Vpu-Deficient HIV Strains Stimulate Innate Immune Signaling Responses in Target Cells

Cell culture and transfections.

All cells were grown under standard conditions as described previously (10). Jurkat R5 (obtained from M. Katze), THP-1, and primary macrophages were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), l-glutamine, and antibiotics. PBMCs were isolated as described previously (10), and macrophages were cultured further using plastic adherence, as described elsewhere (51). Isolation of T cells from healthy vaginal mucosa was carried out as previously described in detail (20, 21) and were harvested from routinely discarded tissues from vaginal-repair surgeries performed in adult women at three medical centers in Seattle, WA. THP-1 cells were differentiated by exposure to 100 nM phorbol myristate acetate (PMA; Sigma) overnight. Tzm-bl cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS, l-glutamine, and antibiotics.

Stable knockdown cell lines.

Knockdown THP-1 cells for IRF3, IPS-1, and MyD88 genes were stably generated using Sigma Mission lentiviral particles. Cells were generated according to the manufacturer's suggested protocol and tested for knockdown by immunoblot analysis of protein levels compared to those in control non-targeting-vector (NTV) cells.

Viral stocks and infection.

HIV-1_LAI was propagated using standard procedures as described previously (10). YU2 point mutant proviral constructs have been described previously (42), and Vpu mutants of JR-CSF were generated via site-directed mutagenesis, as described in the companion paper (8). Generation of point mutant proviral clones was accomplished using a QuikChange mutagenesis kit (Stratagene) following the manufacturer's recommended protocol, optimizing for the large size of the plasmids. The following primers were used: JR-CSF A/C Forward (5′-GCAGTAAGTAGTGCATGTACTGCAACCTTTACAAATATTAGCAATAGTAGC-3′) and Reverse (5′-GCTACTATTGCTAATATTTGTAAAGGTTGCAGTACATGCACTACTTACTGC-3′); HIV-1 strains JR-CSF and YU2 and Vpu-deficient proviral clones were transfected into 293T cells as described previously to generate infectious virus (20, 21). Mock infections represent treatment with conditioned medium, and infections with virus were performed by adding virus to culture medium, with washing performed prior to harvest except where otherwise indicated. The titers of all HIV-1 strains were determined on Tzm-bl cells to determine the concentration of infectious virus. Sendai virus (SeV) strain Cantell was obtained from Charles River Laboratories.

Cell treatments and fractionation.

Synthetic hepatitis C virus (HCV) PAMP RNA was generated as previously described and delivered into cells using a TransIT kit (Mirus) according to the manufacturer's instructions (47). Nuclear and cytoplasmic fractionation was performed using standard methods previously described (28).

Immunoblot analysis, coimmunoprecipitation, and immunofluorescence imaging.

SDS-PAGE and immunoblot analysis were performed using standard procedures as described previously (10). The following antibodies were used in the study: rabbit (Rb) anti-ISG56 (G. Sen), mouse (M) anti-p24, goat (Gt) anti-beta-actin (Santa Cruz), Rb total anti-IRF3 (a gift from Michael David), Rb anti-IRF3-p (Cell Signaling), M total anti-IRF-3 (19), Rb anti-lamin B (Abcam), M antitubulin (Sigma), Rb anti-HIV-1_NL4-3 Vpu, Rb anti-human IPS-1 (Alexis), and Rb anti-human MyD88 (Abcam). For immunoblot detection, the appropriate horseradish peroxidase-conjugated secondary antibody was used (Jackson ImmunoResearch Laboratories), followed by treatment of the membrane with ECL-plus reagent (Roche) and imaging on X-ray film. Densitometry was performed using ImageJ software (NIH) on unsaturated blots.

Statistical analysis.

Differences between groups were analyzed for statistical significance by the Student t test.

Targeted genomics analysis.

RNA was extracted using an RNeasy minikit (Qiagen) according to the manufacturer's instructions. Two hundred nanograms of total RNA was amplified and labeled using an Illumina TotalPrep RNA amplification kit (Ambion). cRNA quality was assessed by capillary electrophoresis on an Agilent 2100 bioanalyzer. Expression levels of mRNA transcripts were assessed by human-HT12 (version 4) expression BeadChips (Illumina). Hybridization was carried out according to the manufacturer's instructions.

Data preprocessing.

Bead summary data were output from Illumina's BeadStudio software without background correction, as this has previously been shown to have detrimental effects (15). Data preprocessing, including a variance-stabilizing transformation (26) and robust-spline normalization, was applied as implemented in the lumi package of R (14). Data were prefiltered for probes that were not expressed (detection P value, ≤0.05) in at least one group (n = 4/group), leaving a set of 17,993 probes for subsequent analysis. Data were further filtered on the basis of an experimentally derived list of virus-induced known ISGs and anti-HIV-1 molecules (Table 1) (7, 53). An additional filter for 74 annotated NF-κB-responsive genes was used to determine specificity of response (Table 1).

Differential expression analysis.

