Keywords:TRIM5,TRIM52, positive selection, dN/dS, restriction factors

CollectingTRIM Orthologs

Human (Homo sapiens)TRIM gene sequences were obtained from Ensembl (Flicek et al. 2012) and GenBank. Chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), orangutan (Pongo abelii), white-cheeked gibbon (Nomascus leucogenys), rhesus macaque (Macaca mulatta), baboon (Papio anubis), squirrel monkey (Saimiri boliviensis), marmoset (Callithrix jacchus), tarsier (Tarsius syrichta), mouse lemur (Microcebus murinus), and bushbaby (Otolemur garnettii) orthologs were obtained when reported from NCBI by BLAST (Altschul et al. 1990) searches of the “RefSeq RNA” databases with the humanTRIM sequence as the query and from Ensembl gene orthology/paralogy predictions (Vilella et al. 2009). Additional primate orthologs were collected when available (e.g., African green monkey [Chlorocebus aethiops]). Subsequent collection ofTRIM sequences, specificallyTRIM52 andTRIM41, via publically available databases were carried out utilizing Ensembl’s genome databases to recover annotated sequences from available animals, including Reptilia, Avian, and Mammalian species.

SequencingTRIM52

To expand our collection of primateTRIM52 sequences to improve the power of downstream evolutionary analysis, we amplifiedTRIM52 using genomic DNA from the following primates: human, chimpanzee, bonobo, gorilla, orangutan, rhesus macaque, African green monkey, talapoin monkey (Miopithecus talapoin), colobus monkey (Colobus guereza), Francois’ leaf monkey (Trachypithecus francoisi), purple-faced langur (Trachypithecus vetulus), and silvery langur (Trachypithecus cristatus). Exon 1 was amplified and sequenced using the following primer pair: Forward: CCACCGATCCCAGAGAGAGG and Reverse: CCTCTGGGGAAGCCAATCTGC. We amplified exon2 by nested PCR with the following primer pairs: Initial primer pair: Forward: GTYGCATGATTTAGAAYTTTACTGACCAA and Reverse: GACAATCCAGGCATCCAGTTATGC. Second, nested primer pair: Forward: ATWATGGTTTATTTAATAYARTATACATTATC and Reverse: GAACTCTAACTCATGGGATGGACAAA. The second, nested primer pair was used to sequence exon2. We used PCR Supermix (Invitrogen, Inc.) for amplification reactions. Reactions used 1 µl of each 10 µM forward primer and 10 µM reverse primer and had a final volume of 12.5 µl. Cycling parameters were 94 °C for 3 min; 40 cycles of 94 °C for 15 s, 55 °C for 15 s, 72 °C for 1 min; 72 °C for 10 min; 10 °C thereafter. Sequencing reactions were carried out using BigDye.TRIM52 sequences have been deposited in the GenBank database (accession numbersJX896135.1–JX896146.1).

Phylogenetic Analysis

NonprimateTRIM52 andTRIM41 sequences were obtained by Blast (Altschul et al. 1990) analysis with the humanTRIM52 protein as query and psi-blast (Altschul, Madden et al. 1997) analysis with the humanTRIM52 RING expansion as query. Psi-blast of the RING expansion recovers onlyTRIM52 and TRIM41 orthologs, suggesting that these are the onlyTRIMs with this expansion. We found no evidence of a protein domain downstream of the B-Box2 domain, with homology to TRIM41, in any of theTRIM52 orthologs. For instance, there is no identifiable Coiled-Coil domain or B30.2 domain downstream of the humanTRIM52 gene in the human genome assembly. All of theTRIM41 sequences are predicted to encode a Coiled-Coil and B30.2 domain. Nonprimate and primateTRIM sequences (TRIM52 andTRIM41) that we recovered from Blast (Altschul et al. 1990) and Ensembl (Flicek et al. 2012) were aligned using ClustalX (Larkin et al. 2007). We only included the RING (omitting the region containing the RING expansion) and B-Box domain. Using this alignment, we constructed a tree using maximum likelihood methodology (Guindon et al. 2010) and used the program Dendroscope (Huson, Richter et al. 2007) to present a phylogram.

