTP

Active site region only Capl4R21CTPsphereAsnB310OD1

(30 A sphere around Asn B310 atom OD1)

Structure 4. Capl4R21G3U

B /Memphis polymerase chains A (PA), B (PB 1), C (PB2) +3 ' vRNA 1-21 (G3U) chain R (R16 G->U)

+5' vRNA 1-14 chain V

+ 14-mer capped RNA primer chain M

Complete structure Capl4R21G3U

Active site region only Cap 14R21 G3UsphereAsnB31 OOD 1

(30 A sphere around Asn B310 atom OD1)

Structure 5. Capl4R21G3UATP

+5' vRNA 1-14 chain V

+ 14-mer capped RNA primer chain M

+ATP chain N Complete structure Capl4R21G3UATP

Active site region only Capl4R21G3UATPsphereAsnB310OD1

(30 Å sphere around Asn B310 atom OD1)

Example 2

Bat influenza A polymerase was co-crystallized with nucleic acid constructs identified under 3) above (SEQ ID NOs: 2, 3 and 8). In an attempt to obtain a higher resolution crystal form, a similar strategy as in Example 1 was applied to crystallize the putative transcription initiation state of A/little-yellow-shouldered-bat/H17N10 polymerase purified as described in Pflug et al, 2014.

The crystallisation conditions are: 5 mg/ml polymerase with 5.1 mg/ml (19.5 mM) with 1.14 x molar excess of each RNA (v5'116, v3'118+3 and capl3+2G) miced in 1 : 1 ratio of 100 mM amino acids, 100 mM Tris/Bicine pH8.5, 8% ethylene glycol (v/v), 4% PEG 8000 (w/v) by hanging drop at room temperature. Crystals are long prisms (dimensions -300 x 30 mM) and were cryo-protected with either an additional 12% ethylene glycol or 25% glycerol before X- ray data collection.

The native crystals are of space-group P212121 with cell dimensions a~91 b~l 19.0 c~251 A. They diffract slightly anisotropically to a maximum resolution of -1.95 A (Table 4). This is the highest resolution crystal form obtained so far for the complete influenza polymerase. The asymmetric unit of the crystal contains the polymerase trimer, with all parts visible although the cap-binding domain and endonuclease are less well ordered, having high B-factors (Figure 13). As shown in Fig. 14, when there is no incoming nucleotide, the motif B loop is in the open position. Consistent with the high resolution in Fig. 14, there are large numbers of ordered water molecules.

The fact that the template 3' end is in the active site suggests that initiating NTPs can be visualized by soaking or co-crystallization. Soaking with GTP and CTP was expected to mimic a replication initiation state. In fact, soaking of GTP and CTP into crystals resulted in a second high-resolution dataset (Table 4). The resulting structure is shown in Fig. 15. This is the first structure showing a priming NTP in the +2 position and the first to show the role of the conserved dibasic motif PA/658-RK and PB1/H649 in stabilizing the triphosphate of the priming NTP (Figure 16). There are two very well defined magnesium ions co-ordinated by the active site aspartates in an apparently catalytic configuration. Because there is CTP in the +1 incoming nucleotide position, the motif B loop flips to the closed conformation with Met409 stacking under the CTP (Figure 16), consistent with what is observed for influenza B in Example 1. The value of this crystal form for RNA synthesis inhibitor development is that unprecedented high resolution is obtained. For instance, the native crystals allow soaking in and visualization of GTP and CTP nucleotide analogues. By modification of the template sequence, other nucleotide analogues can be visualized e.g. the G3U template (SEQ ID NO: 10) would allow ATP analogues to be seen.

Table 4: Crystallographic data collection and refinement statistics high resolution bat influenza polymerase structures.

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SEQUENCES

The following sequences are listed in the description: 1 ) GAAUGCU AUAAUAG (SEQ ID NO : 1 )

2) UUAUCGUCUUCGUCUCCAUAU (SEQ ID NO:2)

3) GAAUGCU AUAAUAGC (SEQ ID NO:3)

4) AGUAGUAACAAGAG (SEQ ID NO:4)

5) AGUAGUAACAAGAG (SEQ ID NO: 5)

6) UAUACCUCUGCUUCUGCU (SEQ ID NO : 6)

7) UAUACCUCUGCUUCUGCUAUU (SEQ ID NO: 7)

8) AGUAGUAACAAGAGGG (SEQ ID NO: 8)

