The ability of primate lentiviruses to degrade the monocyte restriction factor SAMHD1 preceded the birth of the viral accessory protein Vpx

The human SAMHD1 protein potently restricts lentiviral infection in dendritic cells and monocyte/macrophages but is antagonized by the primate lentiviral protein Vpx, which targets SAMHD1 for degradation. However, only two of eight primate lentivirus lineages encode Vpx, whereas its paralog, Vpr, is conserved across all extant primate lentiviruses. We find that not only multiple Vpx but also some Vpr proteins are able to degrade SAMHD1, and such antagonism led to dramatic positive selection of SAMHD1 in the primate subfamily Cercopithecinae. Residues that have evolved under positive selection precisely determine sensitivity to Vpx/Vpr degradation and alter binding specificity. By overlaying these functional analyses on a phylogenetic framework of Vpr and Vpx evolution, we can decipher the chronology of acquisition of SAMHD1-degrading abilities in lentiviruses. We conclude that vpr neofunctionalized to degrade SAMHD1 even prior to the birth of a separate vpx gene, thereby initiating an evolutionary arms race with SAMHD1.     

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.     

Virological characterization of HIV-2 vpx gene mutants in various cell systems

Requirement of intrinsically disordered protein Vpx for HIV-2 replication is cell-type dependent. To define Vpx-dependent conditions, replication ability of HIV-2 vpx mutants was analyzed in various cell lines that differ in cellular type, differentiation state and/or expression level of anti-HIV-1 SAMHD1 degraded by Vpx. Induction of Vpx-sensitive anti-HIV-2 state was not always associated with SAMHD1 expression. Compared with our previous data in lymphocytic cells, growth-defectiveness of the vpx mutants in differentiated THP-1 cells, a newly established multi-cycle infection system, was considerably different. Taken together, our results suggest that Vpx plays cell-type dependent role through its undetermined structure and/or function.     

Ecomorphological specialization leads to loss of evolvability in primate limbs*

Primate limb morphology is often described as either generalized, that is, suited to a range of locomotor and positional behaviors, or specialized for unique locomotor behaviors such as brachiation or bipedalism. The evolution of highly specialized limb morphology may result in loss of evolvability, that is, in a decreased capacity of the locomotor skeleton to evolve in response to selection towards alternative ecomorphological niches. Using evolutionary simulations, I show that the highly specialized limb anatomy of hominoids is associated with a significant loss of evolvability, defined as the number of generations to reach alternative adaptive peaks, and in parallel an increased risk of extinction, particularly in simulated evolution toward generalized quadrupedal limb proportions. Loss of evolvability in apes and humans correlates with three factors: (1) decreased correlation among limb bone lengths (i.e., integration), which slows the rate of change along lines of least evolutionary resistance; (2) limb specialization, which places apes and humans in relatively remote areas of morphospace; and (3) increased skeletal size as a proxy for body size. Thus, locomotor over-specialization can lead to evolutionary dead-ends that significantly increase the probability of hominoid populations going extinct before evolving new adaptive morphologies.     

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.     

Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase

The SAMHD1 protein is an HIV-1 restriction factor that is targeted by the HIV-2 accessory protein Vpx in myeloid lineage cells. Mutations in the SAMHD1 gene cause Aicardi-Goutières syndrome, a genetic disease that mimics congenital viral infection. To determine the physiological function of the SAMHD1 protein, the SAMHD1 gene was cloned, recombinant protein was produced, and the catalytic activity of the purified enzyme was identified. We show that SAMHD1 contains a dGTP-regulated deoxynucleotide triphosphohydrolase. We propose that Vpx targets SAMHD1 for degradation in a viral strategy to control cellular deoxynucleotide levels for efficient replication.     

Evolutionary and functional analyses of the interaction between the myeloid restriction factor SAMHD1 and the lentiviral Vpx protein

SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As?a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in?the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.     

Evolution of the primate lentiviruses: evidence from vpx and vpr.

The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.     

SAMHD1: a new contributor to HIV-1 restriction in resting CD4+ T-cells

How the Human T lymphotropic virus type 1 (HTLV-1) Tax protein stimulates proliferation while triggering cell cycle arrest and senescence remains puzzling. There is also a debate about the ability of Tax to activate or inhibit the DNA damage response. Here, we comment on these different activities and propose a conceptual rationale for the apparently conflicting observations.     

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.     

On the loss of uricolytic activity during primate evolution--I. Silencing of urate oxidase in a hominoid ancestor

Urate oxidase activity is not detectable in liver homogenates from the gibbon, orangutan, chimpanzee, gorilla and human. Liver homogenates from five genera of Old World and two genera of New World monkeys have easily detectable levels of urate oxidase activity. There is no evidence for extant detectable intermediate steps in the loss of urate oxidase activity in the hominoids. Urate oxidase activity from Old World and New World monkeys is stable, a simple observation which debunks a long-standing myth. Urate oxidase activity was silenced in an ancestor to the five living genera of hominoids after divergence from the Old World monkeys.