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

Abstract

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.

Figure 1. Vpx from diverse primate lentiviruses degrade SAMHD1

(A) The ability of Vpx and Vpr to degrade SAMHD1 was assayed by western blot analysis of HA-epitope tagged SAMHD1 from respective primate species co-transfected with 3xFLAG-epitope tagged Vpr or Vpx constructs as indicated. Actin was probed as a loading control. Indicated Vpr and Vpx constructs were expressed in the presence of human SAMHD1 (left) or chimpanzee SAMHD1 (right). (B) Similar western blots as in (A) are shown, analyzing rhesus macaque SAMHD1 (left) and red-capped mangabey (RCM) SAMHD1 (right) expression in the presence of indicated Vpr and Vpx constructs. (C) Similar western blots as in (A) are shown, analyzing mandrill SAMHD1 expression in the presence of indicated Vpr and Vpx from SIVmnd1 or SIVmnd2.

Figure 2. Some Vpr proteins degrade SAMHD1

(A) Unrooted phylogeny of 115 vpr and vpx sequences among diverse primate lentiviruses. Bootstrap values indicate maximum likelihood proportions that are highly supported by Bayesian inference (Figure S1A). Seven phylogenetic clusters are shaded in colors (cutoff at ML bootstrap >75, Bayesian posterior probability > 0.87). Vpx sequences form 2 clades (Light blue and dark blue shaded) that have strong support of monophyly from all other vpr sequences. Functional phenotype of Vpr and Vpx (Table I) that degrade SAMHD1 (Blue stars) or do not degrade SAMHD1 (Red stars) are overlaid on the phylogeny. See also Figure S1B. (B) Western blot analysis of Colobus monkey, De Brazza's monkey, and African green monkey (AGM) SAMHD1 in the presence of indicated Vpr constructs. The AGM SAMHD1 tested is from the Vervet subspecies matching the SIVagmVer 9648 host strain, the Colobus SAMHD1 tested is from the Colobus guereza subspecies. (C) Association of SAMHD1 with Vpr and DDB1 by co-immunoprecipitation was detected by western blot analysis of HA-immunoprecipitated SAMHD1 for FLAG-epitope tagged Vpr and DDB1 association (IP), or input expression (Input). After transfection, cells were treated with 25μM MG-132 for 12 hours prior to immunoprecipitation. SIVdeb Vpr interacts with De Brazza's monkey SAMHD1 (IP), but SAMHD1 expression was not rescued by MG132 treatment (Input). SIVmnd1 Vpr, which fails to degrade mandrill SAMHD1, was assayed as a negative control. Actin and the antibody light chain (Lc) are shown as loading controls.

Figure 3. Primate SAMHD1 has been evolving under positive selection

(A) Cladogram of 31 primate SAMHD1 genes sequenced for the evolutionary analyses. The panel of primates comprised of 8 hominoids, 16 Old World monkeys and 7 New World monkeys. No evidence of recombination was detected by a GARD analysis (Kosakovsky Pond et al., 2006). Values of ω (dN/dS) along each branch were calculated by a free ratio analysis using PAML (Figure S2B). Branches with statistically significant ω values > 1 are highlighted in red, branches highlighted in grey indicate lineages that show ω values > 1, but are not statistically significant based on two-ratio likelihood tests (Figure S2). (B) Likelihood ratio test statistics were used to determine if SAMHD1 evolution across various primate lineages was associated with dN/dS ratios significantly greater than 1 (hence under positive selection). Neutral models (M7) were compared to selection models (M8) under the F61 model of codon substitution. Similar results were obtained in a comparison of M1 (neutral) versus M2 (selection) (data not shown). See also Figure S2. (C) Six positively selected codons were identified (32, 36, 46, 69, 107, 486) with significant posterior probability (Figure S3A) using PAML. The analysis was performed on SAMHD1 sequences from the panel of 16 Old World monkeys, which showed the strongest burst of positive selection in primates (Figure 3B). Likelihood ratio tests were performed between the M7 (neutral) and M8 (selection) models for the full SAMHD1 gene, without the SAM domain or with amino acids 32, 36, 46, 67, 107, 486 omitted from the alignment. Domains were analyzed for signatures of positive selection, with the strongest signals located in the SAM domain. See also Figure S3.

Figure 4. SAMHD1 positive selection residues map to Vpx sensitivity

(A) Alignment of N-terminal and SAM domain regions from indicated primates. Symbols (circle on a stick) represent the positively selected residues marked on the SAMHD1 domains. Sites 46 and 69 which displayed highly significant signals of positive selection are boxed in the alignment. Stars represent the codons under positive selection with strong support (Figure S3A, S3B and S3C). The N terminal region of SAMHD1 from grey mouse lemur is included to represent amino acid residues encoded by a distantly related prosimian primate. (B) Expression of mandrill, AGM and AGM point mutants (AGM D46G and AGM Q69R) were analyzed by western blot, in the presence or absence of SIVmnd2 Vpx expression. (C) Western blot analysis of HA-immunoprecipitated SAMHD1 for FLAG-epitope tagged SIVmnd2 Vpx association. Cells were treated with 25μM MG-132 for 12 hours prior to immunoprecipitation. Heavy chain (Hc) is shown as a loading control. (D) Western blot shows expression of SAMHD1 from mandrill and mandrill-derived mutations (Mnd G46D, Mnd R69Q, and Mnd G46D, R69Q) in the presence or absence of SIVmnd2 Vpx.

Figure 5. SAMHD1 degradation by some Vpr proteins preceded the birth of Vpx

The phylogeny shown in Figure 2A was rooted to common ancestor of SIVolc/SIVwrc, as determined by the phylogenetic positioning of the flanking pol and env genes in relation to pSIVgml (Figure S4), and is consistent with previous reports that the Colobinae SIVs are outgroup to the Cercopithecinae SIVs (Gifford et al., 2008; Gilbert et al., 2009; Liégeois et al., 2009). Important nodes that infer ancestral traits are boxed in numbers.