Vpx enables efficient transduction of monocyte-derived cells, such as macrophages and DC's by SIVsm/mac and HIV-2 viruses; however the mechanism that mediates this effect has not been identified. Our findings link this Vpx function to its ability to interact with components of the ubiquitin proteasome system and identify a ternary protein complex - comprising DDA1, DDB1 and VprBP/DCAF1, a putative substrate receptor for Cullin 4-based E3 ubiquitin ligase - as the immediate downstream effector that Vpx uses to promote macrophage transduction. Importantly, the DDB1-VprBP/DCAF1 module was previously shown to participate in a functional Cullin 4 E3 ubiquitin ligase complex 
. Together, these findings support a model in which Vpx usurps the Cullin 4 E3 ubiquitin ligase utilizing the VprBP/DCAF1 to overcome a block to lentivirus replication upon its entry into monocyte-derived cells.
Our data indicate that Vpx acts early following virion entry into macrophages to allow efficient initiation as well as completion of reverse transcription of the incoming SIVmac RNA genomes. This can be clearly seen from a >10-fold decrease in steady state levels of early reverse transcription products and 103-fold decrease in late reverse transcripts upon challenge with Vpx-deficient virions. These phenotypes could result from defects in virion uncoating and/or in its transit into a permissive cytoplasmic compartment.
A similar, albeit less dramatic, loss of vpx
function phenotypes were previously reported for other SIVsm/mac viral isolates and/or vpx
alleles upon infection of monocyte-derived DCs 
. The finding that Vpx is required for efficient reverse transcription in macrophages was somewhat surprising, because it has been thought that this factor acts at a later stage in the replication cycle by enabling the import of the fully reverse transcribed preintegration complex into the nucleus 
. Our findings, taken together with these previous observations, indicate that SIVmac replication is restricted by the same mechanism in DCs and in macrophages. Thus, it is important to refocus future studies towards post entry events that precede reverse transcription in these monocyte derived cells.
The phenotype of Vpx-deficient virions is reminiscent of that resulting from a block to retrovirus replication imposed by tripartite motif protein 5α (TRIM5α) restriction factors. TRIM5α is a E3 ubiquitin ligase that inactivates the incoming virions, probably by deregulating their uncoating so rapidly that the late reverse transcripts fail to accumulate 
. Also, the observation that proteasome inhibitors partially rescue reverse transcription of Vpx-deficient viruses in DCs is consistent with the idea that SIVmac virions may be targeted by a TRIM5α-like restriction, or by another E3 ubiquitin ligase in monocyte-derived cells 
. Whereas these observations raise the possibility that Vpx could act by counteracting TRIM5α, we note that this is not likely, because TRIM5α is expressed in Jurkat T cells 
, which we found not to restrict Vpx-deficient SIVmac virions.
How does Vpx facilitate reverse transcription in macrophages via its interaction with VprBP? As mentioned above, a recent study suggested that the replication of SIVmac cells could be restricted, at least in part, by an as yet unidentified E3 ubiquitin ligase 
. We initially considered that VprBP-linked Cullin 4 E3 complex could be that enzyme and that Vpx counters the restriction by inhibiting its activity. However, our data from RNAi experiments revealed that VprBP is not required for the restriction to occur and, therefore, do not support this possibility. Furthermore, the incoming virions probably contain at most only several hundred Vpx molecules, similar to Vpr, which also is virion recruited through its interaction with Gag p6 
. Therefore, it is difficult to envision that the limited amounts of virion-bound Vpx would be able to saturate and inhibit the cellular pool of VprBP-associated Cullin 4 E3 complexes, even by a noncompetitive mechanism.
Instead of blocking SIVmac replication, our evidence indicates that VprBP is required for Vpx to overcome the block, implying that Vpx uses VprBP-associated E3 to enable reverse transcription in macrophages. Notably, the same VprBP-associated ubiquitin ligase was shown previously to be targeted by a Vpx paralogue, Vpr, which stimulates the intrinsic catalytic activity ofthis E3 
. The findings that both Vpx and Vpr interact with VprBP in a similar manner via their C-terminal regions, and that both interactions lead to post-translational modification of their associated Cullin 4 subunits suggest that Vpx also usurps the VprBP-associated E3, probably to inactivate a cellular factor that inhibits lentivirus replication in macrophages and DC's. Indeed, viral accessory proteins are known to utilize E3 ubiquitin ligases to direct ubiquitination and proteasomal degradation of cellular proteins that mediate innate immunity to viral infection 
Both Vpx and Vpr bind VprBP through similar molecular interactions, yet the functional outcomes are different. Vpr uses VprBP-associated E3 to activate DNA damage checkpoint controlled by the Ataxia-telangiectasia and Rad3-related (ATR) kinase, while Vpx does not have this function and, instead, enables efficient reverse transcription of SIVmac genome in monocyte-derived cells 
. These different outcomes likely reflect that Vpr and Vpx recruit different sets of substrates for ubiquitination by the same E3 enzyme 
, and that they affect differently the activities of their associated Cullin 4 E3s (see Figure S1
). It will be important in the future to identify cellular proteins whose ubiquitination is altered by Vpx and Vpr in order to advance the understanding of these important virulence factors.
In summary, our findings provide novel insights into the mechanism by which Vpx enables macrophage infection, as they link this function to Vpx interaction with VprBP and its associated Cullin 4 E3 ubiquitin ligase complex. Further studies of how Vpx manipulates protein ubiquitination through its interaction with VprBP should lead to detailed understanding of the biochemical mechanism that limit replication of primate lentiviruses in monocyte-derived cells, and how it is countered by viruses of the HIV-2/SIVmac/sm lineages. This knowledge in turn will likely lead to the conception of new strategies aimed to prevent the virus from establishing reservoirs in these cells.