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18 pages, 2398 KiB

Open AccessArticle

The Ubiquitin Ligase Adaptor NDFIP1 Interacts with TRESK and Negatively Regulates the Background K⁺ Current

by Enikő Pergel, Dániel J. Tóth, Dóra Baukál, Irén Veres and Gábor Czirják

Int. J. Mol. Sci. 2024, 25(16), 8879; https://doi.org/10.3390/ijms25168879 - 15 Aug 2024

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The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the [...] Read more.

The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K⁺ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K⁺ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K⁺ current. Full article

(This article belongs to the Special Issue 25th Anniversary of IJMS: Advances in Biochemistry)

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Figure 1

Figure 1
NDFIP1 reduced the TRESK background K+ current. (A) Two-electrode voltage clamp (TEVC) measurement of three groups of Xenopus oocytes expressing human TRESK (n = 7), coexpressing TRESK with human NDFIP1 (+NDFIP1 (1×), n = 6), or coexpressing the channel with a higher amount of NDFIP1 (+NDFIP1 (2.5×), n = 6; two and half times more NDFIP1 cRNA was microinjected). Inward (negative) currents were measured at −100 mV. Extracellular [K+] was increased from 2 to 80 mM, as shown above the curves, and the oocyte was then challenged with ionomycin (0.5 μM), as indicated by the horizontal black bar. The average currents and SD are plotted. (B) Statistical analysis of the effect of NDFIP1 on the basal TRESK current. The basal TRESK currents were calculated as the difference in 2 and 80 mM [K+], before the application of ionomycin (see panel (A)). (C) Statistical analysis of the effect of NDFIP1 on the TRESK current stimulated with ionomycin. The stimulated TRESK currents were measured at the end of the application of ionomycin in 80 mM [K+] (see panel (A)), and the small currents in 2 mM [K+] were subtracted. * p < 5 × 10−4, ns: not significant (Dunn’s test after Kruskal–Wallis ANOVA). Although not significant with Dunn’s test, the difference in the basal currents between the TRESK and NDFIP1 (1×) groups in panel B was significant with the Mann–Whitney U test (p < 0.005), which gave p < 0.015 after Bonferroni’s correction. TRESK vs. NDFIP1 (1×) in panel (C) gave p < 0.05 with the Mann–Whitney U test after Bonferroni’s correction. Full article ">Figure 2
TRESK was regulated by NDFIP1 more effectively than TREK-1 and GIRK4. (A) K+ currents in two groups of oocytes expressing TRESK, or coexpressing TRESK with NDFIP1 (0.67×), as indicated below the plot. (B) Similar experiment as in panel A with TREK-1. (C) Similar experiment as in panel A with mutant GIRK4. Data are from the same oocyte preparation, n = 5 in all groups. * p < 0.01 with Mann–Whitney U test, ns: not significant. Full article ">Figure 3
Mutation of PY motifs in NDFIP1 prevented the effect of the adaptor protein on TRESK. K+ currents in three groups of oocytes expressing TRESK, coexpressing TRESK with NDFIP1, or coexpressing TRESK with a mutant version of NDFIP1 lacking PY motifs (NDFIP1-noPPxY), as indicated below the plot. Data are from the same oocyte preparation, n = 7 in all groups. * p < 0.04, ** p < 0.02 with Dunn’s test after Kruskal–Wallis ANOVA, ns: not significant. Full article ">Figure 4
Dominant negative Nedd4 counteracted the NDFIP1-mediated reduction in the TRESK current. K+ currents in four groups of oocytes expressing TRESK, coexpressing TRESK with NDFIP1, or coexpressing wild-type (wt) or dominant negative (dn) Nedd4 with TRESK and NDFIP1 (triple coexpression), as indicated below the plot. Data are from the same oocyte preparation, n = 7 in all groups. * p < 0.015, ** p < 10−4 with Dunn’s test after Kruskal–Wallis ANOVA, ns: not significant. Full article ">Figure 5
NDFIP1 reduced the K+ current of all the tested lysine-mutant TRESK constructs. The K+ currents in the oocytes expressing the different lysine-mutant TRESK constructs (see above the panels), in the absence or presence of NDFIP1 coexpression, as indicated below the graphs. Data points on the same panel always come from the same oocyte preparation, n = 5–7 in all groups. * p < 0.02, ** p < 0.01 with Mann–Whitney U test. # RFRK motif of TRESK was mutated to NFNN, including the K168N mutation. In the Δ172–248 and Δ188–275 constructs, the appropriate regions were replaced by a lysine-free linker. In the ΔKK383,384 construct, the C-terminal double lysine was deleted. Full article ">Figure 6
Human TRESK was ubiquitinated in an NDFIP1-dependent manner. The HA2-N70Q-hTRESK protein was pulled down with anti-HA resin from the solubilized membrane preparations of three groups of oocytes expressing HA-tagged TRESK or NDFIP1, or coexpressing both proteins, as indicated below the images. On the left side, a Western blot was performed with an anti-ubiquitin primary antibody (anti-Ub, as indicated above the image). On the right side, an anti-HA immunoblot of the same membrane is shown. Representative of three similar experiments. * monomeric TRESK protein, ** mono-ubiquitinated TRESK in lane 2. Full article ">Figure 7
NDFIP1 is co-immunoprecipitated with TRESK. Three groups of oocytes expressed HA2-N70Q-hTRESK, His8-hNDFIP1, or both proteins, as indicated below the image. The HA2-N70Q-hTRESK protein was pulled down with anti-HA resin from the solubilized membrane preparations of these oocyte groups, and the co-immunoprecipitation of His8-hNDFIP1 was detected with an anti-His immunoblot (upper panel). Aliquots of the solubilized membrane preparations (Input) were also analyzed with an anti-His immunoblot on the same blotting membrane (middle panel). The presence of the bait protein was verified in the samples after anti-HA immunoprecipitation with the anti-HA immunoblot (lower panel; dimeric TRESK was detected). Representative of three similar experiments. IP: immunoprecipitation; WB: Western blot/immunoblot; anti-HA: antibody against the influenza hemagglutinin epitope; anti-His: antibody against the hexahistidine tag. Full article ">Figure 8
Coexpression of NDFIP1 decreased the immunofluorescence signal of HA-tagged TRESK on the oocyte surface. Three groups of oocytes expressed HA2-N70Q-hTRESK, coexpressed this construct with NDFIP1, or remained as a non-injected control (non-inj), as indicated in the representative images and below the graph. White scale bars indicate 100 μM. Data are from the same oocyte preparation, n = 10 in all groups. * p < 0.02, ** p < 0.005 with Dunn’s test after Kruskal–Wallis ANOVA. Full article ">

20 pages, 7578 KiB

Open AccessArticle

PMEPA1/TMEPAI Is a Unique Tumorigenic Activator of AKT Promoting Proteasomal Degradation of PHLPP1 in Triple-Negative Breast Cancer Cells

by Md. Anwarul Haque, Mohammed Abdelaziz, Meidi Utami Puteri, Thanh Thao Vo Nguyen, Kosei Kudo, Yukihide Watanabe and Mitsuyasu Kato

Cancers 2021, 13(19), 4934; https://doi.org/10.3390/cancers13194934 - 30 Sep 2021

Cited by 10 | Viewed by 3003

Abstract

Transmembrane prostate androgen-induced protein (TMEPAI), also known as PMEPA1, is highly expressed in many types of cancer and promotes oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. We previously established TMEPAI-knockout (KO) cells from human triple-negative breast cancer [...] Read more.

