The experiments presented here demonstrate that SIVMAC/HIV-2 Vpx rescues HIV-1 from the antiviral state established by exogenous type I IFN or LPS in MDDCs. This phenotype is truly extraordinary in that Vpx offered complete rescue of HIV-1, after the antiviral state had been fully established, and the magnitude of the rescue approached 1000-fold. Surprisingly, the presence of Vpx in SIVMAC or HIV-2 did not protect these viruses from IFN-β or LPS treatment, even when target cell MDDCs were treated with VLPs bearing additional Vpx prior to challenge with reporter virus. Although Vpx is not normally an HIV-1 accessory protein, it provides a powerful tool that will aid attempts to identify new HIV-1 restriction factors that are elicited by IFN in dendritic cells.
Elucidation of the mechanism by which Vpx rescues HIV-1 from the antiviral state would be aided enormously by an experimental system that exploits a cell line. Among cell lines tested, the most pronounced phenotype was observed with the acute monocytic leukemia cell line THP-1 , which had been treated with phorbol esters to promote differentiation into macrophages, as we reported previously to study Vpx and innate immune signaling [11, 39]. The magnitude inhibition of HIV-1 transduction by LPS or IFN-β in THP-1 macrophages [11, 39] was 10-fold less than that seen in MDDC. Of greater concern, though, rescue of HIV-1 from the antiviral state by Vpx+ VLPs in these cells was only 2 to 10-fold (data not shown). Ongoing mechanistic studies concerning the Vpx phenotype reported here, then, will likely not be possible with a cell line.
HIV-1 transduction of monocyte-derived macrophages (MDMs) was also greatly stimulated by Vpx; although, in the absence of exogenous IFN, HIV-1 transduction efficiency was lower in these cells than in MDDCs (data not shown). A necessary consequence is that a smaller proportion of the Vpx effect in MDMs was specific to the antiviral state. In other words, the magnitude rescue of HIV-1 by Vpx following establishment of the antiviral state with exogenous IFN was most evident in MDDCs. In the presence of exogenous type 1 IFN, MDDCs might express an HIV-1-specific, Vpx-sensitive, anti-viral effector at higher levels than do MDMs. Alternatively, constitutive expression levels of this putative factor might be higher in MDMs.
Viruses often encode factors that prevent establishment of the antiviral state. For example, hepatitis C virus, poliovirus, and rhinovirus proteases degrade MDA-5, RIG-I, IPS-1, and TRIF [48–53]. In the experimental system reported here, Vpx was administered after the antiviral state was fully established. Therefore, Vpx does not act by blocking induction of the antiviral state. This is consistent with the observation that vpx had no significant effect on the transcriptional induction of luciferase reporters for critical innate immune factors, including IFN-β, NF-κB, or AP-1 (additional file 4, Figure S4).
Additionally, Vpx appears not to launch a global shutdown of the antiviral state. It caused no change in levels of MDDC cell surface markers for maturation, in IFN-β secretion and steady-state protein levels for MX1 and APOBEC3A, or in steady-state levels of mRNAs produced by 8 ISGs (additional file 1, Figure S1). More importantly, Vpx did not rescue SIVMAC or HIV-2, indicating that the antiviral state was very much intact following exposure to Vpx. More likely, Vpx inactivates an HIV-1-specific antiviral effector that is induced by IFN. This inactivation might involve degradation, the same way that Vif promotes the degradation of APOBEC3G [30–32] or Vpu promotes the degradation of TETHERIN [54–56]. Alternatively, Vpx might sequester the putative factor, blocking it without assistance from ubiquitination machinery, as may also be the case with Vif and Vpu [57, 58].
Though it has been known for over 20 years that type 1 IFN and LPS block HIV-1 infection of myeloid cells , the effector proteins responsible for the block to HIV-1 transduction of IFN-treated MDDCs is not known. Several ISG-encoded proteins inhibit HIV-1, APOBEC3G  and Tetherin [60, 61] being prominent among them. These host restriction factors pose obstacles to infection of sufficient importance that HIV-1 maintains two of its nine genes - vif and vpu, respectively - to counteract them. Neither Vif nor Vpu is required for the phenotype reported here since Vpx rescued minimal HIV-1 vectors lacking all viral accessory proteins as efficiently as it rescued full HIV-1 virus. Additionally, the best-characterized phenotypes of Vif and Vpu require their presence during virion assembly and the experiments reported here likely involve effects of Vpx that are restricted to the target cell.
TRIM5, another restriction factor encoded by an ISG, is required for establishment of an antiviral state by LPS in MDDCs . Nonetheless, endogenous human TRIM5 is unlikely to be a direct antiviral effector in the experiments reported here since inhibition of HIV-1 transduction by exogenous type 1 IFN is not reversed by TRIM5 knockdown . Other TRIM proteins are encoded by ISGs , and some of these exhibit antiviral activity . TRIM22, for example, blocks HIV-1 LTR-directed transcription , but the putative antiviral effector in IFN-treated MDDCs acts before integration, as documented by Alu-PCR (Figure 7). Additionally, TRIM22 does not block transcription from the heterologous promoter (SFFVp) used in the transduction vectors here .