A Bayesian statistical framework that was developed by Pierre Baldi and Tony Long called Cyber-T was used to test for the effect of infected (JR-CSF, JR-CSF A/C) versus mock-infected samples on gene expression (27). We used a false discovery rate (FDR) Benjamini-Hochberg method to control for multiple testing (3). Probes selected for further analysis had an FDR-adjusted P value of ≤0.05. A log₂-fold change was calculated as the difference between infected and mock-infected samples. All statistical testing for gene expression was performed in FlexArray, a front-end wrapper for R (4). To compare samples from infected and mock-infected groups, we clustered the expression profiles for a select gene set using unsupervised hierarchical clustering with Euclidean average linkage clustering with both gene and sample leaf ordering as implemented in the TMEV program (45, 46).

The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Tzm-bl cells were from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc., monoclonal antibody to HIV-1 p24 (AG3.0) was from Jonathan Allan, and HIV-1_NL4-3 Vpu antiserum was from Klaus Strebel.

Vpu-deficient HIV-1 strains stimulate the production of ISGs upon infection of target cells.

Vpu-deficient strains of HIV-1 such as HIV_YU2 are unable to downmodulate IRF3-dependent signaling or drive relocalization of IRF3 to the lysosomal compartment (8). To evaluate the role of Vpu in regulating IRF3-dependent signaling of innate immunity during HIV-1 infection, we examined the response of cells acutely infected with HIV-1_JR-CSF (Vpu-positive) or HIV-1_YU2 (Vpu-negative) virus strains. Infection of differentiated THP-1 cells and R5-permissive Jurkat cells with HIV-1_JR-CSF failed to induce innate immune signaling responses marked by induction of expression of ISG56 (Fig. 1A and B, respectively), an IRF3 target gene (18). Conversely, HIV-1_YU2 infection triggered the robust induction of ISG56 production in both cell types (Fig. 1A and B). Moreover, HIV-1_YU2 induced the innate immune signaling of ISG56 production upon infection of primary macrophages (Fig. 1C), and importantly, ex vivo infection of cultures of T cells harvested from human vaginal epithelium samples by HIV-1_YU2 induced an innate immune response characterized by ISG expression, whereas infection by HIV-1_JR-CSF did not induce this response (Fig. 1D). To control for the effects of other genetic differences between the HIV-1_YU2 and HIV-1_JR-CSF strains, we took advantage of congenic HIV-1_JR-CSF and HIV-1_{JR-CSF A/C} mutant viruses for direct comparison of their innate immune stimulatory capacity. The mutant HIV-1_JR-CSF containing an A/C transversion in the Vpu start codon triggered a robust innate immune response in THP-1 cells in a dose-dependent manner (Fig. 1E) which greatly exceeded the response to the wild-type (wt) HIV-1 strain. It is important to note that we have previously shown, as indicated in the companion paper (8), that these Vpu-deficient proviruses' ability to block innate immune signaling can be rescued by providing Vpu in trans. Additionally, when Vpu is ectopically expressed in producer cells along with Vpu-deficient provirus, the resulting virus produced is robustly stimulatory for innate immune induction (data not shown). These observations reveal that T cells and macrophages are fully competent to recognize HIV-1 through a pathogen-sensing pathway(s) that directs the innate antiviral response against infection but this response is blocked by HIV-1 in a Vpu-dependent manner.

IRF3-dependent but IPS-1- and MyD88-independent induction of ISGs in response to Vpu-deficient HIV-1 infection.

The inability of HIV-1_YU2 to produce Vpu and suppress IRF3 provides a novel model system to examine the features of natural innate immune triggering during HIV-1 infection. We therefore assessed the role of canonical IRF3-dependent PRR pathways in viral recognition and innate immune signaling during acute infection of THP-1 cells. Infection with HIV-1_YU2 induced IRF3 phosphorylation, similar to that induced by infection with SeV (visible as laddering and slower-migrating IRF3 bands), but infection with HIV-1_JR-CSF did not induce this response (Fig. 2A). Moreover, infection by HIV-1_YU2 resulted in the nuclear accumulation of phospho-IRF3 and concomitant expression of ISG56 (Fig. 2B and C). To assess the requirement for IRF3 and the possible roles of RLR- and MyD88-dependent PRR signaling pathways in this response, we examined signaling in THP-1 cells stably expressing a nontargeting control short hairpin RNA (shRNA) or shRNA targeting IRF3, IPS-1, or MyD88, respectively. Each cell line exhibited stable knockdown of target gene expression compared to nontargeting controls (Fig. 2D). Compared to the control cells, IRF3-knockdown cells no longer induced ISG56 expression in response to HIV-1_YU2 or SeV infection (Fig. 2C). However, IPS-1-knockdown cells and MyD88-knockdown cells both retained their response and induced ISG56 expression when infected with HIV-1_YU2. As expected, MyD88-knockdown cells also responded to SeV, a specific RLR agonist (28), but this response was ablated in IPS-1-knockdown cells (Fig. 2C). Thus, we conclude that virus recognition and innate immune signaling of HIV-1 infection occur through an IRF3-dependent but IPS-1- and MyD88-independent cellular pathway(s).

Rescue of Vpu-deficient HIV-1 by knockdown of IRF3.