Delineation of TRIM Protein Domain Boundaries and Secondary Structure

RING, B-box1, and B-box2 domains were identified based on the consensus sequences (Meroni and Diez-Roux 2005). Coiled-Coil domain boundaries were identified by predicting secondary protein structure with PSIPRED (McGuffin, Bryson et al. 2000) and identifying the long alpha helix that is associated with this motif (Lupas 1996). B30.2 or other C-terminal domains were identified by using the CDD (Marchler-Bauer, Anderson et al. 2005) and SMART (Schultz, Copley et al. 2000) domain databases, and the N-terminal boundary of B30.2 domains was aided by secondary structure prediction, as the B30.2 domain consists entirely of sequential tandem beta-strands (Seto, Liu et al. 1999;Masters, Yao et al. 2006).

Computational Analysis of Positive Selection

Detection of recurrent positive selection by multiple alignment comparisons was carried out using the CODEML program from the PAML package (Yang 1997). Constrained model M7 was tested against unconstrained model M8 under the following parameters: f61 (codon frequencies of 61 nonstop codons are calculated), starting omega: 0.4 and 1.5. All simulations converged and results are consistent between both codon models (2lnλ;P values were calculated assuming two degrees of freedom). We present the percentage of sites estimated to evolve under positive selection and the average dN/dS for those sites. Posterior probabilities were calculated according to the Naive Empirical Bayes model (Yang 1997). Positive selection, as detected by PAML, was further supported by Fast Unbiased Bayesian AppRoximation (FUBAR) and Random Effects Likelihood (REL), implemented through the Datamonkey suite of phylogenetic analysis tools (Delport et al. 2010).TRIM genes were required to exhibit overlapping sites of positive selection by PAML and Datamonkey to be identified as under positive selection. Specific sites of positive selection identified by both PAML and Datamonkey were denoted by underline (table 1).

TRIM52 Restriction Assays

We generated CRFK cell lines that stably express HA-tagged human and rhesusTRIM52 by transduction of a retrovirus vector (LPCX) encoding human and rhesusTRIM52 as described (Sawyer et al. 2005). Stable cell lines, including a negative control empty vector CRFK cell line, were plated on 12-well plates (0.8 × 10⁵ cells/well). These were allowed to incubate overnight and then infected with the following GFP-encoding retroviruses: HIV-1, HIV-2 (ROD9), and FIV. We used a virus titer determined to give us at least 15% infection. Three days after infection, cells were fixed with paraformaldehyde and GFP expression was measured by flow cytometry.

Positive Selection Has Acted on SeveralTRIM Genes in Primates

To screen theTRIM gene family for signatures of having participated in an evolutionary arms race, we evaluatedTRIM orthologs from primates for recurrent positive selection via maximum likelihood analyses using the CODEML program from the PAML package (Yang 2007). We comparedTRIM orthologs from human, chimpanzee, orangutan, rhesus, and marmoset reference genomes. In some instances, we were able to identify additional orthologs from other primate genome sequencing projects that are underway via Ensembl (Vilella et al. 2009) or from previous gene-directed sequencing efforts. For a fewTRIM genes, we were unable to identify the full complement of orthologs, as the genes were either absent or not intact in the available genome assemblies. Using this collection of orthologs, we identified 17 out of 67TRIM genes as having evolved under positive selection using aP value cutoff of 0.05:TRIM2,TRIML2,TRIM5,TRIM7,TRIM10,TRIM15,TRIM21,TRIM22,TRIM25,TRIM31,TRIM38,TRIM52,TRIM58,TRIM60,TRIM69,TRIM75, andTRIM76 (table 1 andsupplementary table S1,Supplementary Material online). This list includesTRIM5 andTRIM22, restriction factors that were previously reported to show strong evidence of positive selection (Stremlau et al. 2004;Sawyer et al. 2005;Sawyer et al. 2007;Barr et al. 2008).

