SIVSM/HIV-2 Vpx proteins promote retroviral escape from a proteasome-dependent restriction pathway present in human dendritic cells

Abstract

Background: Vpx is a non-structural protein coded by members of the SIVSM/HIV-2 lineage that is believed to have originated by duplication of the common vpr gene present in primate lentiviruses. Vpx is incorporated into virion particles and is thus present during the early steps of viral infection, where it is thought to drive nuclear import of viral nucleoprotein complexes. We have previously shown that Vpx is required for SIVMAC-derived lentiviral vectors (LVs) infection of human monocyte-derived dendritic cells (DCs). However, since the requirement for Vpx is specific for DCs and not for other non-dividing cell types, this suggests that Vpx may play a role other than nuclear import.

Results: Here, we show that the function of Vpx in the infection of DCs is conserved exclusively within the SIVSM/HIV-2 lineage. At a molecular level, Vpx acts by promoting the accumulation of full length viral DNA. Furthermore, when supplied in target cells prior to infection, Vpx exerts a similar effect following infection of DCs with retroviruses as divergent as primate and feline lentiviruses and gammaretroviruses. Lastly, the effect of Vpx overlaps with that of the proteasome inhibitor MG132 in DCs.

Conclusion: Overall, our results support the notion that Vpx modifies the intracellular milieu of target DCs to facilitate lentiviral infection. The data suggest that this is achieved by promoting viral escape from a proteasome-dependent pathway especially detrimental to viral infection in DCs.

Figure 1

The function of Vpx in the infection of DCs is conserved uniquely in members of the SIV_SM/HIV-2 lineage. A) HIV-2 and SIV_MAC LVs rely on Vpx for the infection of DCs. VSVg-pseudotyped SIV_MAC and HIV-2 LVs (coding or not for Vpx) were produced in 293T cells, purified by ultracentrifugation and used to infect DCs at a multiplicity of infection (MOI) of 3. Vpx-containing SIV_MAC VLPs (Vpx-VLPs) similarly produced were added onto DCs at MOI equivalent of 2 (as measured by exo-RT test with standards of known infectivity) for 2 hrs prior to infection with the above-mentioned LVs. GFP⁺ cells were scored 3 days later by flow cytometry. B) Only Vpx proteins from the SIV_SM/HIV-2 lineage rescue the infectivity defect of Vpx-deficient SIV_MAC LVs. VSVg-pseudotyped SIV_MAC LVs (SIV15^-, coding gag-pro-pol) were produced in presence or absence of Flag-tagged Vpx proteins derived from SIV_MAC, SIV_RCM and HIV-2. Virions were then normalized for their infectious titer on HeLa cells and used to infect DCs at MOI 0.3 or 3. The incorporation of Flag-Vpx proteins into virion particles was assessed by Western blot (right panel). The different migration on SDS-PAGE of HIV-2 and SIV_MAC Vpx proteins has already been reported [40]. C) Only Vpx proteins from the SIV_SM/HIV-2 lineage increase WT HIV-1 LVs infection in a pre-incubation assay. Non-infectious SIV_MAC VLPs containing the different Flag-Vpx proteins were produced and used as described in A in a pre-incubation assay to test their effect on WT HIV-1 LVs infectivity (used at a constant MOI of 3). Incorporation of Flag-Vpx proteins into VLPs was assessed by Western blot (right panel). One representative data set out of 3 to 5 independent experiments is shown for each panel.

Figure 2

Vpx allows the accumulation of full length viral DNA following SIV_MAC infection of DCs. DCs were infected with normalized amounts of SIV_MAC LVs containing or not Vpx (MOI of 2). Cell aliquots were harvested at 4 and 24 hrs post-infection and analyzed by semi-quantitative PCR (serial five-fold sample DNA dilutions) using primers that recognized specifically early (MSSS) and late (FL and 2LTRs) products of reverse transcription. The amount of sample added in the PCR reaction decreases from right to left, as represented by triangles: sample amount). Amplification of mitochondrial DNA (mtDNA) was used for normalization. PCR products were transferred onto a nylon membrane and hybridized with ³²P-labelled specific probes prior to phosphor imager analysis and quantification. One representative data set out of 4 independent experiments is shown here.