Transmembrane prostate androgen-induced protein (TMEPAI), also known as PMEPA1, is highly expressed in many types of cancer and promotes oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. We previously established TMEPAI-knockout (KO) cells from human triple-negative breast cancer (TNBC) cell lines and found that TMEPAI-KO cells showed reduced tumorigenic abilities. Here, we report that TMEPAI-KO cells upregulated the expression of pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and suppressed AKT Ser473 phosphorylation, which was consistent with TCGA dataset analysis. Additionally, the knockdown (KD) of PHLPP1 in TMEPAI-KO cells partially but significantly rescued AKT Ser473 phosphorylation, as well as in vitro and in vivo tumorigenic activities, thus showing that TMEPAI functions as an oncogenic protein through the regulation of PHLPP1 subsequent to AKT activation. Furthermore, we demonstrated that TMEPAI PPxY (PY) motifs are essential for binding to NEDD4-2, an E3 ubiquitin ligase, and PHLPP1-downregulatory ability. Moreover, TMEPAI enhanced the complex formation of PHLPP1 with NEDD4-2 and PHLPP1 polyubiquitination, which leads to its proteasomal degradation. These findings indicate that the PY motifs of TMEPAI suppress the amount of PHLPP1 and maintain AKT Ser473 phosphorylation at high levels to enhance the tumorigenic potentiality of TNBC. Full article

(This article belongs to the Section Cancer Causes, Screening and Diagnosis)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Roles of TMEPAI in breast cancer cell proliferation, tumor sphere formation, and cell migration. (A,B), Colony formation assays were performed using parental and TMEPAI-KO TNBC cell clones (Hs578T #5 and #18(A) and BT-549 #22 and #29 (B), respectively). Representative photographs of colonies and calculated colony formation rate. (C,D), Representative tumor spheres of parental and TMEPAI-KO cells (Hs578T (C) and BT-549 (D), respectively). The scale bar indicates 100 mm. The bar charts depict the numbers of large spheres formed by the parental and TMEPAI-KO cells (Hs578T and BT-549, respectively). (E), The migration ability of parental and TMEPAI-KO cells (MDA-MB-231) was determined by a transwell cell migration assay and photographs were taken to calculate the migrated cell numbers. The scale bar indicates 100 mm. The values presented here are the means ± SDs of 3 independent experiments. The bars with asterisks indicate significant differences between the parental cells at p ≤ 0.01 (**). Full article ">Figure 2
Association between altered expression of TMEPAI mRNA and AKT Ser473 or AKT Thr308 in TCGA-invasive breast cancer cases. (A), Top biological pathways with the highest normalized enrichment score (NES) identified on gene set enrichment analysis (GSEA) of TMEPAI.High samples (303) compared with TMEPAI.Low samples (303) in TCGA invasive breast cancer cases (BRCA) at FDR q-value < 0.05. (B,C), Enrichment plot of signatures of the TGF-β receptor signaling (B) and PI3K/AKT signaling (C) pathways showing the profile of the running enrichment score and the positions of gene set members on the rank-ordered list of upregulated genes in TMEPAI.High TCGA invasive breast cancer cases (BRCA). (D,E), Comparison of ranks of reverse phase protein array values (RPPA) of AKT Ser473 (D) or AKT Thr308 (E) between TMEPAI.High and TMEPAI.Low samples in cases that have matched single mRNAseq and RPPA samples (193 samples each); Mann–Whitney test, **** p value < 0.0001, figures show Mean ranks and standard deviation. Full article ">Figure 3
Effects of TMEPAI on AKT signal activation. (A,B), Lysates from TGF-β stimulated parental and TMEPAI-KO cells [Hs578T (A) and BT-549 (B)] were subjected to Western blot analysis to detect the amounts of TMEPAI, PHLPP1, pAKT(S473), and AKT. The bar charts express the relative PHLPP1 and pAKT band intensity normalized to the corresponding β-actin band intensity. (C,D), PHLPP1-knockdown efficiency was confirmed and the amounts of pAKT(S473) and AKT were detected (Hs578T (C) and BT-549 (D), respectively) by means of Western blot analysis. β-actin was used as the loading control. The bar graphs depict the relative amount of pAKT (S473) in the control and PHLPP1 KD cells. The values presented here are the means ± SDs of 3 independent experiments. The bars with asterisks indicate significant difference from the parental vs. the TMEPAI-KO cells at p ≤ 0.01 (**) and from the siControl vs. the siPHLPP1 of TMEPAI-KO cells at p ≤ 0.01 (##). The uncropped Western Blot images can be found in <a href="#app1-cancers-13-04934" class="html-app">Figure S6</a>. Full article ">Figure 4
Knockdown of PHLPP1 can rescue colony numbers, tumor sphere growth, and cell migration in TMEPAI-KO breast cancer cells. (A,B), Colonies of PHLPP1-knockdown cells (Hs578T (A) and BT-549 (B), respectively) and the colony formation rates are shown in the bar graphs. (C,D), Tumor sphere-forming ability of PHLPP1-knockdown cells (Hs578T (C) and BT-549 (D)). The scale bar indicates 100 mm. (E) Migratory ability of PHLPP1-KD and control cells. The representative photos are shown, The scale bar indicates 100 mm. The values presented here are the means ± SDs of 3 independent experiments. The bars with asterisks indicate significant difference from the parental vs. the TMEPAI-KO cells at p ≤ 0.01 (**) and from the siControl (#EV) vs. the siPHLPP1(sh#PHLPP1) of the TMEPAI-KO cells at p ≤ 0.01 (##). Full article ">Figure 5
Knockdown of PHLPP1 promotes xenograft tumor formation in both parental and TMEPAI KO cells. (A), Macroscopic view of the tumors. PHLPP1-knockdown cells from parental and TMEPAI-KO MDA-MB-231 cells (1 × 106) were subcutaneously injected into the nude mice. After one and a half months, the mice were sacrificed and the tumors collected from each group. (B), The collected tumors were weighed. (C), The tumor tissues were crushed with a homogenizer, and lysate samples were prepared for Western blot analysis. TMEPAI, PHLPP1, pAKT(S473), and total AKT were detected as indicated. β-actin was used as the loading control. (D,E), Representative pictures and calculated proliferative index (respectively) of tumor xenografts stained with anti Ki-67 antibody. The scale bar indicates 100 mm, Differences between each datum were evaluated using one-way ANOVA and the t test; p ≤ 0.001 (****). The uncropped Western Blot images can be found in <a href="#app1-cancers-13-04934" class="html-app">Figure S6</a>. Full article ">Figure 6
PY motifs of TMEPAI are required for downregulation of PHLPP1 to sustain AKT phosphorylation. (A), Schematic representation of mutations in SIM and PY motifs of human TMEPAI. (B), Lysates from TMEPAI re-expressed cells were subjected to Western blot analysis to detect TMEPAI, PHLPP1, pAKT(S473), and AKT as indicated. The bar chart depicts the relative densities of the pAKT(S473) bands. (C), The visualized colonies and their respective colony-forming rates are shown. The values presented here are the means ± SDs of 3 independent experiments; p ≤ 0.01 (**), where n.s. means not significant. The uncropped Western Blot images can be found in <a href="#app1-cancers-13-04934" class="html-app">Figure S6</a>. Full article ">Figure 7
TMEPAI destabilizes PHLPP1 by enhancing the complex formation of NEDD4-2 and PHLPP1 and promotes PHLPP1 ubiquitination. (A,B), KD efficiency of NEDD4-2 (A) and SMURF1 (B) and their effects on PHLPP1 amounts in Hs578T cells. The values presented here are the means ± SDs of 3 independent experiments; p ≤ 0.05 (*) where n.s. means not significant. (C), Co-immunoprecipitation of TMEPAI and NEDD4-2. WT, wild type; 4A, a SIM 4A mutant; P186A, a SIM P186A mutant; dPY, a double PY mutant. (D), Co-immunoprecipitation of NEDD4-2 and PHLPP1 in the presence of MG132. (E), Co-immunoprecipitation of TMEPAI and PHLPP1 in the presence of MG132 showing no detectable interaction between TMEPAI and PHLPP1. (F), Co-precipitated PHLPP1 with FLAG-NEDD4-2 was detected with increasing amounts of TMEPAI-V5. (G), HEK-293T cells stably expressing HA-ubiquitin (HA-Ub-HEK-293T) were transfected with TMEPAI-V5, FLAG-PHLPP1, and 6xMyc-NEDD4-2, and ubiquitinated PHLPP1 was detected by immunoprecipitation of FLAG-PHLPP1 and blotting with anti-HA antibody. The cell lysates were directly subjected to Western blot analysis to detect the total amounts of the expressed proteins (Input). The uncropped Western Blot images can be found in <a href="#app1-cancers-13-04934" class="html-app">Figure S6</a>. Full article ">

20 pages, 2279 KiB

Open AccessReview

Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses

by Lisa Welker, Jean-Christophe Paillart and Serena Bernacchi

Viruses 2021, 13(8), 1559; https://doi.org/10.3390/v13081559 - 6 Aug 2021

Cited by 21 | Viewed by 4496

Abstract

Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of [...] Read more.

Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses. Full article

(This article belongs to the Special Issue RNA Viruses and Membranes)

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Figure 1

Figure 1
Retroviral Gag organization domains. All retroviruses display matrix domain (MA, in violet), capsid (CA, in green), and nucleocapsid (NC, in orange). Spacer peptides (SP) and peptide sequences containing L domains are indicated within structural precursors of HIV-1, EIAV, FIV, HIV-2, RSV, MMTV, M-PMV, MLV, PERV, HTLV-I, HML-2, and PFV. Full article ">Figure 2
L domains in the structural proteins of RNA viruses including filoviruses (EBOV and MARV), rhabdoviruses (RABV and VSV), arenaviruses (LASV), paramyoviruses (PIV5), and picornavirus (HAV). Full article ">Figure 3
Schematic representation of the domains in HIV-1 Pr55Gag precursor. The myristoyl moiety is indicated at the N-terminus of the precursor. The MA domain drives the interaction between the Pr55Gag and the PM through a bipartite signal consisting of a HBR domain and the covalently attached myristic acid moiety. The CA domain of Pr55Gag mediates Pr55Gag oligomerization and ensures formation of the core of the mature viral particles, and the NC domain, which contains two zinc-finger motifs, corresponds to the primary binding motif to nucleic acids, and contributes to the Pr55Gag multimerization. The p6 domain contributes to the specificity of Pr55Gag–gRNA interactions and is essential for viral budding. Pr55Gag contains also two spacer peptides which are SP1 and SP2. Full article ">Figure 4
The recruitment of the ESCRT cellular factors by L domains in HIV Pr55Gag ensures viral particles release. The cellular factor angiomotin (AMOT) promotes the recruitment of the ubiquitin ligase NEDD4L at HIV-1 budding sites. The ESCRT machinery is then recruited by NEDD4 family, and this interaction seems to be related to the ubiquitination of viral structural proteins. The P(T/S)AP L domain of Pr55Gag recruits the ESCRT-I factor TSG101 at viral assembly sites by direct binding to its ubiquitin E2 variant (UEV) domain, and the YPXnL motif recruits the ESCRT-III-associated factor ALIX by binding to its V domain. The ESCRT-III proteins drive the interaction with the VPS4 ATPase. This late-acting factor leads to membrane remodeling and its fission, and it drives the disassembling of the ESCRT-III filaments. The process ends with the release of the newly formed viral particle. Full article ">

13 pages, 1954 KiB

Open AccessArticle

Ubiquitin Ligase SMURF2 Interacts with Filovirus VP40 and Promotes Egress of VP40 VLPs

by Ariel Shepley-McTaggart, Michael Patrick Schwoerer, Cari A. Sagum, Mark T. Bedford, Chaitanya K. Jaladanki, Hao Fan, Joel Cassel and Ronald N. Harty

Viruses 2021, 13(2), 288; https://doi.org/10.3390/v13020288 - 12 Feb 2021

Cited by 9 | Viewed by 3401

Abstract

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly [...] Read more.

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack specific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus–cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40–SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of identified host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections. Full article

(This article belongs to the Special Issue Ubiquitin and Ubiquitin-Like Pathways in Viral Infection)

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Figure 1

Figure 1
WW domain array screen. Purified GST/WW domain fusion proteins were arrayed in duplicate in Boxes A–N in numbered squares 1–12 as shown. The GST/WW domain fusion proteins present in Box D are shown. The array was screened with the following biotin-labeled peptides: EB-WT (MRRVILPTAPPEYMEAI-K-biotin), EB-Mut (MRRVILPTAAAEAMEAI-K-biotin), MV-WT (MQYLNPPPYADHGGANQL-K-biotin), or MV-Mut (MQYLNAAPAADHGANQL-K-biotin). A positive interaction was observed between SMURF2-3 (position #5) and both EB-WT and MV-WT peptides. An anti-GST control for protein expression is shown. The center M square contains GST alone. Full article ">Figure 2
Docking Models of PPxY/WW interactions. Docking models showing PPxY/WW interactions for: (A) eVP40-WT, (B) mVP40-WT, and (C) SMAD7 PPxY-containing peptides with WW domain #3 from SMURF2. (D) Protein–peptide docking scores. Full article ">Figure 3
Surface plasmon resonance. Graphical evaluation of eVP40-WT and PPxY mutant peptides binding to GST/WW domain #3 of SMURF2 with response units (RU) plotted on the Y-axis and peptide concentration in uM plotted on the X-axis. The Kd for the WT peptide was calculated to be 78 uM with a Bmax of 39 RU. Full article ">Figure 4
GST pulldown between SMURF2 WW domains and full length VP40. (A) GST pulldown assay using purified GST-SMURF2 WW domains 1, 2, and 3 with HEK293T cell extracts containing eVP40 WT (lanes 1, 4, and 7), eVP40 PPxY mutant (lanes 2, 5, and 8), or GST alone (lanes 3, 6, and 9). (B) GST pulldown assay using purified GST-SMURF2 WW domains 1, 2, and 3 with HEK293T cell extracts containing mVP40 WT (lanes 1, 4, and 7), mVP40 PPxY mutant (lanes 2, 5, and 8), or GST alone (lanes 3, 6, and 9). Input levels of eVP40, mVP40, GST, and actin are shown. Full article ">Figure 5
Enzymatically active SMURF2 positively regulates eVP40 VLP budding. (A) Representative Western blot of HEK293T cell extracts and VLPs of eVP40 alone (lane 1), eVP40 + SMURF2-WT (lane 2), and eVP40 + SMURF2-C716A mutant (lane 3). (B) Graph showing the relative budding efficiency of eVP40 VLPs under the indicated conditions from three independent experiments; student t test, ** = p <0.01, *** = p <0.005. Full article ">Figure 6
SiRNA knockdown of SMURF2 decreases VP40 VLP budding efficiency. (A) HEK293T cells were transfected as indicated and the indicated proteins were detected in cell extracts and VLPs by Western blotting. (B) Graphical representation of the relative budding efficiency of eVP40 under the indicated conditions from three independent experiments; student t test, ** = p <0.005. Full article ">

21 pages, 3570 KiB

Open AccessArticle

The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

by Wei Zhang, Ayaka Muramatsu, Rina Matsuo, Naoki Teranishi, Yui Kahara, Terunao Takahara, Hideki Shibata and Masatoshi Maki

Int. J. Mol. Sci. 2020, 21(17), 6315; https://doi.org/10.3390/ijms21176315 - 31 Aug 2020

Cited by 8 | Viewed by 3316

Abstract

ALG-2 is a penta-EF-hand Ca²⁺-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). [...] Read more.

ALG-2 is a penta-EF-hand Ca²⁺-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca²⁺ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca²⁺-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs. Full article

(This article belongs to the Special Issue Calcium-Binding Proteins and Cell Signaling 2.0)

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Graphical abstract

19 pages, 3620 KiB

Open AccessArticle

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal ¹⁸¹L-¹⁸⁵L and Plays a Negative Regulation Role during Viral Lytic Infection

by Chuang Lyu, Wei-Dong Li, Shu-Wen Wang, Jin-Mei Peng, Yong-Bo Yang, Zhi-Jun Tian and Xue-Hui Cai

Int. J. Mol. Sci. 2020, 21(9), 3148; https://doi.org/10.3390/ijms21093148 - 29 Apr 2020

Cited by 4 | Viewed by 2597

Abstract

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on [...] Read more.