9) Gly Gly Tyr Thr lie Asp Thr Val lie Arg Thr His Glu Tyr Ser Pro Ser Ala Leu Asn Thr Met Thr Lys Asp Glu Arg Lys Lys Arg Arg Ala IIe Ala Thr Ala IIe Arg Val Glu Leu Asp Asn Thr Lys Trp Asn Glu Cys Leu Asn Pro Arg IIe Phe Phe Ser Lys IIe Ala Arg Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Gin Ser Ser Asp Asp Phe Leu Gly IIe Asn Met Ser Lys Thr Lys (SEQ ID NO: 9)

10) UUAUCUUCUUCGUCUCCAUAU (SEQ ID NO: 10)

H) X₁-Xm

12) Y_1-Y_n

13) X₁X_m.5AAUAG

14) UUAUCY₆-Y_n

15) X₁X_m.6AAUAGC

Claims

1. A nucleic acid construct comprising a primer strand and a template strand, the primer strand having the sequence 5'-X₁-X_m-3' and the template strand having the sequence 3'-Yi-Y_n-5', wherein X and Y are independently nucleotides, wherein m is an integer of from 12 to 16 and n is an integer of from 13 to 50, wherein between five and six consecutive nucleotides of the primer strand and of the template strand are complementary to each other such that

(ii) nucleotides X_m-5, X_m-4, X_m-3, X_m-2, X_m-1, and X_m of the primer strand are complementary to nucleotides Y₁, Y₂, Y₃, Y4, Y5, and Y₆ of the template strand.

2. The nucleic acid construct according to claim 1, wherein at least 50 % of the nucleotides of the primer strand and of the template strand are ribonucleotides.

3. The nucleic acid construct of claim 1 or 2, wherein the primer strand has a m⁷Gppp-cap connected to the 5' end, wherein m⁷Gppp is 7-methyl-guanosine linked to a triphosphate bridge.

4. The nucleic acid construct of any one of claims 1 to 3, wherein m is an integer of from 14 to 15 and n is an integer of from 16 to 30.

5. The nucleic acid construct of any one of claims 1 to 4, further comprising an activator strand having the sequence 5'-Z₁-Z₀-3', wherein Z is independently selected from the group consisting of adenine, uracil, guanine and cytosine, wherein o is an integer of from 12 to 18, and wherein

(a) in case of alternative (i) of claim 1 at least two consecutive nucleotides of the activator strand beginning with Z ₁₁ are complementary to at least two consecutive nucleotides of the template strand beginning with Y₁₂; and

(b) in case of alternative (ii) of claim 1 at least two consecutive nucleotides of the activator strand beginning with Z_u are complementary to at least two consecutive nucleotides of the template strand beginning with Y₁₃.

6. The nucleic acid construct of claim 5, wherein o is an integer of from 13 to 17, preferably of from 14 to 16.

7. The nucleic acid construct of any of claims 5 to 6, wherein at least four consecutive nucleotides of the activator strand beginning with Z_u are complementary to at least four consecutive nucleotides of the template strand beginning with Y₁₃.

8. The nucleic acid construct of any of claims 5 to 7, wherein nucleotides Zi to Z_w of the activator strand together form a stem-loop structure.

9. The nucleic acid construct of any of claims 5 to 8, wherein the activator strand has a triphosphate bridge connected to its 5' end.

10. The nucleic acid construct of any of claims 5 to 9, wherein the 3' end of the activator strand is connected to the 5' end of the template strand.

11. The nucleic acid construct of any of claims 1 to 10, wherein the primer strand having the sequence as denoted in SEQ ID NO: 1 or 3 or a sequence at least 80% identical to SEQ ID NO: l or 3, and wherein the template strand having the sequence as denoted in SEQ ID NO:2 or a sequence at least 80% identical to SEQ ID NO:2; optionally wherein the nucleic acid construct further comprises an activator strand having the sequence as denoted in SEQ ID NO:4 or a sequence at least 80% identical to SEQ ID NO:4.

12. A polynucleotide encoding the nucleic acid construct of any of claims 1 to 11.

13. A recombinant vector comprising the polynucleotide of claim 12.

14. A recombinant host cell comprising the polynucleotide of claim 12 or the recombinant vector of claim 12.

15. A construct comprising an influenza PB1 RNA synthesis active site and the nucleic acid construct of any one of claims 1 to 11 bound thereto.

16. A construct comprising an influenza PB2 cap binding site and the nucleic acid construct of any one of claims 1 to 11 or 15 bound thereto.

17. A construct comprising an influenza polymerase and the nucleic acid construct of any one of claims 1 to 11 bound to the PB1 RNA synthesis active site of the influenza polymerase.

18. The construct according to claim 17, wherein the nucleic acid construct is also bound to the PB2 cap binding site of the influenza polymerase.