Our screen also recovered known restriction factorsTRIM15,TRIM21,TRIM25, andTRIM38 for which no evolutionary analyses had been previously conducted.TRIM15 was discovered in a knockdown screen to inhibit the release of retroviruses (Uchil et al. 2008) and later found to have a role in the RIG-I sensing pathway (Uchil et al. 2013). We find sites of positive selection within the RING, Coiled-Coil, and B30.2 domains ofTRIM15 (fig. 1).TRIM21 is able to degrade viruses via an intracellular antibody-mediated mechanism (Mallery et al. 2010). Positive selection was detected within the B-Box and B30.2 domains of TRIM21 (fig. 1).TRIM25 activates RIG-I signaling via ubiquitination (Gack et al. 2007), andTRIM25-mediated signal transduction is known to be inhibited by the direct interaction of influenza A protein NS1 to the Coiled-Coil domain of TRIM25 (Gack et al. 2009). We find thatTRIM25 exhibits a number of sites under positive selection, clustered between the Coiled-Coil and B30.2 domains (fig. 1).TRIM38 is known to negatively regulate innate immunity by targeting TRIF (Xue et al. 2012), NAP1 (Zhao, Wang, Zhang, Yuan et al. 2012), and TRAF6 (Zhao, Wang, Zhang, Wang et al. 2012) for ubiquitination and degradation.TRIM38 has also been shown to improve the fitness of HIV-1 during entry by an unknown mechanism (Uchil et al. 2008). Only a single site of positive selection residing between the Coiled-Coil and B30.2 domains was detected (fig. 1).

TheTRIM genes with known antiviral activity that we recovered in our positive selection screen (TRIM5,TRIM15,TRIM21,TRIM22,TRIM25,and TRIM38) belong to the C-IV family ofTRIM genes based on their domain structure (Ozato et al. 2008). Our screen highlighted six additional genes from the C-IV family that have not been previously implicated in antiviral defense:TRIM7,TRIM10,TRIM58,TRIM60,TRIM69, andTRIM75. We found modest signatures of positive selection for three of these genes,TRIM7,TRIM10,and TRIM75. A single site was found to be under positive selection inTRIM7 in the Coiled-Coil domain (fig. 1). Similarly,TRIM75 has a single site of positive selection in its RING domain associated with E3 ubiquitin ligase activity. ForTRIM10, positive selection was found in the Coiled-Coil and B30.2 domains, which may reflect changes in target recognition, similar toTRIM5 (Sawyer et al. 2005;Maillard et al. 2010). We further found evidence of robust positive selection forTRIM58,TRIM60, andTRIM69, which have multiple sites with high dN/dS values (fig. 1). Indeed, among all known primateTRIM genes, the signature of positive selection for these three genes appears to be on par with what we see for the bona fide restriction factorTRIM5. ForTRIM58, we find a cluster of positively selected sites in the B30.2 domain and a single sight highlighted in the Coiled-Coil domain.TRIM60 has positively selected sites in each of the RING, B-Box, Coiled-Coil, and B30.2 domains (with significant clustering in the latter two domains).TRIM69, similarly, exhibits a cluster of sites under positive selection in the Coiled-Coil and B30.2 domains. Thus, this evolutionary screen of primateTRIM genes identified multiple new candidate restriction factors belonging to the same subfamily (C-IV) already known to harbor known antiviral genes.

We also identified candidate restriction factors outside of the C-IV family of TRIM genes:TRIM2 (C-VII),TRIML2 (UC),TRIM31 (C-V),TRIM52 (C-V), andTRIM76 (UC).TRIM2 contains a Filamin-type immunoglobulin domain and array of NHL repeats. Two sites of positive selection were found inTRIM2, with neither of these residing within the known domains ofTRIM2 (fig. 1).TRIML2 is a highly unusualTRIM-like gene. It lacks canonical RING and B-box domains, being solely composed of Coiled-Coil and B30.2 domains. Formally, it does not meet the criteria of being an RBCC-typeTRIM gene. However, given the propensity ofTRIM proteins to homo- and heterodimerize, we also included such noncanonical genes within our analysis. We find a single site of positive selection within the C-terminus B30.2 domain ofTRIML2 (fig. 1).TRIM31 is a suspected retroviral restriction factor that acts at the stages of entry and release for HIV-1 and MLV, respectively (Uchil et al. 2008). We identified three sites exhibiting signatures of positive selection inTRIM31; two of these sites are in the C-terminal region, which is not homologous to any knownTRIM-associated domains but may represent an analogous virus-interacting domain (fig. 1).TRIM52 is unique among the restriction factor candidates as it only encodes the RING and B-Box domains. We identified the majority of positive selection within the RING domain and a single site immediately upstream of the B-Box domain. Intriguingly, the RING domain ofTRIM52 has expanded and is the largest among the genes recovered by our screen (fig. 1); this expansion also appears to contain the positively selected sites.TRIM76 is the final candidate identified. It encodes for a large ∼3,500 amino-acid protein that contains B-Box, Coiled-Coil, Fibronectin III, and B30.2 domain in its C-terminus region. The remainder of the protein does not contain homology to any annotated domains but contains the six sites of positive selection we identified.