In the course of examining ISG expression levels in MDDCs it was observed that, in response to exogenous type 1 IFN or LPS, APOBEC3A mRNA levels increased nearly 10,000-fold and the protein levels also increased to an impressive extent (additional file 1, Figure S1C). APOBEC3A is a nuclear protein [65, 66] and therefore a reasonable candidate for the Vpx-sensitive, IFN-stimulated, anti-HIV-1 effector protein. Specific association of APOBEC3A with Vpx was not detected in co-transfection experiments in 293T cells, and no effect on inhibition of HIV-1 was observed when APOBEC3A knockdown was attempted with lentiviral vectors or with transfected double-stranded RNA oligonucleotides (data not shown). These findings are in contrast to reports that Vpx associates with APOBEC3A and that a vpx mutant that does not bind to APOBEC3A failed to stimulate HIV-1 infection of monocytes . APOBEC3A knockdown was also reported to render monocytes more permissive for HIV-1 . These discrepancies with the results reported here might be due to cell type differences, i.e, monocytes versus MDDCs, or other differences in methodology.
Vpx was recently shown to bind to SAMHD1 and promote the degradation of this myeloid cell protein [69, 70]. While SAMHD1 is clearly a Vpx-sensitive inhibitor of HIV-1 replication in myeloid cells, it does not appear to be the IFN-stimulated HIV-1 inhibitor described here. SAMHD1 knockdown in THP-1 cells results in more than 10-fold increase in HIV-1 replication ; in contrast to the enormous effect of Vpx in IFN-treated MDDCs, HIV-1 infection of IFN-treated THP-1 cells increases only two to three-fold in response to Vpx.
Both Vpr and Vpx bind DCAF1 (VPRBP) and associate with the DDB1/RBX1/CUL4A E3 ubiquitin ligase complex [12, 13, 15, 33–37, 71, 72]. Vpr might, therefore, be expected to interfere with Vpx binding to DCAF1 and the E3 complex. However, the presence of HIV-1 Vpr or SIVMAC Vpr did not significantly alter the ability of SIVMAC Vpx to protect HIV-1 from the antiviral state, underlying the unique ability of Vpx to protect HIV-1.
The unexpected finding that Vpx mutant proteins that do not bind to DCAF1 (Figure 5A and references [12, 13, 15, 35]) retain the ability to rescue HIV-1 from exogenous IFN indicates that the DCAF1/DDB1/RBX1/CUL4A E3 ubiquitin ligase complex is dispensable for the phenotype reported here. Consistent with this result was the demonstration that Vpx rescued HIV-1 in the presence of an effective DCAF1 knockdown (Figure 6). While the DCAF1/DDB1/RBX1/CUL4A E3 ubiquitin ligase complex, and Vpx, is clearly required for SIVMAC to infect human macrophages in the absence of exogenous type 1 IFN , Vpx interaction with DCAF1 was also not required for HIV-1 transduction of THP-1 macrophages . These results indicate that, if Vpx rescues HIV-1 from the antiviral state by promoting the degradation of an antiviral effector, it does so by recruiting a yet-to-be-identified E3 ubiquitin ligase complex.
As previously reported [10, 13, 14], Vpx had a large effect on HIV-1 reverse transcription in transduced MDDCs (Figure 7). An additional effect of Vpx was observed, though, that was specific to the cells that had been treated with exogenous type 1 IFN: Vpx overcame a block to HIV-1 transduction that occurred after the virus had entered the target cell nucleus (Figure 7). Thus, it may be that Vpx protects HIV-1 from more than one antiviral factor. The first factor is constitutively expressed in myeloid cells and blocks reverse transcription. The second factor is induced by IFN and acts in the nucleus to block transduction. HIV-1 CA and IN, two proteins essential at this stage of the HIV-1 replication cycle [28, 73, 74], would be likely targets of this antiviral factor. To date, attempts to demonstrate the importance of these proteins by transferring Vpx-responsiveness using chimeric viruses have not been successful due to the poor infectivity of these constructs in highly permissive cell lines, let alone in MDDCs.
Why does Vpx protect HIV-1, and not SIVMAC or HIV-2, from the antiviral state in MDDCs? A number of scenarios are possible. It might be that there is an IFN-inducible, HIV-1-specific inhibitor, which is suppressed by Vpx. This factor might be induced by the recently reported HIV-1-specific, cryptic sensor in MDDCs . In this case, one would need to invoke an additional, IFN-induced, SIVMAC-specific factor, which is not suppressed by Vpx. Alternatively, there might be a single IFN-induced inhibitor of both viruses, from which Vpx offers protection to HIV-1 but not to SIVMAC. Whichever scenario is correct, identification of antiviral factors such as these has the potential to guide development of new drugs for inhibiting HIV-1 replication in the clinical context. Additionally, given the critical role of dendritic cells at the interface between the innate and acquired immune systems [76, 77], identification of such factors may aid attempts to understand how the innate immune system detects HIV-1, and assist efforts to stimulate acquired immune responses to HIV-1 [39, 78].