Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection

Abstract

Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4–based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

Author Summary

Monocyte-derived tissue macrophages play crucial roles in infection by primate lentiviruses. Human and simian lentiviruses of the HIV-2 and SIVsm/mac lineages encode a virion-bound virulence factor termed Vpx. Vpx is required to establish infection specifically of monocyte-derived cells, but the underlying molecular mechanism is unclear. In this study we characterize how the replication of SIVmac is blocked in the absence of Vpx and how Vpx overcomes this block. We find that Vpx is required for efficient reverse transcription of the incoming RNA genome, suggesting that Vpx acts early following virion entry into the macrophage, probably on events linked to virion uncoating and/or reverse transcription. We also identified a Vpx-associated ternary protein complex that is the key mediator of Vpx function specifically in macrophages. This complex links Vpx to the cellular machinery that mediates protein ubiquitination and degradation. Together, we describe the immediate downstream effector, the molecular machinery and a tentative mechanism that lentiviral Vpx uses to enable reverse transcription in macrophages. Our findings should lead to the conception of new strategies to control macrophage infection by human and simian lentiviruses.