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of ¹⁸¹L/A, ¹⁸⁵L/A, or ¹⁸¹L/A-¹⁸⁵L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells. Full article

(This article belongs to the Section Molecular Microbiology)

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Figure 1

Figure 1
Pseudorabies virus (PRV) pUL56 interacts and co-localizes with BAG3. (A) Co-IP of pUL56 and BAG3. Each of the indicated plasmids is transfected into HEK293T cells, respectively. At 48 hpt, the whole cell lysates are obtained and mixed with the indicated combinations and immunoprecipitated with FLAG M2 beads. A 20% aliquot of whole cell lysates is used to confirm protein expression. The asterisk indicates a IgG light chain in the co-IP assay. (B) HEK293T cells in a 12-well plate are transfected with 120 pmol of the indicated siRNAs targeting to BAG3 for 48 h. The specificity of the anti-BAG3 polyclonal antibody is confirmed by a Western blot. (C) A co-localization analysis of endogenous BAG3 with GFP-pUL56 in the co-transfected Vero cells. BAG3 distributes evenly in the cytoplasm (asterisk, upper panel) in non-transfected Vero cells. The distribution of BAG3 is not affected by GFP (arrowheads, upper panel), whereas it is intensively restricted and co-localized with GFP-pUL56 in the perinuclear region (arrowheads, lower panel). (D) BAG3 can be detected in both the peripheral (dorsal root ganglion, DRG) and central (spinal cord and brain) nervous system in C57/BL6 mice. The scale bar indicates 10 μm (C). Full article ">Figure 2
The pUL56 interacts with BAG3 through the PPxY motifs. (A) The schematic diagram shows four pUL56 PPxY mutants. (B) The co-localization analysis shows that pUL56 PYM1−3 co-localize with BAG3 in the transfected Vero cells, respectively. The pUL56 PYM4 has no apparent co-localization with BAG3 in the transfected Vero cells. The inset enlargements show the co-localized fluorescence in the cells. The arrowheads indicate co-localization. (C) Co-IP of BAG3 and pUL56 PYM1−4, respectively. A 20% aliquot of the whole cell lysates is used to confirm protein expression. The scale bar indicates 20 μm (B). Full article ">Figure 3
BAG3 is relocated to the Golgi via an interaction with pUL56. The plasmids pCMV-HA-mBAG3 and pAcGFP-UL56 are co-transfected into HEK293T cells. At 24 hpt, the HA-mBAG3 and Golgi are detected with anti-HA and anti-GM130 antibodies, which is followed by staining with Alexa Fluor 568-conjugate goat anti-mouse and Alexa Fluor 647-conjugate goat anti-rabbit secondary antibodies. (A) The HA-mBAG3 shows a cytoplasmic distribution in HEK293T cells. (B) A representative micrograph shows an intensive co-localization of HA-mBAG3 and GFP-pUL56, and both are targeted to the Golgi (arrows). In addition, partial GFP-pUL56 also targets HA-mBAG3 to the nuclear membrane (arrowheads). Nuclei are stained with 4′,6-Diamidino-2-phenylindole (DAPI). The scale bar indicates 5 μm (A,B). Full article ">Figure 4
BAG3 downregulation is induced by the pUL56 C-terminus. (A) HEK293T cells are transfected with pAcGFP or pAcGFP-UL56, respectively. At 48 hpt, the endogenous BAG3 is significantly downregulated in pAcGFP-UL56, but not pAcGFP transfected or non-transfected cells. (B) Co-transfection of pCMV-HA-mBAG3 with pAcGFP, pAcGFP-UL56, or pAcGFP-UL56 PYM4 into HEK293T cells, respectively. At 48 hpt, HA-mBAG3 is significantly downregulated in pAcGFP-UL56 and pAcGFP-UL56 PYM4 as compared to pAcGFP transfected cells. In addition, there is no significant difference in the relative HA-mBAG3/β-actin levels between pAcGFP-UL56 and pAcGFP-UL56 PYM4 transfected cells. (C) The pUL56 is truncated into three segments S1 (1−67 aa), S2 (68−134 aa, containing four PPxY motifs), and S3 (135−207 aa, containing one predicted transmembrane domain, TMD). Co-transfection of pCMV-HA-mBAG3 with pAcGFP-UL56 S1, S2 and S3 into HEK293T cells, respectively. At 48 hpt, HA-mBAG3 is significantly downregulated in pAcGFP-UL56 S3, but not in S1 or S2 transfected cells as compared to the empty vector. (D) A pUL56 C-terminal mutant (M13) that loses Golgi localization in Vero cells. (E) Co-transfection of pCMV-HA-mBAG3 with pAcGFP-UL56 M13 does not affect relative HA-mBAG3/β-actin levels as compared to empty vector (right panel). The statistical analyses are performed using an ordinary one-way ANOVA post-Tukey’s multiple comparisons test (**: p < 0.01, ***: p < 0.001, ns: no statistical significance). Each statistical data represents three independent experiments. Scale bar indicates 10 μm (D). Full article ">Figure 5
The C-terminal 181L-185L is responsible for pUL56-induced BAG3 degradation. (A) A series of pUL56 C-terminal mutants (pUL56 M1−13) were constructed in our previous study in which the M1−9 were shown to reside at the Golgi, whereas M10−13 resided in the cytoplasm. (B) HEK293T cells are co-transfected with 1 μg pCMV-HA-mBAG3 with an equal amount of pAcGFP, pAcGFP-UL56, or pAcGFP-UL56 M1−12, respectively. At 48 hpt, the whole cell lysates are collected and subjected to a Western blot analysis with the corresponding antibodies. The stars indicate the pUL56 mutants do not mediate HA-mBAG3 downregulation. (C) The mutations L181A, L185A, and L181/5A abrogate pUL56-induced BAG3 degradation. (D) The mutants pUL56 L181A, L185A, and L181/5A localize at the Golgi. The arrowheads indicate Golgi localization. (E) Analysis of amino acid composition of the predicted C-terminal transmembrane helix (177−203 aa) in pUL56. (F) The 1.5 μg pCMV-HA-mBAG3 is co-transfected with 1 μg pAcGFP or pAcGFP-UL56 into HEK293T cells, respectively. At 12 hpt, the cells are treated with the indicated inhibitors for 12 h. At 24 hpt, the cell lysates are collected and subjected to a Western blot analysis. (G) A schematic diagram depicts the molecular basis underlying pUL56-induced BAG3 degradation. Statistical analyses are performed using an ordinary one-way ANOVA post Tukey’s multiple comparisons test (*: p < 0.05, **: p < 0.01, ns: no statistical significance). Each statistical analysis represents three independent experiments. The scale bars indicate 5 μm (D). Full article ">Figure 6
Cleaved BAG3 products are induced in pseudorabies virus (PRV) infected cells in a species dependent manner. The Vero, HEK293T, BHK-21, PC12, and MEF cells are infected with WT and ΔUL56 PRV at an MOI of 0.05, respectively. The mock infected cells are used as a control. At 24 hpi, the whole cell lysates are collected and subjected to a Western blot analysis with anti-BAG3 and anti-PRV gB antibodies. Full article ">Figure 7
Overexpression of BAG3 suppresses PRV proliferation during lytic infection in HEK293T cells. (A,B) HEK293T cells are non-transfected or transfected with indicated amounts of pCMV-HA-mBAG3 for 24 h, which is followed by a mock-infection or infection with WT (A) or ΔUL56 (B) PRV at an MOI of 0.05. At 24 hpi, the whole cell lysates are collected and prepared for Western blot analyses. (C) HEK293T cells are non-transfected or transfected with 0.5 or 2 μg pCMV-HA-mBAG3 for 24 h, which is followed by infection with WT PRV at an MOI of 0.05. The intracellular viruses are harvested at 12 and 24 hpi, respectively. The viruses are titrated in Vero cells. The statistical analysis is performed using two-way ANOVA post Sidak’s multiple comparison test (****: p < 0.0001, ns: no statistical significance), and represents three independent experiments for titer determination. Full article ">Figure 8
Knockdown of BAG3 promotes PRV proliferation during lytic infection in HEK293T cells. (A) HEK293T cells grown in a 12-well plate are transfected with siBAG3 or siNC at a final amount of 120 pmol for 48 h, which was followed by infection with WT or ΔUL56 PRV at an MOI of 0.05, respectively. At 24 hpi, the whole cell lysates are collected and prepared for Western blot analyses. (B) HEK293T cells grown in a 12-well plate are transfected with siBAG3 or siNC at a final amount of 120 pmol for 48 h, which is followed by infection with WT PRV and MOI of 0.05. The intracellular viral titers are determined by TCID50 in Vero cells at 12 and 24 hpi, respectively. The statistical analysis is performed using two-way ANOVA post Sidak’s multiple comparison test (***: p < 0.001, ns: no statistical significance) and it represents three independent experiments for titer determination. Full article ">

