Coevolutionary dynamics between tribe Cercopithecini tetherins and their lentiviruses

Abstract

Human immunodeficiency virus, a primate lentivirus (PLV), causes AIDS in humans, whereas most PLVs are less or not pathogenic in monkeys. These notions suggest that the co-evolutionary process of PLVs and their hosts associates with viral pathogenicity, and therefore, that elucidating the history of virus-host co-evolution is one of the most intriguing topics in the field of virology. To address this, recent studies have focused on the interplay between intrinsic anti-viral proteins, such as tetherin, and viral antagonists. Through an experimental-phylogenetic approach, here we investigate the co-evolutionary interplay between tribe Cercopithecini tetherin and viral antagonists, Nef and Vpu. We reveal that tribe Cercopithecini tetherins are positively selected, possibly triggered by ancient Nef-like factor(s). We reconstruct the ancestral sequence of tribe Cercopithecini tetherin and demonstrate that all Nef proteins are capable of antagonizing ancestral Cercopithecini tetherin. Further, we consider the significance of evolutionary arms race between tribe Cercopithecini and their PLVs.

Figure 1. Molecular phylogenetic analyses of primate tetherin and CD4.

(a,b) Phylogenic trees of 58 primate tetherins (a) and 22 primate CD4s (b) reconstructed using NJ method. Both trees were rerooted with the NWM clade. The species belonging to Tribe Cercopithecini are shown in pink. The species indicated in bold are the sequences newly identified in this study. GenBank accession numbers are listed in Tables 1 and 2. In panel a, the number (8.2) indicates the age of diversification (million years ago) that is estimated in a previous study. A phylogenetic tree of 58 primate tetherins reconstructed using ML method is shown in Supplementary Fig. 1. (c,d) The positive selection detected in different regions of tetherin gene (c) and CD4 gene (d). The regions inferred to be under positive selection with statistical significance are represented in bold. ND, not detected. (e,f) Positively selected sites identified from tetherin gene (e) and CD4 gene (f). The codons under positive selection identified by PAML with posterior probability >0.95 are shown in red. All PAML analyses were performed under two models of codon usage, F61 and F3×4, and they yield consistent results.

Figure 2. Molecular phylogenetic analyses of tribe Cercopithecini tetherin.

(a) Phylogenic tree of 22 Tribe Cercopithecini tetherins reconstructed using NJ method. The species indicated in bold are the sequences newly identified in this study. The hosts of vpu-positive SIV are shown in orange, and those of vpu-negative SIV are shown in cyan. The numbers indicate the dN/dS value for each branch inferred by the branch model in the PAML analysis. The red star indicates the ancestral tetherin of tribe Cercopithecini. (b,c) The positive selection detected in different regions of tetherin gene. In panel b, the regions inferred to be under positive selection with statistical significance are represented in bold. In panel (c), positively selected sites identified by the PAML analysis are shown. The codons under positive selection identified by PAML with posterior probability >0.95 are shown in red. All PAML analyses were performed under two models of codon usage, F61 and F3x4, and they yield consistent results.

Figure 3. Molecular phylogenetic and structural analyses of tetherins of SIV-infected monkeys.

(a) The result obtained from the three branch-site analyses for Tribe Cercopithtecini (n = 22), the hosts of vpu-negative SIV (cyan, n = 14), and those of vpu-positive SIV (orange, n = 6). The clades inferred to be under positive selection with statistical significance are represented in bold. (b) The positive selection detected in different regions of tetherin gene of the hosts of vpu-negative SIV (left, n = 14) and those of vpu-positive SIV (right, n = 6). The regions inferred to be under positive selection with statistical significance are represented in bold. (c,d) Positively selected sites identified in our analyses. In panel (c), the codons under positive selection identified by PAML with posterior probability >0.95 are shown in red. In panel d, the codons under positive selection inferred by HyPhy with Bayes factor >50 are shown in red, and the codons identified as positively selected sites by PAML are indicated with asterisks. (e) Structure modeling of the ancestral tetherin of tribe Cercopithtecini. The transparent surface with the ribbon diagram of the extracellular domain (ECD) of ancestral Cercopithtecini tetherin, which is generated by SWISS-MODEL server based on the ECD of human tetherin (PDB code: 3MQB), is shown. The two views of monomer (top) and tetramer (bottom) models, rotated by 180°, are respectively shown. The 5 positively selected sites in the ECD of tetherins of vpu-negative SIV hosts (codons 63, 67, 99, 100, and 159) are indicated in red. (f) Genetic diversity analysis. The values indicate the overall mean genetic distance, which is calculated by using Tamura-Nei model in MEGA6, with standard error.

Figure 4. Experimental analyses of the anti-viral activity of ancestral Cercopithtecini tetherin and antagonistic ability of SIV Nefs.

(a) Western blotting of cell lysates. Representative results are shown. Blots have been cropped; full uncropped blots are available as Supplementary Fig. 3. (b) TZM-bl assay. The data represents the percentage of infectivity compared to the values without tetherin with standard deviation. The assay was performed in triplicate. The statistic differences (*P < 0.05; **P < 0.01) versus the values of No Nef are determined by Student’s t test.

Figure 5. Evolution and diversification of SIV.

(a) Dating the divergence times of 34 SIV lineages. The MCC tree constructed using BEAST is shown. This analysis was conducted by using the amino acid sequences of Gag, Pol, Vif, and Env. Cyan, vpu-negative SIVs; orange, vpu-positive SIVs; black, SIVs identified in western red colobus; and grey, SIVs identified in black-and-white colobus. The orange stars (nodes 8 and 13) indicate the time of vpu gene acquisition, and the green star (node 30) indicates the time of Vpr neofunction. X-axis indicates the year before present. GenBank accession numbers of the SIV sequences used in this analysis are listed in Table 4. The estimated divergence time, posterior probability, and bootstrap value of each node of the tree are listed in Table 3. (b) Distribution of the monkeys infected with vpu-positive SIV. The data is extracted from the reference. The image is created using Illustrator (Adobe) by overlaying the maps shown in reference. GSN, greater spot-nosed monkey; MON, mona monkey; MUS, mustached monkey; DEN, dent’s mona monkey. (c) The nucleotide length of SIV. The nucleotide length between the end of each viral gene (tat1, vpr, rev1, and vif) and the initiation codon of env are measured. Statistic differences between SIVdeb and the other vpu-negative SIV are determined by Welch’s t test. PSIV, prosimian endogenous lentivirus.