Co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in HIV-1 ancestor

Cross-species transmission and adaptation of simian immunodeficiency viruses (SIVs) to humans have given rise to human immunodeficiency viruses (HIVs). HIV type 1 (HIV-1) and type 2 (HIV-2) were derived from SIVs that infected chimpanzee (SIVcpz) and sooty mangabey (SIVsm), respectively. The HIV-1 restriction factor SAMHD1 inhibits HIV-1 infection in human myeloid cells and can be counteracted by the Vpx protein of HIV-2 and the SIVsm lineage. However, HIV-1 and its ancestor SIVcpz do not encode a Vpx protein and HIV-1 has not evolved a mechanism to overcome SAMHD1-mediated restriction. Here we show that the co-evolution of primate SAMHD1 and lentivirus Vpx leads to the loss of the vpx gene in SIVcpz and HIV-1. We found evidence for positive selection of SAMHD1 in orangutan, gibbon, rhesus macaque, and marmoset, but not in human, chimpanzee and gorilla that are natural hosts of Vpx-negative HIV-1, SIVcpz and SIVgor, respectively, indicating that vpx drives the evolution of primate SAMHD1. Ancestral host state reconstruction and temporal dynamic analyses suggest that the most recent common ancestor of SIVrcm, SIVmnd, SIVcpz, SIVgor and HIV-1 was a SIV that had a vpx gene; however, the vpx gene of SIVcpz was lost approximately 3643 to 2969 years ago during the infection of chimpanzees. Thus, HIV-1 could not inherit the lost vpx gene from its ancestor SIVcpz. The lack of Vpx in HIV-1 results in restricted infection in myeloid cells that are important for antiviral immunity, which could contribute to the AIDS pandemic by escaping the immune responses.     

Functional Analysis of the Relationship between Vpx and the Restriction Factor SAMHD1

SAMHD1 is a newly identified restriction factor that targets lentiviruses in myeloid cells and is countered by the SIV_SM/HIV-2 Vpx protein. By analyzing a large panel of Vpx mutants, we identify several residues throughout the 3-helix bundle predicted for Vpx that impair both its functionality and its ability to degrade SAMHD1. We determine that SAMHD1 is a strictly non-shuttling nuclear protein and that as expected WT Vpx localizes with it in the nucleus. However, we also identify a functional Vpx mutant with predominant cytoplasmic distribution that colocalizes with SAMHD1 in this location, suggesting that Vpx may also retain SAMHD1 in the cell cytoplasm, prior to its entry into the nucleus. Several mutations in Vpx were shown to affect the stability of Vpx, as well as Vpx:Vpx interactions. However, no strict correlation was observed between these parameters and the functionality of Vpx, implying that neither properties is absolutely required for this function and indicating that even unstable Vpx mutants may be very efficient in inducing SAMHD1 degradation. Overall, our analysis identifies several Vpx residues required for SAMHD1 degradation and points to a very efficient and plastic mechanism through which Vpx depletes this restriction factor.     

Functional region mapping of HIV-2 Vpx protein

To determine functional regions of HIV-2 Vpx, we analyzed a series of site-specific vpx-mutants for their growth potentials in lymphocytic cells and compared the results with those in macrophages. We found that amino acid residues important for virus growth in lymphocytic cells, in macrophages, and in both are clustered separately in Vpx. Through generation and characterization of new vpx-mutants, we further demonstrated that a remarkable proline-stretch present at the C-terminus of Vpx is critical for its stable expression, thereby contributing to its functional activity. Taken together, there can be functionally distinct regions in HIV-2 Vpx.     

Assembly with the Cul4A-DDB1DCAF1 ubiquitin ligase protects HIV-1 Vpr from proteasomal degradation

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.     

A novel DCAF1‐binding motif required for Vpx‐mediated degradation of nuclear SAMHD1 and Vpr‐induced G2 arrest

HIV‐2 and closely related SIV Vpx proteins are essential for viral replication in macrophages and dendritic cells. Vpx hijacks DCAF1–DDB1–Cul4 E3 ubiquitin ligase to promote viral replication. DCAF1 is essential for cell proliferation and embryonic development and is responsible for the polyubiquitination of poorly defined cellular proteins. How substrate receptors recruit the DCAF1‐containing E3 ubiquitin ligase to induce protein degradation is still poorly understood. Here we identify a highly conserved motif (Wx4Φx2Φx3AΦxH) that is present in diverse Vpx and Vpr proteins of primate lentiviruses. We demonstrate that the Wx4Φx2Φx3AΦxH motif in SIVmac Vpx is required for both the Vpx–DCAF1 interaction and/or Vpx‐mediated degradation of SAMHD1. DCAF1‐binding defective Vpx mutants also have impaired ability to promote SIVΔVpx virus infection of myeloid cells. Critical amino acids in the Wx4Φx2Φx3AΦxH motif of SIV Vpx that are important for DCAF1 interaction maintained the ability to bind SAMHD1, indicating that the DCAF1 and SAMHD1 interactions involve distinctive interfaces in Vpx. Surprisingly, VpxW24A mutant proteins that were still capable of binding DCAF1 and SAMHD1 lost the ability to induce SAMHD1 degradation, suggesting that Vpx is not a simple linker between the DCAF1–DDB1–Cul4 E3 ubiquitin ligase and its substrate, SAMHD1.VpxW24A maintained the ability to accumulate in the nucleus despite the fact that nuclear, but not cytoplasmic, mutant forms of SAMHD1 were more sensitive to Vpx‐mediated degradation. The Wx4Φx2Φx3AΦxH motif in HIV‐1 Vpr is also required for the Vpr–DCAF1 interaction and Vpr‐induced G2 cell cycle arrest. Thus, our data reveal previously unrecognized functional interactions involved in the assembly of virally hijacked DCAF1–DDB1‐based E3 ubiquitin ligase complex.     