20 pages, 1754 KiB

Open AccessArticle

E3 Ligase ITCH Interacts with the Z Matrix Protein of Lassa and Mopeia Viruses and Is Required for the Release of Infectious Particles

by Nicolas Baillet, Sophie Krieger, Xavier Carnec, Mathieu Mateo, Alexandra Journeaux, Othmann Merabet, Valérie Caro, Frédéric Tangy, Pierre-Olivier Vidalain and Sylvain Baize

Viruses 2020, 12(1), 49; https://doi.org/10.3390/v12010049 - 31 Dec 2019

Cited by 12 | Viewed by 3247

Abstract

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential [...] Read more.

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release. Full article

(This article belongs to the Section Animal Viruses)

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Figure 1

Figure 1
siRNA screen of ITCH, WWP1 and WWP2 during old-world arenavirus infection. (A) A549 cells were transfected with siRNA against ITCH (si-ITCH), WWP1 (si-WWP1), WWP2 (si-WWP2) or with a non-targeting siRNA (si-CTL) for 72 h before analysis of silencing efficiency by western blotting (n = 4 independent experiments). (B) A549 cells were transfected with the indicated siRNA for 72 h before infection with the indicated virus at a MOI of 0.01. Two days after infection, cell supernatants were harvested and titrated on Vero cells. The viral titer was calculated as FFU/mL before normalization to control the conditions. The dotted line corresponds to the threshold at which a mean drop of 70% of infectious particles production was observed. The results show the mean percentages ± SEM pooled from four independent experiments. ** p < 0.01, and *** p < 0.001 as determined by a Kruskal–Wallis test followed by Dunn’s post hoc test for multiple comparisons. Full article ">Figure 2
Confirmation of the interaction between various arenavirus Z proteins and the cellular protein ITCH. (A) 293T cells were co-transfected with HA-tagged ITCH and/or FLAG-tagged Z protein of MOPV (ZMop-FLAG), LASV (ZLas-FLAG), LCMV (ZLcm-FLAG), LUJV (ZLuj-FLAG), JUNV (ZJun-FLAG). After 15 h, cells were lysed and protein extracts were readily analyzed (input) or incubated with anti-HA magnetic beads for ITCH immunoprecipitation (IP (HA)). FLAG-tagged Z co-immunoprecipitated proteins were detected by western blotting (n = 3 independent experiments). (B) 293T cells were co-transfected with HA-tagged ITCH and/or FLAG-tagged Z protein of MOPV (ZMop-FLAG), LASV (ZLas-FLAG), LCMV (ZLcm-FLAG), LUJV (ZLuj-FLAG), JUNV (ZJun-FLAG). After 15 h, the cells were lysed (input) or immunoprecipitated with FLAG magnetic beads (IP FLAG) and exogenous HA-tagged ITCH proteins were detected by western blotting (n = 3 independent experiments). (C) HeLa cells were co-transfected with the indicated plasmids and fixed after 15 h of culture for confocal microscopy. Exogenous eGFP-ITCH is shown in green and the MOPV and LASV Z-mCherry viral proteins are shown in red. Nuclei (blue) were visualized using DAPI reagent and colocalization between eGFP-ITCH and Z-mCherry is shown in yellow. All images were taken on a confocal Zeiss LSM 510 with an Axioscope 63× oil immersion lens objective. Scale bar represents 30 µm. (D) Schematic representation of Arenavirus Z matrix protein domains and the generation of mutants by alanine-scanning directed mutagenesis. The scheme represents the position of alanine mutations (in gray) for all MOPV and LASV Z mutants. (E) 293T cells were transfected with empty vector, vectors encoding eGFP-ITCH, LASV, or MOPV Z WT FLAG-tagged protein or their relative mutants obtained from directed alanine-scanning mutagenesis experiments. After 15 h of culture, cells were lysed and cell lysates were directly harvested (cells) or incubated with anti-FLAG magnetic beads (IP (FLAG)). ITCH and FLAG-tagged Z proteins were detected by western blotting (n = 3 independent experiments). Full article ">Figure 3
ITCH directly binds to the Z PPxY motif of the LASV and MOPV Z proteins but is not involved in Z ubiquitination. (A) The 293T cells were transfected with a non-targeting si-CTL RNA or with si-ITCH for 72 h and then transfected with a HA-tagged ubiquitin and/or with plasmids encoding the FLAG-tagged Z protein of LASV or MOPV (ZL, ZM) or their respective mutants of the late domain (ZL-A19, ZM-A20). A total of 24 h after the second transfection, cells were harvested and total protein extracts (Cells) or proteins co-immunoprecipitated using anti-FLAG magnetic beads (IP (FLAG)) were analyzed by western-blot (n = 2 independent experiments). (B) The 293T cells were transfected with the wild type ITCH (ITCH-WT) or with the enzymatically inactive mutant ITCH (ITCH-C830A). The same cells were co-transfected with an empty plasmid (CTL), or with the MOPV or LASV Z protein (ZM or ZL). After 15 h of culture, cell extracts were harvested (cells) or incubated with anti-FLAG magnetic beads (IP (FLAG)). Anti-HA, anti-ITCH, anti-FLAG, and anti-actin were used for western blotting analysis (n = 2 independent experiments). (C) The A549 cells were transfected with the indicated siRNA and cultured for 72 h before being infected with a recombinant MOPV expressing a Z-FLAG tagged protein (MOPV-ZF) at a MOI of 0.1 for 48 h. Cell extracts (cells) were harvested or incubated with anti-FLAG magnetic beads (IP (FLAG)) before western blotting analysis. Full article ">Figure 4
Effect of ITCH on MOPV and LASV infection. (A) A549 and HeLa cells were transfected with the indicated siRNA and cultured for 72 h before analysis of silencing efficiency by western blotting (n = 4 independent experiments). (B–D) The same cells as in (A) were then infected with LASV or MOPV with a MOI of 0.01 (A549 cells) or with a MOI of 0.1 (HeLa cells) for 1 h before being cultured for two days (A549 cells) or three days (HeLa cells) at 37 °C. Viral RNA was then extracted from the cells (B) and supernatants (C) for quantification by RTqPCR. Infectious particles from the supernatants were also titrated on Vero cells (D). Error bars represent the standard error of the means from four independent experiments. * p < 0.05, as determined by the Mann–Whitney test. (E) The 293T cells were transfected with the indicated siRNA and cultured for 72 h before being transfected with an empty plasmid (CTL), or with the FLAG-tagged Z protein of MOPV or LASV (ZM-FLAG or ZL-FLAG). After 15 h of culture, cell extracts were directly analyzed by western blotting (cells) and supernatants were harvested, deposited on a sucrose cushion, and ultracentrifuged at 56K rpm for 90 min, before analysis of the pellet by western blotting (VLPs). Representative results are shown, along with a graph representing the intensity of the VLP Z bands. Error bars represent the standard error of the means from four independent experiments. * p < 0.05, as determined by the Mann–Whitney test. (F) A549 cells were transfected with non-targeting siRNA or ITCH targeting siRNA for 72 h before infection with recombinant MOPV-WT (MOPV-WT) or MOPV-ZFLAG (MOPV-ZF) at a MOI of 2 for 24 h. Intracellular ZFLAG expression was then analyzed by flow cytometry. The bar corresponds to FLAG positive A549 cells. Data shown come from one experiment representative of three replicates. Full article ">

20 pages, 1831 KiB

Open AccessArticle

Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms

by Lloyd C. Wahl, Jessica E. Watt, Hiu T. T. Yim, Danielle De Bourcier, James Tolchard, Surinder M. Soond, Tharin M. A. Blumenschein and Andrew Chantry

Int. J. Mol. Sci. 2019, 20(19), 4682; https://doi.org/10.3390/ijms20194682 - 21 Sep 2019

Cited by 10 | Viewed by 4036

Abstract

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein–protein interaction WW domains. The WW4 domain-containing [...] Read more.