Thus, our evolutionary screen for novel restriction factors among theTRIM gene family identified 15 members not previously known to be under positive selection. Most excitingly, our screen identifies as many as 10 novel candidates for antiviral function. Although all of theseTRIM genes may not participate in host–pathogen interactions, several exciting canonical candidates (e.g.,TRIM58 andTRIM60) emerged from our screen. In addition, we identified several noncanonical candidates (e.g.,TRIML2 andTRIM52) that may have otherwise been overlooked as potential restriction factors because they are missing some of the key RBCC domains. We decided to pick one of these noncanonical candidates,TRIM52, for a more in-depth analysis to confirm and expand the findings of our initial screen.

Rapid Evolution of theTRIM52 RING Domain in Primates

We decided to evaluateTRIM52 in more detail because it structurally deviated the most from the canonicalTRIM restriction factors (i.e.,TRIM5 andTRIM22). For example,TRIM52 lacks the viral recognition (B30.2) domain and displays signatures of rapid evolution within the RING domain. Moreover,TRIM52 appears to lack an intact Coiled-Coil domain within its coding region (fig. 1). Thus,TRIM52 is comprised solely of the RING and B-Box2 domains, making it a highly unusual member of the TRIM multigene family. Even the RING domain ofTRIM52 is highly unusual. RING domains of theTRIM family are generally defined by the consensus sequenceCx2Cx9-45Cx1-3Hx2-3Cx2Cx4-48Cx2[C/D], where eight cysteine, histidine, or aspartic acid residues coordinate two zinc atoms (Meroni and Diez-Roux 2005). The region between the sixth and the seventh coordinating residues is referred to as the “loop 2” region of the RING tertiary structure using the precedent of the human c-cbl RING-containing E3 ubiquitin ligase (Zheng et al. 2000). The majority ofTRIM genes encode between 4 and 48 amino acids in their loop 2 region, with the mode being 13 amino acids (fig. 2). However, severalTRIM genes were found to deviate from the consensus range. Most notably,TRIM52 encodes 139 amino acids in its loop 2 region (fig. 2). Thus,TRIM52 encodes the largest RING domain of any humanTRIM gene. BLAST (Altschul et al. 1990) analysis of this region reveals similarity only to mammalianTRIM52 andTRIM41 genes, both of which have exceptionally large RING domain expansions.

In order to elucidate the evolutionary relationship betweenTRIM52 andTRIM41 and to deduce when this large loop 2 RING expansion occurred, we carried out phylogenetic analyses ofTRIM52 andTRIM41 sequences that were obtained from Blast (Altschul et al. 1990) searches of vertebrate genomes. Our analyses revealed thatTRIM52 andTRIM41 are close paralogs that are found in close proximity to each other in most mammalian genomes (fig. 3). We found that the reptile (anole lizard), avian (chicken and wild turkey), and marsupial (Tasmanian devil and opossum) genomes have only singleTRIM41-like genes, which are phylogenetic outgroups to both theTRIM52 andTRIM41 clades from eutherian mammals (fig. 3). This suggests thatTRIM52 was born in eutherian mammals ∼190 Ma via a partial duplication ofTRIM41, having lost both the Coiled-Coil and B30.2 domains at birth (Meredith et al. 2011).

Despite their evolutionary relationship, our screen for positive selection in primates highlightedTRIM52 but notTRIM41. To further evaluate the evolutionary history ofTRIM52, we repeated our analysis of recurrent, site-based positive selection via maximum likelihood analyses using primateTRIM52 orthologs obtained by our additional sequencing efforts. From this in-depth analysis, we refined the sites of recurrent, codon-based positive selection (figs. 1 and4B). The sites of positive selection reside primarily within the expanded “loop 2” region ofTRIM52. This rapid evolution of the RING domain is especially evident in an evolutionary comparison focused on the “loop 2” expansion unique toTRIM41 andTRIM52, which highlights the dramatic acceleration of amino acid replacements inTRIM52 (fig. 4A andB). In contrast toTRIM52, we found no evidence of positive selection having acted on theTRIM41 using available primate sequences from databases (fig. 4A). Thus, in stark contrast toTRIM41 and its RING domain that has been evolving under constraint, we find thatTRIM52 has been rapidly evolving throughout primate history, with much of that selection acting on the expanded RING domain.

