Our data support the notion that Vpx of the SIVSM/HIV-2 lineage allows the efficient accumulation of complete viral DNA by counteracting a proteasome-dependent restriction pathway specifically in DCs. We have not observed a similar role of Vpx in the infection of other non-dividing cell types such as macrophages or IL7-stimulated PBLs, although Vpx had a minor stimulating effect on the former cell type . This suggests that the restriction pathway that is targeted by Vpx is particularly active in DCs with respect to other cell types.
We believe that multiple evidences support the hypothesis that Vpx modifies DCs by counteracting a specific restriction mechanism. Vpx provided in trans in target DCs induces the accumulation of viral DNAs following infection with quite distantly related retroviruses, excluding the possibility that Vpx acts on conserved viral elements. This function of Vpx is specific to immature DCs (as well as mature DCs, not shown). Lastly, the positive effect of Vpx is maintained if Vpx-VLPs and infectious LVs enter DCs via distinct entry pathways (RD114, GALV and VSVg, not shown), supporting the notion that Vpx targets cellular rather than viral components.
The kinetic analysis of full length viral DNA accumulation following HIV-1 infection revealed that Vpx speeds up the completion of the RT process, a reaction that seems relatively slow in DCs. Indeed, the majority of viral DNA is synthesized by 7 hrs in presence of Vpx as opposed to 48 hrs in its absence. Despite the fact that at 48 hrs post-infection equivalent amounts of viral DNA accumulated in both conditions, the viral DNA synthesized in presence of Vpx is by far more infectious. This suggests that viral genomes that are not completed in a short time are more likely to be targeted by anti-viral cellular defense mechanisms that diminish their infectivity. Given that the viral DNA is contained within a nucleoprotein complex that chaperones it through its life cycle, such defenses may act at multiple steps. In this respect, by promoting the completion of viral DNA synthesis by RT, Vpx may drive structural rearrangements in viral complexes that alter their stability or their trafficking within the cytoplasm with the result of protecting them.
Several results shown here argue that the block relieved by Vpx in DCs utilizes the proteasome. In fact, the proteasome inhibitor MG132 partially rescued the accumulation of full length viral DNA after infection with SIVMAC LVs lacking Vpx. MG132 had an effect of the same order of magnitude of Vpx on HIV-1 infection but the two effects were not additive. Lastly, the positive effect of proteasome inhibitors on viral infection of most cell types appears much milder than the one observed here in DCs ([24–28] between 3 to 7 fold, as opposed to 30 fold on average in DCs). Due to their high antigen processing ability, the possibility that DCs display high levels of proteasome activity is not unlikely. Although this hypothesis remains to be tested, it may explain why Vpx is required specifically in DCs.
An alternative explanation for the phenomenon observed here is that Vpx does not target a restriction pathway but simply increases the overall efficiency of RT synthesis by altering the intracellular dNTP pool. Although such hypothesis has not been tested directly, we believe it unlikely because early RT products (MSSS) are unaffected by Vpx.
A Vif-insensitive restriction block specified by APOBEC3G molecules present in the form of low molecular weight complexes has been described in cells resistant to HIV-1 infection, such as quiescent lymphocytes, monocytes and more recently DCs [29, 30]. However, Vpx doesn't restore HIV-1 infection in quiescent lymphocytes nor monocytes (not shown) making it unlikely, although formally possible, that Vpx acts by inhibiting APOBEC.
Our data may be reminiscent of the tripartite motif protein 5alpha-induced restriction (TRIM5α) and of its negative impact on lentiviral infection [31, 32]. However, we believe that the effect described here are independent from TRIM5α-mediated restriction. Indeed, the defect of Vpx-deficient SIVMAC LVs is not relieved with increasing amounts of viral targets and human TRIM5α is not known to target HIV-1 nor SIVMAC infection. This suggests that Vpx may act by counteracting a distinct restriction pathway that remains to be identified.