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFβ signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein–protein interaction WW domains. The WW4 domain-containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial–mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3–WW4 tandem domains and a truncated HECT domain that can inhibit TGFβ signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis. Full article

(This article belongs to the Section Biochemistry)

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Figure 1

Figure 1
The three-stranded anti-parallel β-sheet structure of WWP2 WW4 solved by NMR. (A) A schematic of the B1 domain of streptococcal protein G (GB1)–WW4 recombinant protein. (B) Ribbon diagram depictions of the WWP2 WW4 domain from the most representative model (model 1) of the refined 20-model structural ensemble. Full article ">Figure 2
The interaction between WWP2 WW4 and Smad7/S206-phosphorylated Smad7 (pSmad7). (A) The Smad7 ligand, with the PPxY motif in bold and the putative S206 phosphorylation site in red. (B) The superimposed WW4/Smad7 titration heteronuclear single quantum coherence (HSQCs). Lower ligand concentrations are in light grey, and higher ligand concentrations are in dark grey. (C) The superimposed WW4/pSmad7 titration HSQCs. Lower ligand concentrations are in light blue, and higher ligand concentrations are in dark blue. (D) The WW4/Smad7 (1.19 ± 0.21 mM) and WW4/pSmad7 (0.5 ± 0.07 mM) dissociation constants. (E) The shift distances of the WW4/Smad7 and WW4/pSmad7 titration HSQC amide peaks in ppm. The WW4 secondary structure is aligned to the residue number along the x-axis, with β-strands represented as orange boxes. (F) The WWP2 WW4 domain structure with Smad7 binding site residues colour-coded for titration shift distances. The labelled residues are the XP-binding groove (472Phe and 461Tyr) and the second specificity pocket (463Val, 465His and 468Arg), which determine specificity for the PPxY recognition motif. Full article ">Figure 3
Splice variation at the wwp2 gene. (A) A Western blot of HEK293A cells transfected with combinations of mouse and/or human epithelial splicing regulatory proteins (ESRPs) as indicated and stimulated with TGFβ; the membrane was probed with anti-WWP2C antibody or anti-β-actin antibody as a control. (B) The wwp2 gene locus (not to scale) with aligned transcripts and expressed sequence tag (ESTs) as annotated. (C) Semi-quantitative RT-PCR using primers targeting intron 9/10 of wwp2 mRNA extracted from A375 cells treated with TGFβ over 18 h. (D) Semi-quantitative RT-PCR using primers targeting exon 17–intron 19/20 of wwp2 mRNA extracted from COLO-357 and VCaP cells treated with TGFβ over 8 h and from A375 and SK-MEL28 cells treated over 6 h. (E) A schematic showing the domain composition of WWP2-FL, WWP2-C and the putative new isoform WWP2C-ΔHECT. (F) Fold change in luciferase activity in HEK293A cells co-transfected with WWP2C-ΔHECT and the Smad3-dependent CAGA12-luciferase reporter, treated and untreated with TGFβ, with standard error bars. Full article ">Figure 4
The interaction between tandem WWP2 WW3–4 and Smad7. (A) The superimposed WW3–4/Smad7 titration HSQCs. Lower ligand concentrations are in light orange, and higher ligand concentrations are in dark orange. (B) The superimposed WW3/Smad7 titration HSQCs. Lower ligand concentrations are in light green, and higher ligand concentrations are in dark green. (C) The superimposed WW4/Smad7 titration HSQCs. Lower ligand concentrations are in light purple, and higher ligand concentrations are in dark purple. (D) The shift distances of the WW3–4/Smad7 (orange), WW3/Smad7 (green) and WW4/Smad7 (purple) titration HSQC amide peaks in ppm. The WW3–4 secondary structure is aligned to the residue number along the x-axis, with β-strands represented as yellow boxes. (E) The WW3–4/Smad7 binding curve KD fit. (F) The WW3/Smad7 (139 ± 14.4 µM), WW3(tandem)/Smad7 (20.57 ± 26.24 µM), WW4/Smad7 (237 ± 15.7 µM) and WW4(tandem)/Smad7 (249.3 ± 68.1 µM) dissociation constants. (G) HSQC peaks belonging to the WW3 domain at different chemical shifts for WW3–4 (orange) and WW3 (green). Full article ">

14 pages, 2423 KiB

Open AccessEditor’s ChoiceCommunication

Host-Driven Phosphorylation Appears to Regulate the Budding Activity of the Lassa Virus Matrix Protein

by Christopher M. Ziegler, Philip Eisenhauer, Inessa Manuelyan, Marion E. Weir, Emily A. Bruce, Bryan A. Ballif and Jason Botten

Pathogens 2018, 7(4), 97; https://doi.org/10.3390/pathogens7040097 - 9 Dec 2018

Cited by 6 | Viewed by 4513

Abstract

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the [...] Read more.

Lassa mammarenavirus (LASV) is an enveloped RNA virus that can cause Lassa fever, an acute hemorrhagic fever syndrome associated with significant morbidity and high rates of fatality in endemic regions of western Africa. The arenavirus matrix protein Z has several functions during the virus life cycle, including coordinating viral assembly, driving the release of new virus particles, regulating viral polymerase activity, and antagonizing the host antiviral response. There is limited knowledge regarding how the various functions of Z are regulated. To investigate possible means of regulation, mass spectrometry was used to identify potential sites of phosphorylation in the LASV Z protein. This analysis revealed that two serines (S18, S98) and one tyrosine (Y97) are phosphorylated in the flexible N- and C-terminal regions of the protein. Notably, two of these sites, Y97 and S98, are located in (Y97) or directly adjacent to (S98) the PPXY late domain, an important motif for virus release. Studies with non-phosphorylatable and phosphomimetic Z proteins revealed that these sites are important regulators of the release of LASV particles and that host-driven, reversible phosphorylation may play an important role in the regulation of LASV Z protein function. Full article

(This article belongs to the Special Issue Lassa Fever: Viral Replication, Disease Pathogenesis, and Host Immune Modulations)