Repeated Loss/Pseudogenization ofTRIM52 in Mammals

Our sequencing survey also revealed at least two instances ofTRIM52 loss or pseudogenization over the course of primate evolution (fig. 5). For instance, within marmoset and other New World monkey genomes, we were only able to identify exon 2 using a combination of Blast (Altschul et al. 1990) and BLAT (Kent 2002) searches (supplementary fig. S1,Supplementary Material online) and our own polymerase chain reaction (PCR) analyses. These analyses suggested thatTRIM52 is present but pseudogenized throughout the New World monkey lineage. We were also unable to detectTRIM52 from gibbon genomes (N. leucogenys,Hylobates agilis, andSymphalangus syndactylus) via PCR with genomic DNA, despite using PCR primers that amplifiedTRIM52 from all other Hominoids and Old World monkeys. Blast (Altschul et al. 1990) and BLAT (Kent 2002) analyses support the absence ofTRIM52 from publicly available gibbon genomes.

This pattern of stochasticTRIM52 loss was also evident in other mammalian orders. We identifiedTRIM52 pseudogenization or loss in African elephant (Loxodonta africana), horse (Equus caballus), microbat (Myotis lucifugus), and megabats (Pteropus vampyrus) (supplementary fig. S1,Supplementary Material online). We were also unable to identifyTRIM52 throughout the glires (Rodentia and Lagomorpha) lineage of mammals, suggesting that it has been deleted early within this lineage. However, utilizing UCSC (Kent et al. 2002) and Ensembl (Flicek et al. 2012) predictions, we were able to recoverTRIM52 from the genomes of the mouse and rat. Sequence analysis of these predicted mouse and ratTRIM52 revealed that they do not encode a B-Box domain. Therefore, the annotated mouse and ratTRIM52 comprised only a RING domain. Furthermore, theTRIM52 orthologs we did identify in mouse and rat genomes were not located proximal toTRIM41 and are therefore the only nonsyntenicTRIM52 orthologs in mammals (supplementary fig. S2,Supplementary Material online). When we included mouse and ratTRIM52 in our phylogenetic analysis (supplementary fig. S3,Supplementary Material online), branch support at the node separating theTRIM41 andTRIM52 clades was lowered (even though mouse and ratTRIM52 genes localized within theTRIM52 clade). Due to the apparent loss ofTRIM52 throughout glires, the truncated structure of mouse and ratTRIM52, and their ambiguous phylogenetic placement, we therefore cannot confidently assign these mouse and ratTRIM genes as bona fideTRIM52 orthologs, labeling themTRIM52-like instead (supplementary fig. S1,Supplementary Material online). Given this uncertainty, we had omitted the mouse and ratTRIM52-like sequences from our phylogenetic analysis (fig. 3). Additional genome sequencing within eutherian mammals may reveal still additional instances ofTRIM52 loss or pseudogenization, suggestive of episodes of relaxed selective pressure among individual lineages.

Human and RhesusTRIM52 Do Not Restrict Lentiviruses

The history of positive selection uncovered among primateTRIM52 orthologs indicates that its function has been adaptively evolving. Although many members of theTRIM family positively and negatively impact retroviruses (Uchil et al. 2008),TRIM52 was not tested in previous analyses. Indeed, the degree of adaptive evolution within the RING domain ofTRIM52 suggests that the role of viral recognition has shifted in the absence of the B30.2 domain to the RING domain. Thus, we evaluated human and rhesusTRIM52 orthologs for antiviral activity against a limited panel of lentiviruses (supplementary fig. S4,Supplementary Material online). Although many of these viruses are restricted by otherTRIM proteins, we found no evidence of restriction by either human or rhesusTRIM52. Thus, although the evolutionary patterns of positive selection and episodic loss strongly implicateTRIM52 in some form of host defense, the targets of this activity are still unknown and likely not retroviral.