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4(DCAF1) E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4(DCAF1-Vpx) E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4(DCAF1) and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.     

Phosphorylation of SAMHD1 by Cyclin A2/CDK1 Regulates Its Restriction Activity toward HIV-1

1. Download : Download full-size image

     

Variation of Two Primate Lineage-Specific Residues in Human SAMHD1 Confers Resistance to N Terminus-Targeted SIV Vpx Proteins

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection in myeloid cells but is inactivated by certain classes of simian immunodeficiency virus (SIV) Vpx proteins. Vpx proteins recruit the DCAF1-CRL4 E3 ubiquitin ligase to trigger species-specific SAMHD1 degradation. Determinants of SIV Vpx-mediated primate SAMHD1 degradation have been mapped to its C terminus. In this study, we have identified the N terminus of human SAMHD1 as a major species-specific determinant of Vpx-mediated suppression. The SIVmnd2 and SIVrcm Vpx proteins recognize the N terminus of rhesus, but not human, SAMHD1. We have also demonstrated that variation of two primate lineage-specific residues between human and rhesus SAMHD1 proteins determine resistance to SIVmnd2 and SIVrcm Vpx proteins. These residues (Cys15 and Ser52) are sequentially mutated to Phe in different lineages of Old World monkeys. Consequently, SIVmnd2 and SIVrcm Vpx proteins that could recognize Phe15- and Phe52-containing SAMHD1 could not inactivate human SAMHD1, which contains Cys15 and Ser52. In contrast, SIVmac Vpx, which targets the C terminus of SAMHD1 molecules, could inactivate various primate SAMHD1 molecules with divergent C-terminal sequences. Both C terminus-targeted SIVmac Vpx and N terminus-targeted SIVrcm Vpx require DCAF1 for the induction of SAMHD1 degradation. The ability of SIV Vpx to restrict SAMHD1 among different primate species is a manifestation of the SAMHD1 evolutionary pattern among those species.     

Restriction of HIV-1 Replication in Monocytes Is Abolished by Vpx of SIVsmmPBj

Background

Human primary monocytes are refractory to infection with the human immunodeficiency virus 1 (HIV-1) or transduction with HIV-1-derived vectors. In contrast, efficient single round transduction of monocytes is mediated by vectors derived from simian immunodeficiency virus of sooty mangabeys (SIVsmmPBj), depending on the presence of the viral accessory protein Vpx.

Methods and Findings

Here we analyzed whether Vpx of SIVsmmPBj is sufficient for transduction of primary monocytes by HIV-1-derived vectors. To enable incorporation of PBj Vpx into HIV-1 vector particles, a HA-Vpr/Vpx fusion protein was generated. Supplementation of HIV-1 vector particles with this fusion protein was not sufficient to facilitate transduction of human monocytes. However, monocyte transduction with HIV-1-derived vectors was significantly enhanced after delivery of Vpx proteins by virus-like particles (VLPs) derived from SIVsmmPBj. Moreover, pre-incubation with Vpx-containing VLPs restored replication capacity of infectious HIV-1 in human monocytes. In monocytes of non-human primates, single-round transduction with HIV-1 vectors was enabled.

Conclusion

Vpx enhances transduction of primary human and even non-human monocytes with HIV-1-derived vectors, only if delivered in the background of SIVsmmPBj-derived virus-like particles. Thus, for accurate Vpx function the presence of SIVsmmPBj capsid proteins might be required. Vpx is essential to overcome a block of early infection steps in primary monocytes.     

HIV-1 Matrix Protein Binding to RNA

The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein plays multiple roles in the viral replication cycle. One essential role is to target PrGag proteins to their lipid raft-associated phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] assembly sites at the plasma membranes of infected cells. In addition to this role, several reports have implicated nucleic acid binding properties to retroviral MAs. Evidence indicates that RNA binding enhances the binding specificity of MA to PI(4,5)P₂-containing membranes and supports a hypothesis in which RNA binding to MA acts as a chaperone that protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to plasma membrane assembly sites. To gain a better understanding of HIV-1 MA-RNA interactions, we have analyzed the interaction of HIV MA with RNA ligands that were selected previously for their high affinities to MA. Binding interactions were characterized via bead binding, fluorescence anisotropy, gel shift, and analytical ultracentrifugation methods. Moreover, MA residues that are involved in RNA binding were identified from NMR chemical shift data. Our results indicate that the MA RNA and PI(4,5)P₂ binding sites overlap and suggest models for Gag-membrane and Gag-RNA interactions and for the HIV assembly pathway.