19. A method for crystallizing an influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site, comprising the steps of:

(i) contacting the nucleic acid construct of any one of claims 1 to 11 with an influenza polymerase or variant or fragment thereof and allowing binding of the nucleic acid construct to the PB1 RNA synthesis active site, and

(ii) crystallizing the influenza polymerase or variant or fragment thereof with the bound nucleic acid construct.

20. The method of claim 19, further comprising the step of obtaining crystal diffraction data.

21. The method of any one of claims 19 and 20, wherein the nucleic acid construct is further allowed to bind to the PB2 cap binding site.

22. A method for cryogenic electron microscopy of an influenza polymerase or variant or fragment thereof comprising the PB1 RNA synthesis active site, comprising the steps of:

(i) contacting the nucleic acid construct of any one of claims 1 to 11 with the influenza polymerase or variant or fragment thereof to allow binding of the nucleic acid construct to the PB1 RNA synthesis active site, and

23. The method of claim 22, wherein the nucleic acid construct is further allowed to bind to the PB2 cap binding site.

24. A method for in vitro testing of a function of influenza polymerase comprising: (i) providing an influenza polymerase or variant or fragment thereof comprising the PB 1 RNA synthesis active site;

(ii) adding to the influenza polymerase or variant or fragment thereof the nucleic acid construct of any one of claims 1 to 11 and allowing binding of the nucleic acid construct to the PB 1 RNA synthesis active site;

(iii) adding nucleosides or nucleoside analogues;

(iv) evaluating the polymerase activity of the influenza polymerase.

25. A method for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, comprising the steps of:

(i) contacting an influenza polymerase or variant or fragment thereof comprising at least the PB1 RNA synthesis active site with (a) the nucleic acid construct of any one of claims 1 to 11 and (b) a test compound, and

26. The method according to claim 25, wherein contacting the influenza polymerase or variant or fragment thereof with the nucleic acid construct comprises contacting the influenza polymerase or variant or fragment thereof consecutively with different nucleic acid constructs having different sequences and/or lengths of the primer strand, the template strand, and/or the activator strand.

27. The method according to claim 25 or 26, wherein the strands of the nucleic acid construct are contacted with the influenza polymerase or variant or fragment thereof prior to, concomitantly with, or after addition of said test compound.

28. A method for identifying, selecting or designing a compound which inhibits influenza RNA polymerase, comprising the steps of:

(a) constructing a computer model of the influenza polymerase with the nucleic acid construct bound to the PB 1 RNA synthesis active site based on the crystal diffraction data obtained in claim 20;

(i) assembling molecular fragments into said compound,

(ii) selecting a compound from a small molecule database, and

(iii) de novo ligand design of said compound; (c) employing computational means to perform a fitting program operation between computer models of the said compound and the PB1 RNA synthesis active site with the bound nucleic acid construct; and

29. The method of any of claims 25 to 28 comprising the further step of synthesizing said compound and optionally formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s) and/or carrier(s).

30. The method of any of claims 25 to 29, wherein said compound is selected from the group consisting of a small molecule, a peptide or a protein.

31. A compound identified or obtained by a method according to any one of claims 25 to 30.

32. The compound according to claim 31 for use in medicine.

33. A pharmaceutical composition comprising the compound of claim 31, and optionally a pharmaceutically acceptable carrier.

34. The compound according to claim 31, the compound for use according to claim 32, or the pharmaceutical composition according to claim 33, for use in treating, ameliorating, or preventing a disease caused by viral infections with negative-sense ssRNA viruses.

35. The compound or the pharmaceutical composition for use according to claim 34, wherein the disease is caused by viral infections of the Mononegavirales order comprising the Bomaviridae, Filoviridae, Paramyxoviridae, and Rhabdoviridae families, preferably wherein said disease condition is caused by the Orthomyxoviridae, Arenaviridae, or Bunyaviridae families.

36. The compound or the pharmaceutical composition for use according to claim 35, wherein the disease is caused by a virus selected from the group consisting of Borna disease virus, Marburg virus, Ebola virus, Sendai virus, Mumps virus, Measles virus, Human respiratory syncytial virus, Turkey rhinotracheitis virus, Vesicular stomatitis Indiana virus, Nipah virus, Henda virus, Rabies virus, Bovine ephemeral fever virus, Infectious hematopoietic necrosis virus, Thogoto virus, Influenza A virus, Influenza B virus, Influenza C virus, Hantaan virus, Crimean-congo hemorrhagic fever virus, Rift Valley fever virus, and La Crosse virus.

37. The compound or the pharmaceutical composition for use according to claim 36, wherein the disease is selected from the group consisting of hemorrhagic fever, rabies, influenza, mumps and measles.