An evolutionary screen highlights canonical and noncanonical candidate antiviral genes within the primate TRIM gene family

Abstract

Recurrent viral pressure has acted on host-encoded antiviral genes during primate and mammalian evolution. This selective pressure has resulted in dramatic episodes of adaptation in host antiviral genes, often detected via positive selection. These evolutionary signatures of adaptation have the potential to highlight previously unrecognized antiviral genes (also called restriction factors). Although the TRIM multigene family is recognized for encoding several bona fide restriction factors (e.g., TRIM5alpha), most members of this expansive gene family remain uncharacterized. Here, we investigated the TRIM multigene family for signatures of positive selection to identify novel candidate antiviral genes. Our analysis reveals previously undocumented signatures of positive selection in 17 TRIM genes, 10 of which represent novel candidate restriction factors. These include the unusual TRIM52 gene, which has evolved under strong positive selection despite its encoded protein lacking a putative viral recognition (B30.2) domain. We show that TRIM52 arose via gene duplication from the TRIM41 gene. Both TRIM52 and TRIM41 have dramatically expanded RING domains compared with the rest of the TRIM multigene family, yet this domain has evolved under positive selection only in primate TRIM52, suggesting that it represents a novel host-virus interaction interface. Our evolutionary-based screen not only documents positive selection in known TRIM restriction factors but also highlights candidate novel restriction factors, providing insight into the interfaces of host-pathogen interactions mediated by the TRIM multigene family.

Keywords: TRIM5; TRIM52; dN/dS; positive selection; restriction factors.

Fig. 1.—

Architectures of TRIM family members exhibiting positive selection in primates. We present a domain schematic for all the proteins with signatures of positive selection in our evolutionary survey (table 1) along with their total length in amino acids (parentheses); in cases of alternate splicing, we represent the largest possible protein isoform encoded by a given TRIM gene. The schematized protein domains are based on GenBank and Ensembl reports. Sites of recurrent positive selection are marked with lollipops above the protein representation. The sites identified by a more in-depth analysis of TRIM52 are shown as lollipops below the protein representation.

Fig. 2.—

Variability in the length of the RING domain. The RING domains from 67 annotated human TRIM genes were collected from Ensembl (Flicek et al. 2012) and GenBank and evaluated to determine the length of the variable loop 2 region located within the domain. Alignments of homologous regions were built using ClustalX (Larkin et al. 2007) and the number of residues residing in the variable region were counted. The predicted length of this variable region ranges from 4 to 48 amino acids. TRIM52 and TRIM41 have the largest expansion of their RING domains.

Fig. 3.—

Phylogenetic relationship of TRIM52 and TRIM41. A phylogram of homologous regions of the RING and B-Box2 domains from TRIM52 and TRIM41 orthologs was built using a maximum likelihood based approach via PhyML (Guindon et al. 2010). Statistical support is represented by bootstrap values, collected from 100 iterations. The * symbol denotes the presence of nonsense mutations that result in pseudogenization.

Fig. 4.—

Positive selection within the RING domain of TRIM52. (A) More than half the sites predicted to be evolving under positive selection (fig. 1 and table 1) are located within the RING domain of TRIM52. To further highlight this, we identified the number of synonymous (S) and nonsynonymous (N) substitutions that have occurred in the expanded loop 2 region of TRIM52 in primate evolution (the equivalent domain of TRIM41 is shown for comparison). Examples of dramatic episodes of lineage-specific positive selection in TRIM52’s RING domain are highlighted in bold. (B) The differences in evolutionary signals are further demonstrated by an alignment of a 90 amino acid-long stretch of the loop 2 region from primate TRIM41 and TRIM52. Sites of positive selection are highlighted with a star and boldface.

Fig. 5.—

Presence/absence of TRIM52 in primates. We evaluated TRIM52 from a range of Hominoids, Old World monkeys, and New World monkeys, using sequences collected from 1) Ensembl (Flicek et al. 2012) and GenBank and via 2) PCR. Primates surveyed by our analysis are presented in a guide tree of the well-accepted primate phylogeny (Perelman et al. 2011). 3) We were unable to amplify TRIM52 from the gibbon lineage of Hominoids (represented by dotted branches), despite the use of primers that we used to amplify orthologs from other Hominoids and Old World monkeys. Grayed branches represent lineages where we observed TRIM52 to be pseudogenized.

Fig. 6.—

Implicated restriction factors. Of 67TRIM genes, several members have been implicated as restriction factors, either positively or negatively impacting viral fitness. In many of these cases, direct interactions with viral proteins has not been detected (top) (reviewed in Ozato et al. 2008; Kawai and Akira 2011). Seven of these genes have evolved under positive selection in primates—two that were previously published, TRIM5 and TRIM22 (Sawyer et al. 2007; Sawyer et al. 2005), in addition to TRIM25 (Gack et al. 2009), TRIM21 (Mallery et al. 2010), TRIM15, TRIM31, and TRIM38 (Uchil et al. 2008) (Overlap). We believe these restriction factors likely act via a direct interaction interface to recognize or evade viral proteins. In addition, we found ten TRIM genes to be rapidly evolving that represent novel restriction factor candidates which may also act via direct host–virus interactions (bottom).