Figure 3

Vpx exerts a general positive effect on lentiviral infection and results in an increased accumulation of full length viral DNA. A) Infections of DCs were carried out with VSVg-pseudotyped retroviral vectors bearing a CMV-GFP expression cassette (RVs, MOI 5) with or without Vpx-VLPs pre-incubation (MOI equivalent of 2, measured by exo-RT activity in comparison with standards of known infectivity). The percentage of infected cells was determined by flow cytometry 72 hours afterwards. B) DCs were pre-incubated with Vpx-VLPs at an MOI equivalent of 2 for 2 hrs prior to infection with a constant amount of HIV-1 (B), FIV (C) and MLV retroviral vectors (RV, D) at MOI 5. Cell aliquots were harvested at 4 and 24 hrs post-infection for HIV-1 and at 24 hrs only for FIV and MLV and analyzed by semi-quantitative PCR on serial five-fold sample dilutions (sample amount represented by triangles, as in the legend to Fig. 2), using primers that recognized specifically early and late products of reverse transcription. Amplification of actin DNA (actin) was used for normalization. For MLV, the positive control for 2LTRs amplification is represented by cell lysates of HeLa cells obtained 24 hrs post-infection with 10 fold less MLV vector than was used for DCs. PCR products were transferred onto a nylon membrane and hybridized with ³²P-labelled specific probes prior to phosphor imager analysis and quantification. One representative data set out of 3 to 4 independent experiments is shown here.

Figure 4

Vpx allows faster rates of complete and infectious viral DNA accumulation following HIV-1 infection. A) DCs were infected with a constant amount of HIV-1 LVs with or without Vpx-VLPs pre-incubation. Cell aliquots were harvested at times comprised between 4 and 48 hrs post-infection and the accumulation of FL viral DNA analyzed by semi-quantitative PCR. PCR products were quantified by phosphor imager (ordinate) after southern blot and hybridization analysis and input DNA normalization and are presented here in function of time (abscissa). B) DCs were infected with HIV-1 LVs with or without Vpx-VLPs pre-incubation and infection's rates obtained under normal conditions were compared with those obtained by inhibiting RT synthesis by addition of the nonnucleoside RT inhibitor Nevirapine at 7 hrs post-infection (10 μg/ml, this concentration inhibits completely viral infection if provided at the time of infection, not shown). GFP positive cells were analyzed by flow cytometry 5 days post infection. One representative data out of 2 independent experiments are presented for each panel.

Figure 5

Vpx and the proteasome inhibitor MG132, but not Arsenate, display similar effects on the infection of DCs. A) DCs were either infected with Vpx-deficient SIV_MAC or HIV-1 LVs (A and B, respectively, at MOI of 0.5 and 5) and treated singularly or in combination with 1 μM arsenic acid (As₂O₃) and Vpx-VLPs (at MOI equivalents of 0,5 and 2,5). The efficiency of infection was evaluated 72 hrs after by flow cytometry analysis. C) DCs were infected with Vpx containing or deficient SIV_MAC LVs (MOI 5) in presence or absence of MG132 (1 μg/ml). The drug was added 30 min prior to infection then left for a total of 7 hrs prior to cell washing and media replacement. DCs were then lysed at 24 hrs post-infection for FL PCR analysis as described in the legend of Fig. 5A. Results are presented as a fold increase in FL DNA for each condition with respect to the amount produced upon infection with Vpx-deficient SIV_MAC LVs. D) DCs were similarly infected with a constant amount of HIV-1 LVs in presence or absence of MG132 and 2 different amounts of Vpx-VLPs. Media was replaced 7 hrs post-drug addition and cells analyzed 2 days afterwards by flow cytometry. One representative experiment out of 3 is shown here for each panel.