TRIM52, A Candidate Antiviral Gene

A screen for positive selection identified 15 new members of the primateTRIM gene family. Of these,TRIM52 was the most unusual. Indeed, in the absence of these evolutionary analyses,TRIM52 might not draw attention as a candidate antiviral factor because it lacks a canonical virus-interaction domain (fig. 1). AlthoughTRIM52 lacks B30.2 and Coiled-Coil domains, the gene bears an ancient expansion of the RING domain that exhibits positive selection. TRIM52 is not the first candidate antiviral TRIM gene that lacks a canonical viral capsid-binding domain, however. In a previous analysis of rodentTRIM5 paralogs, we identified mouse (Mus musculus)TRIM12, which only encodes RING, B-Box2, and Coiled-Coil domains (Tareen et al. 2009). Similar toTRIM52, mouseTRIM12 exhibits signatures of positive selection despite the absence of a recognized interaction interface (i.e., B30.2 domain). Indeed, our finding of positive selection within the RING domain leads to the intriguing model whereby the antiviral interaction interface ofTRIM52 may have now shifted to within its RING domain. This is an unusual exception to the highly modular arrangement of the mammalian and fishTRIM gene family in which the target interaction interface is usually restricted to the Coiled-Coil or B30.2 domains, which are also the hotspots for positive selection (Reymond et al. 2001;Meroni and Diez-Roux 2005;Nisole et al. 2005;Song, Gold et al. 2005;Yap et al. 2005;Sawyer et al. 2007;van der Aa et al. 2009).

Despite the strong signature of positive selection, we identified at least six independent losses ofTRIM52 within mammals, including two events in primates. The absence ofTRIM52 from gibbon genomes may reflect its genomic position, proximal to the telomeric region in Hominoids and Old World monkeys. However, this genomic positioning is not shared in other mammals (supplementary fig. S2,Supplementary Material online) and therefore cannot account for the multiple loss events we have observed. Furthermore, we found no evidence for either loss or pseudogenization of the proximally locatedTRIM41 gene. This suggests that the parental gene is under strong functional constraint, whereas the episodes ofTRIM52 loss strongly suggest that thisTRIM gene does not carry out a conserved, housekeeping function in mammalian genomes, further supporting its proposed role as an antiviral factor. Intriguingly, the recurrent loss ofTRIM52 is reminiscent of the dynamic evolutionary history observed by otherTRIM genes with antiviral function. For instance, the dogTRIM5 ortholog is pseudogenized (Sawyer et al. 2007), whereas cats encode a truncated form ofTRIM5 with a disrupted B30.2 domain; both lineages are unable to express TRIM5alpha (McEwan et al. 2009). Both rodent (mouse and rat) and cow genomes lackTRIM22 orthologs but contain expanded sets ofTRIM5 paralogs (Sawyer et al. 2007;Tareen et al. 2009). Expansions are not unique toTRIM5.Han et al (2011) identified severalTRIM genes that are copy number variable in human genomes. Similar dynamics have also been observed in several teleost species, where uniqueTRIM genes (fintrims) have expanded and diversified in each lineage (van der Aa et al. 2009). We have previously suggested that positive selection and the expansion ofTRIM genes is driven by new or continuous selective pressure, likely provided by viral pathogens (Sawyer et al. 2007;Tareen et al. 2009;Han et al. 2011). Similarly, the loss or relaxation of such a selective pressure could result in the loss of aTRIM gene (Sawyer et al. 2006;Sawyer et al. 2007). Thus, considering the dynamic history ofTRIM52 and our evidence of positive selection, we posit that this unusualTRIM gene is involved in genome defense but can be lost either due to relaxed selection or because of the high costs borne by encoding such a defense (Sawyer et al. 2006).

It is also possible thatTRIM52 is under positive selection not because of antiviral activity but instead to maintain its interaction with a host target substrate that is also adaptively evolving. However, we find this coevolutionary scenario unlikely because such host–host interaction surfaces are not typically found to evolve under positive selection unless they are challenged by a pathogenic influence (Koyanagi et al. 2010;Daugherty and Malik 2012). Furthermore, this scenario would posit that the many incidences ofTRIM52 loss we have documented would have to coincide with the simultaneous loss of the target substrate or the requirement to maintain the interaction.