While Vpu-negative HIV-1 is replication competent (42), we observed a reduction in viral protein expression in cells infected with Vpu-negative HIV-1 relative to those infected with congenic wt virus, and this reduction associated with the stimulation of the host cell innate immune response (see Fig. 1D as an example). We hypothesized that the loss of IRF3 regulation by Vpu-negative viruses may impart cellular defenses that suppress HIV-1, similar to the outcome of expressing constitutively active IRF3 (10). Of note is the fact that HIV-1_JR-CSF directs the rapid degradation of IRF3 during acute infection (10), and accordingly, we found that it replicated (measured by p24 accumulation at 24 h postinfection [hpi] compared to input measured at 8 hpi) to similar levels in both control and IRF3-knockdown THP-1 cells over the first 24 h of infection (Fig. 3A). In contrast, Vpu-negative HIV-1_{JR-CSF A/C} replication was reduced in control cells compared to wt virus replication, but replication was partially rescued during acute infection in IRF3-knockdown cells (Fig. 4A and B). This resulted in a significant increase in viral growth of the HIV-1_{JR-CSF A/C} mutant in the IRF3-knockdown cells, with no change observed for the wt strain (Fig. 4B). Importantly, these experiments focused only on the first round of HIV-1 infection/replication, therefore avoiding any contribution of tetherin/BST2 in suppressing virus production at the level of viral egress. Thus, HIV-1 triggering of IRF3-dependent innate immune defenses restricts viral replication fitness, wherein Vpu mediates a blockade of IRF3 signaling to support efficient HIV-1 replication.

Vpu-deficient HIV strains stimulate the induced expression of many anti-HIV ISGs.

The lysosomal targeting and degradation of IRF3 by Vpu might alter IRF3-dependent innate immune responses within the infected cell, thus providing a permissive environment to support acute HIV-1 infection (see the companion paper [8]). To address this idea, we performed targeted genomics analyses of innate immune gene expression at 8 or 16 h posttreatment to compare the responses of mock-infected cells to those of cells infected with congenic wt HIV-1_JR-CSF or HIV-1_{JR-CSF A/C} as well as HIV-1_YU2 during an acute infection. As a positive control for innate immune induction by IRF3-dependent PAMP signaling, we assessed similar cells that were transfected with a well-characterized synthetic PAMP motif encoded within the RNA genome of hepatitis C virus (47). As shown in Fig. 4A, while THP-1 cells responded to PAMP RNA to induce the expression of innate immune response genes and ISGs, acute infection of THP-1 cells with HIV-1_JR-CSF failed to induce a response at 8 h postinfection and induced only a very weak response at 16 h postinfection. Remarkably however, infection of THP-1 cells with HIV-1_{JR-CSF A/C} or HIV-1_YU2 lacking Vpu expression resulted in a marked induction of innate immune response gene expression at 8 h postinfection and a highly robust response by 16 h postinfection. By comparison, the innate immune response induced by HIV-1_JR-CSF at 16 h postinfection was substantially weaker than that of congenic HIV-1_{JR-CSF A/C} occurring within 8 h postinfection (Fig. 4A). The majority of the HIV-1-induced genes encoded known antiviral factors and immune signaling molecules (PKR, MX1, STAT1) (44) and, importantly, included many ISGs known to be HIV-1 restriction factors, including tetherin/BST2, viperin, ISG15, ISG20, TRIM5, and APOBEC3G (Fig. 4B) (16, 36, 39, 43). Surprisingly, we did not detect the expression of IFN-β in either the PAMP-treated cells or Vpu-deficient HIV-1-infected cells, possibly reflecting the possibility that IFN-β induction occurred temporally outside our sampling time points or that it was present below our limit of detection.

Our previous work has shown that lab strains of HIV-1 have no effect toward suppressing the NF-κB signaling capacity of infected cells (10). To determine if the differential ISG induction profile was specific for IRF3 signaling of the Vpu-deficient viral strains, we compared these results with the induction profile of known NF-κB-regulated genes, by analyzing the gene induction profile of over 70 annotated NF-κB-responsive genes (Table 1) from the same infection samples for HIV-1_JR-CSF, HIV-1_{JR-CSF A/C}, or HIV-1_YU2. Indeed, we found that several known NF-κB-dependent genes were upregulated during infection with wt HIV-1_JR-CSF and Vpu-deficient HIV-1 strains (Fig. 4C). To compare these results directly, we examined the 10 most upregulated NF-κB-dependent genes from the HIV-1_{JR-CSF A/C} infection and compared the expression induced by all three viral strains at 16 h postinfection (Fig. 4C). We found that all 10 genes were significantly upregulated in response to infection by all three viruses. With the exception of TNFSF14, the NF-κB-responsive genes were induced to similar levels. Thus, deletion of Vpu releases a specific IRF3 blockade imposed by HIV-1, without alteration of virus-induced NF-κB programs, to reveal an endogenous innate immune signaling program induced by HIV-1 infection. IRF3 suppression by Vpu therefore serves an important role to prevent the specific induction of innate immune gene expression, including the expression of well-characterized anti-HIV-1 restriction factors. These results define the Vpu-IRF3 interaction as a regulatory node that governs the innate immune response of the host cell to HIV-1 infection.