Positive selection of primate TRIM5alpha identifies a critical species-specific retroviral restriction domain

Abstract

Primate genomes encode a variety of innate immune strategies to defend themselves against retroviruses. One of these, TRIM5alpha, can restrict diverse retroviruses in a species-specific manner. Thus, whereas rhesus TRIM5alpha can strongly restrict HIV-1, human TRIM5alpha only has weak HIV-1 restriction. The biology of TRIM5alpha restriction suggests that it is locked in an antagonistic conflict with the proteins encoding the viral capsid. Such antagonistic interactions frequently result in rapid amino acid replacements at the protein-protein interface, as each genetic entity vies for evolutionary dominance. By analyzing its evolutionary history, we find strong evidence for ancient positive selection in the primate TRIM5alpha gene. This selection is strikingly variable with some of the strongest selection occurring in the human lineage. This history suggests that TRIM5alpha evolution has been driven by antagonistic interactions with a wide variety of viruses and endogenous retroviruses that predate the origin of primate lentiviruses. A 13-aa "patch" in the SPRY protein domain bears a dense concentration of positively selected residues, potentially implicating it as an antiviral interface. By using functional studies of chimeric TRIM5alpha genes, we show that this patch is generally essential for retroviral restriction and is responsible for most of the species-specific antiretroviral restriction activity. Our study highlights the power of evolutionary analyses, in which positive selection identifies not only the age of genetic conflict but also the interaction interface where this conflict plays out.

Fig. 1.

Ancient and variable positive selection has shaped TRIM5α evolution. (A) TRIM5α is the longest of six reported transcripts of the eight-exon human TRIM5 gene. It encodes a 493-aa protein consisting of a RING finger, a B-Box2, and a coiled-coil domain (signature domains of the TRIM family; ref. 24), as well as an α-isoform-specific SPRY domain. (B) TRIM5α was sequenced from a panel of primates representing 33 million years of evolutionary divergence. Values of dN/dS along each branch were calculated by using the free-ratio model of paml for either the whole gene or for the SPRY domain alone (in parentheses), as shown on a cladogram of the accepted primate phylogeny (38). A dN/dS value of >1 suggests that positive selection has acted along that lineage. inf refers to cases where dS = 0.

Fig. 2.

The SPRY domain is a hot spot for insertions/deletions and positive selection. (A) Codons highlighted by paml as being subject to positive selection with high posterior probabilities (P > 0.95) in an analysis of all primates are indicated as circles on stems above the TRIM5α protein schematic. In a more limited analysis of just the hominids and Old World monkeys (OWMs), only five residues in the entire protein were identified as evolving under positive selection (triangles on stems below the TRIM5α schematic). Representative SPRY domains from OWMs and New World monkeys are schematized, along with notable exceptions that have internal duplications (indicated by tandem arrowheads). These duplications appear to have accumulated more replacement (R) changes than synonymous (S) changes (R:S ratios indicated above). In owl monkeys, a CyclophilinA insertion (TRIM-Cyp) occurred between exons 7 and 8 (26), and it is believed that the 8th exon of TRIM5 containing the SPRY domain is not transcribed. (B) Codons identified as being under positive selection are indicated in gray background and by using the same symbols as in A. Changes relative to human are indicated in bold. NWMs have a large deletion in the area of the patch (see Data Set 1, which is published as supporting information on the PNAS web site). Secondary structure predictions and confidence values (0, low; 9,high) were made by using the psipred server (19). Arrows indicate β-strands, and lines indicate coils. According to this prediction, most of the TRIM5α protein is predicted to be comprised of α-helices, whereas the entire SPRY domain is predicted with high confidence to be β-strands and coils with no α-helices.

Fig. 3.

The positively selected patch is critical for the species-specific retroviral restriction by TRIM5α. (A) Retroviral vectors (LPCX) containing hemagglutinin-tagged TRIM5α alleles from human and rhesus genomes were modified to delete the rhesus SPRY patch (Rhesus DEL), swap the human patch into rhesus TRIM5α (Rhesus SWAP), or swap the rhesus patch into the human TRIM5α (Human SWAP). The sequences in the region of the swaps are shown. (B) A Western blot (using a hemagglutinin antibody) shows stable CRFK lines expressing each construct at roughly equivalent levels. (C) Variants of the rhesus TRIM5α protein were assessed for their ability to restrict HIV-1. Single-cycle assays for HIV-1 infectivity were carried out in CRFK (feline renal fibroblasts) cells expressing exogenous TRIM5α genes. Experiments were performed by using 5-fold dilutions of an HIV-1 provirus that was deleted for env and where GFP was inserted into the nef region (20). HIV-positive cells were detected by flow cytometry for GFP expression. (D) A variant of the human TRIM5α (human SWAP) was tested in single-cycle assays for HIV-1 infectivity as in C. (E) CRFK cells that express the TRIM5α alleles were challenged with SIVagm virus, which expresses luciferase in place of the nef gene. Preliminary experiments were performed to establish a dose of virus within the linear range of infection (data not shown). All infections were performed in triplicate, and luciferase activity was measured 2 days after infection. The relative light units are shown on the y-axis, and the percent infection (compared with the CRFK control) is shown above the bar. SD of the data also are shown.