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Figure 1

Figure 1
Identification of phosphorylation sites in the Lassa mammarenavirus (LASV) Z matrix protein. (A) Coomassie-stained polyacrylamide gel of affinity-purified LASV Z. Streptavidin-coated magnetic beads were used to affinity purify Z from virus-like particles (VLPs) released from cells co-transfected with plasmids encoding a biotin ligase and LASV Z C-terminally tagged with a biotin acceptor peptide (BAP). The band corresponding to the LASV Z protein (indicated by the red box) was excised from the gel and subjected to proteolytic digestion with trypsin or a combination of trypsin and chymotrypsin. The resultant peptides were extracted and subjected to liquid chromatography–tandem mass spectrometry analysis. The asterisk denotes the band of monomeric streptavidin that is eluted from streptavidin beads following boiling. (B) The protein nuclear magnetic resonance structure of the Josiah strain of the LASV Z matrix (PDB 2M1S) is shown. The side chains of the phosphorylated residues (S18, Y97, and S98) are highlighted in green on the protein structure. The two late domains found in LASV Z, PTAP and PPPY, are colored orange. Zinc ions, which are coordinated by the central really interesting new gene (RING) domain of Z, are shown as yellow spheres in the protein structure. The myristoylated glycine residue (at position 2) is also indicated. (C) Protein sequence alignment of select mammarenavirus Z proteins. The Clustal Omega multiple sequence alignment tool was used to align the sequences of selected mammarenavirus Z proteins. The portion of the C-terminal region of each Z protein (the amino acids to the right of the dashed vertical line) containing the late domains (designated as orange, underlined amino acids) was aligned with LASV Z strain Josiah, starting with the most C-terminal amino acid. The following accession numbers were used: GU481069.1 (Lassa mammarenavirus, strain Nig08-04), AAO59514.1 (Lassa mammarenavirus, strain CSF), NP_694871.1 (Lassa mammarenavirus, strain Josiah), AAO59510.1 (Lassa mammarenavirus, strain NL), AAO59508.1 (Lassa mammarenavirus, strain AV), MF990887.1 (Lassa mammarenavirus, strain TGO) AAD03395.1 (Lymphocytic choriomeningitis mammarenavirus, strain WE), ABC96003.1 (Lymphocytic choriomeningitis mammarenavirus, strain Armstrong 53b), ABY20731.1 (Dandenong virus), ABC71138.1 (Mobala mammarenavirus), ABC71136.1 (Mopeia mammarenavirus, strain Mozambique), ABC71142.1 (Ippy mammarenavirus), YP_002929492.1 (Lujo mammarenavirus), NP_899216.1 (Junín mammarenavirus, strain XJ13), NP_899220.1 (Guanarito mammarenavirus), NP_899214.1 (Machupo mammarenavirus), ABY59837.1 (Brazilian mammarenavirus), YP_138535.1 (Pichindé mammarenavirus), YP_001649224.1 (Bear Canyon mammarenavirus), YP_001649215.1 (Oliveros mammarenavirus). For each LASV isolate, the corresponding lineage is listed after the strain. Full article ">Figure 2
Confirmation of Y97 phosphorylation site in LASV Z. (A,B) HEK293T cells were transfected with plasmids encoding the indicated streptavidin-binding peptide (SBP)-tagged lymphocytic choriomeningitis virus (LCMV) Z or LASV Z, and two days later streptavidin-coated magnetic beads were used to affinity purify (AP) intracellular Z from cells that had been treated with water or hydrogen peroxide (H2O2). Levels of phosphotyrosine and Z-SBP in affinity-purified samples (and unpurified cellular input for Z-SBP) were determined by western blotting with anti-phosphotyrosine or anti-SBP antibodies, respectively. Levels of phosphotyrosine are shown for wild-type (WT) LCMV Z and LASV Z (A) as well as for WT and phosphosite-mutant (Y97F) LASV Z proteins (B). Western blots are representative of five (A) or four (B) independent experiments. Full article ">Figure 3
VLP release assay of WT and phosphomutant LASV Z proteins. (A–C) HEK293T cells were transfected with plasmids encoding the WT LASV Z protein or the LASV Z containing mutations that prevent (S to A; Y to F) or mimic (S to D; Y to E) phosphorylation at the serine phosphorylation sites (A) or the tyrosine phosphorylation site (B). The glycine-to-alanine mutant (G2A) served as a negative control as it prevents myristoylation of Z, resulting in drastic inhibition of Z’s budding activity. Quantitative, fluorescent western blotting was used to quantify the amount of intracellular and VLP-derived Z protein. A representative western blot of intracellular or VLP-derived SBP-tagged Z protein is shown in (C). The VLP release activity was determined by dividing the quantity of Z in VLPs by the quantity of intracellular Z, then normalized to the amount of wild-type Z. The values represent the mean ± standard error of the mean from three (A) or four (B) independent experiments. Mean values were compared using a one-way ANOVA with the Holm–Sidak’s test for multiple comparisons. (A,B), n.s. (not significant), ** p < 0.01, **** p < 0.0001. Full article ">

12 pages, 3049 KiB

Open AccessFeature PaperArticle

Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress

by Ziying Han, Michael P. Schwoerer, Philip Hicks, Jingjing Liang, Gordon Ruthel, Corbett T. Berry, Bruce D. Freedman, Cari A. Sagum, Mark T. Bedford, Sachdev S. Sidhu, Marius Sudol and Ronald N. Harty

Diseases 2018, 6(3), 64; https://doi.org/10.3390/diseases6030064 - 13 Jul 2018

Cited by 6 | Viewed by 4554

Abstract

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like [...] Read more.

Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins. Full article

(This article belongs to the Special Issue Host-pathogen Interactions in Ebola, Chikungunya, and Zika Viruses)

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Figure 1

Figure 1
Identification of an interaction between host BAG3 and LFV-Z. Schematic diagram of the GST-WW and GST-SH3 array chip is shown in the top panel. Each lettered square contains one mock (M) and 12 numbered GST-WW or -SH3 domain fusion proteins in duplicate. Fluorescence labeled biotinylated LFV-Z-WT (TAPPEIPPSQNPPPYSP-K-Biotin) and LFV-Z PY mutant (TAPPEIPPSQNAAPASP-K-Biotin) peptides were used to screen the array. A strong interaction between the LFV-Z-WT peptide and the WW-domain of BAG3 was indicated by bright green fluorescent spots shown in the red squares and indicated by the red arrows. No interaction was detected between LFV-Z-PY mutant peptide and BAG3 as shown in the bottom right panel (red squares and arrows). Full article ">Figure 2
Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. (A) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; (B) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; (C) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments. Full article ">Figure 3
BAG3-WT protein inhibits LFV-Z VLP budding in a PPxY/WW-domain dependent manner. (A) Western blot analysis of cell extracts and VLPs from HEK293T cells transfected with LFV-Z alone (lane 1), or LFV-Z + BAG3-WT (lane 2) or BAG3-ΔN mutant (lane 3); (B) Relative budding efficiency of LFV-Z VLPs in HEK293T cells transfected as indicated. Error bars represent the standard deviation of the mean from three independent experiments (n = 3). Full article ">Figure 4
Budding of LFV-Z VLPs, eVP40 VLPs, and VSV-M40 virus from BAG3-WT or BAG3 knockout (KO) cells. (A) HAP1-BAG3-WT (WT) or HAP1-BAG3 knockout cells (KO) were transfected with LFV-Z, and the indicated proteins were detected in cell extracts and VLPs by Western blot analysis as shown in two independent experiments; (B) HAP1-BAG3-WT (WT) or HAP1-BAG3 knockout cells (KO) were transfected with eVP40, and the indicated proteins were detected in cell extracts and VLPs by Western blot analysis as shown in two independent experiments. The red arrows indicate the fold increase in budding of LFV-Z and eVP40 VLPs in BAG3-KO cells compared to BAG3-WT cells; (C) HAP1-BAG3-WT (WT) or HAP1-BAG3 knockout cells (KO) were infected with VSV-M40 recombinant virus, and the indicated proteins were detected in cell extracts by Western blot analysis as shown in three independent experiments. The bar graph depicts the average titers of infectious VSV-M40 virus from three independent experiments. VSV-M40 titers were log10 transformed before checking normality (via Shapiro Wilks normality test) and assessing equality of variance (via F-test). (** p = 0.003). Full article ">Figure 5
Intracellular localization of LFV-Z and BAG3-WT using confocal microscopy. HEK293T cells were co-transfected with LFV-Z and BAG3-WT (A–C, top row), or LFV-Z with BAG3-ΔN mutant (D–F, bottom row). Abundant LFV-Z VLPs (red) were observed to be released from cells co-expressing the BAG3-ΔN mutant (bottom row, merge (F), white dotted squares), compared to that released from cells expressing LFV-Z + BAG3-WT (top row, merge (C), solid white square). Scale bar = 10 μm. Full article ">

4181 KiB

Open AccessArticle

Influence of Cellular Trafficking Pathway on Bluetongue Virus Infection in Ovine Cells

by Bishnupriya Bhattacharya, Cristina C. Celma and Polly Roy

Viruses 2015, 7(5), 2378-2403; https://doi.org/10.3390/v7052378 - 13 May 2015

Cited by 17 | Viewed by 6296

Abstract

Bluetongue virus (BTV), a non-enveloped arbovirus, causes hemorrhagic disease in ruminants. However, the influence of natural host cell proteins on BTV replication process is not defined. In addition to cell lysis, BTV also exits non-ovine cultured cells by non-lytic pathways mediated by nonstructural [...] Read more.

Bluetongue virus (BTV), a non-enveloped arbovirus, causes hemorrhagic disease in ruminants. However, the influence of natural host cell proteins on BTV replication process is not defined. In addition to cell lysis, BTV also exits non-ovine cultured cells by non-lytic pathways mediated by nonstructural protein NS3 that interacts with virus capsid and cellular proteins belonging to calpactin and ESCRT family. The PPXY late domain motif known to recruit NEDD4 family of HECT ubiquitin E3 ligases is also highly conserved in NS3. In this study using a mixture of molecular, biochemical and microscopic techniques we have analyzed the importance of ovine cellular proteins and vesicles in BTV infection. Electron microscopic analysis of BTV infected ovine cells demonstrated close association of mature particles with intracellular vesicles. Inhibition of Multi Vesicular Body (MVB) resident lipid phosphatidylinositol-3-phosphate resulted in decreased total virus titre suggesting that the vesicles might be MVBs. Proteasome mediated inhibition of ubiquitin or modification of virus lacking the PPXY in NS3 reduced virus growth. Thus, our study demonstrated that cellular components comprising of MVB and exocytic pathways proteins are involved in BTV replication in ovine cells. Full article

(This article belongs to the Section Animal Viruses)

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Figure 1

Figure 1
Electron Microscope analysis of infected cell sections. Both PT and control BSR cells were infected with Bluetongue virus (BTV), fixed and processed for cell sectioning. Virus particles in PT (A,B) and BSR (C,D) cells are associated with vesicles (thin black arrows) or released and attached to membranes (open black arrows). In BSR cells the particles can also be seen released by the perturbation of the plasma membrane (broken arrows). Bar, 500 nm. Full article ">Figure 2
Effect of MVB disruption on BTV. (A,B) PT and BSR cells infected with WT BTV at 0.1 MOI were incubated at 12 h pi with LY294002, a specific inhibitor of PI 3-kinase. The total virus (A) and relative (B) titres at 16 h pi were plotted. The plaque assays were done in triplicate and bars represent standard error. Significant difference in the virus titres were designated by the asterisk (*); (C,D) Expression of NS2, VP5 and actin in infected PT (C) and BSR (D) cells. The cells were treated similar to A. Lysates were analyzed by SDS-PAGE and Western blotting. Molecular masses and proteins are indicated on left and right, respectively. Presence or absence of diluent DMSO and inhibitors has been indicated by (+) or (−). NI signified uninfected cells. Full article ">Figure 3
Influence of proteosome inhibitors on BTV growth in PT cells. (A,B) Cells infected with WT BTV at 0.1 MOI were incubated with MG132 or lactacystin. The total (A) and relative (B) titres were assessed at 12 h pi. The plaque assays were done in triplicate and bars represent standard error. Significant difference in the virus titres were designated by the asterisk (*). Presence or absence of diluent DMSO and inhibitors have been indicated below the graph; (C) Expression of NS2, actin and ubiquitin in treated and infected PT cells. The cells were treated similar to A. Lysates were analyzed by SDS-PAGE and Western blotting. Molecular masses and proteins are indicated on left and right, respectively. Arrows represent presence of ubiquitin in untreated cells. Presence or absence of diluent DMSO and inhibitors has been indicated on the top of the panel. NI signified uninfected cells. Full article ">Figure 4
Influence of proteosome inhibitors on virus release. (A) PT cells infected with WT BTV at 0.1 MOI were incubated for 10 or 14 h and treated with MG132 or lactacystin for 2 h. Total and released virus titres were assessed by plaque assay and the significant difference in the virus titres were designated by asterisk (*). The plaque assays were done in triplicate and bars represent standard error; (B) Relative virus titres of (A); (C–F) Expression of free ubiquitin (C,D) and VP5, NS2, actin (E,F) in treated and infected PT cells. The cells were treated similar to A,B. Lysates were analyzed by SDS-PAGE and Western blotting. Molecular masses and proteins are indicated on left and right, respectively. Presence or absence of diluent DMSO and inhibitors has been indicated by (+) or (−), respectively. Arrows represent presence of ubiquitin in untreated cells. NI signified uninfected cells. Full article ">Figure 5
Mutation of PPXY late domain in NS3. (A) Schematic representation depicting position of the PPRY motif in NS3. The numbers designate amino acid positions in NS3 sequence; TM and EC signifies transmembrane domain and extra cellular regions (B,C). Electron Microscope analysis of PT cell sections infected with WT BTV (B) and BTVΔPPRY (C) analyzed at 20 h pi. Virus particles under the plasma membrane and within vesicles (black arrows) been indicated. Bar, 500 nm. Full article ">Figure 6
Characterization of BTVΔPPRY and BTVΔPTAP. Total titres at different times pi of either BTVΔPPRY, BTVΔPSAP or BTV in PT (A); BSR (B) and BSR-NS3 (C) cells were determined, expressed as PFU/mL, and plotted on logarithmic scales. Asterisk (*) indicates that the decrease in titres of BTVΔPPRY or BTVΔPSAP at 12 and 24 h pi is statistically significant to BTV (p < 0.05). The MOI of infection, cell types and viruses used have been indicated in each graph. Full article ">Figure 7
Effect of PPRY domain mutation on NS3 distribution. Localization (A,C) and expression (B,D) of NS3 in (A,B) PT and (C,D) BSR cells infected with BTVΔPPRY or WT BTV1. NS3 was visualized in green and nucleus has been labelled with Hoechst (blue). Scale included in each panel represents 20 µm. The expression of NS2, NS3 and actin in infected (B) PT and (D) BSR cells were analysed at 12 and 24 h pi. Lysates were analyzed by SDS-PAGE and Western blotting. Molecular masses and virus proteins are indicated on left and right, respectively. Full article ">Figure 7 Cont.
Effect of PPRY domain mutation on NS3 distribution. Localization (A,C) and expression (B,D) of NS3 in (A,B) PT and (C,D) BSR cells infected with BTVΔPPRY or WT BTV1. NS3 was visualized in green and nucleus has been labelled with Hoechst (blue). Scale included in each panel represents 20 µm. The expression of NS2, NS3 and actin in infected (B) PT and (D) BSR cells were analysed at 12 and 24 h pi. Lysates were analyzed by SDS-PAGE and Western blotting. Molecular masses and virus proteins are indicated on left and right, respectively. Full article ">Figure 8
Release of BTV influenced by mutation of PPRY domain. The total and cell free titres in PT (A, left) and BSRs (B, left) were plotted on a logarithmic scale. The titre of each fraction was determined by plaque assay. (A,B, right) The relative release was calculated as the ratio of cell free to total virus titre and normalized to 100% for control virus release. Bars represent the standard errors from three sets of replicates; (C) Release of virus particles from PT cells infected with BTVΔPPRY or BTV. Monolayers of PT cells were infected with mutant or control virus. The supernatant and whole cell lysates were harvested 12 h pi (left) and 24 h pi (right) and analyzed by immunoblotting with antibodies against VP5, NS2, NS3/NS3A and tubulin. The protein bands belong to different regions of the same blot. The glycosylation pattern is indicated by asterisk; (D) Analysis of NS2 and actin in BSR cells infected with supernatants of PT cells infected with mutant and control viruses, harvested at 12 and 24 h pi. Full article ">

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