Positive Selection within theTRIM Gene Family

Based on the unbiased approach of our screen, we predicted the recovery of several known restriction factors. In particular, there was an expectation of identifyingTRIM5 andTRIM22, both previously highlighted for their positive selection (Sawyer et al. 2005;Sawyer et al. 2007). In addition to these, we recovered other known or suspected restriction factors:TRIM15,TRIM21,TRIM25, andTRIM38. We detected positive selection occurring all alongTRIM25, in particular within the Coiled-Coil and B30.2 domains (fig. 1).TRIM25 plays a role in influenza infection, where its activity is critical for the activation of the RIG-I-dependent signaling cascade (Gack et al. 2007). Specifically, influenza A encodes protein NS1 that directly interacts and inhibitsTRIM25 at the Coiled-Coil domain inhibiting the ubiquitination and activation of RIG-I. This is reminiscent of adaptive evolution in other known restriction factors, such as in the case of MAVS to evade protease cleavage by hepatitis C virus (Patel et al. 2012) or in tetherin to evade lentivirus Nef or Vpu (Lim et al. 2010). In both cases, positive selection highlights regions of the host-encoded protein targeted by viral antagonists and provided insight into mechanisms of host evasion. Thus, it is likely that the sites of positive selection exhibited byTRIM25 reveal adaptation during primate history to evade NS1 or NS1-like antagonists.TRIM15 similarly plays a role regulating innate immune signaling, and its positive selection may reflect a similar constraint asTRIM25. In contrast,TRIM21 is able to target cytosolic antibodies bound to viruses and auto-ubiqutinate, leading to the proteasomal degradation of theTRIM21-bound complex (Mallery et al. 2010). As this complex forms viaTRIM21 binding to the invariant region of antibodies (James et al. 2007), it is unlikely that the interaction between host-encoded products is responsible for the positive selection we detected. Instead, it is much more likely that a novel viral antagonist targets TRIM21 and that positive selection is reflective of evasion from such an antagonist. Unique among these recoveredTRIM genes,TRIM38 has been found to assist HIV-1 during entry (Uchil et al. 2008).TRIM38 has recently been recognized for having a role in negatively regulating innate immunity by targeting components of innate immunity for degradation (Xue et al. 2012;Zhao, Wang, Zhang, Yuan et al. 2012;Zhao, Wang, Zhang, Wang et al. 2012). Positive selection onTRIM38 may therefore also reflect its escape from viral-mediated antagonism of innate immunity. Thus, our analysis of positive selection provides insight into the interface and nature of host–pathogen interactions in cases of known restriction factors (fig. 6). Specifically, we expect these sites of positive selection to be affecting structure, either indirectly altering the host–pathogen interface or at the direct interface contacting viral proteins. In specialized cases likeTRIM5, this direct interaction is predicted of a rapidly evolving antiviralTRIM gene. Alternatively, as we predict ofTRIM25, positive selection is reflective of evading viral antagonism.

OneTRIM gene that did not show a signature of positive selection at all isTRIM19/PML. This is in agreement with an extended sequencing of primateTRIM19/PML orthologs which concluded that there was no evidence for positive selection of this gene (Ortiz et al. 2006). This may be surprising in light of the evidence that PML functions in antiviral defense (reviewed inNisole et al. 2005). However, positive selection would only be expected to act on genes encoding proteins that directly interact with viral proteins, and so any upstream or downstream effector may not present such a signal. It is interesting thatTRIM1 (MID2) also shows no adaptive signature, given that the humanTRIM1 protein has been shown to have anti-MLV activity (Yap et al. 2004). This may reflect a retroviral restriction that is not currently being utilized by humans or chimpanzees, because our screen was especially focused on this lineage of mammals. Nonetheless, it is important to point out that the absence of positive selection does not precludeTRIM genes from being candidate restriction factors, but thoseTRIM genes that have evolved under positive selection represent the most likely candidates for having an antiviral role via direct interaction (fig. 6).

As many of theTRIM genes remain largely uncharacterized, our evolutionary screen is able to highlight candidate restriction factors based on exhibition of positive selection, a hallmark of antiviral genes at the direct interface of the host–viral pathogen arms race (Daugherty and Malik 2012). Based on the extent of rapid evolution observed among them, we propose thatTRIM58,TRIM60, andTRIM69 represent the best uncharacterized candidates for novel restriction factors within the primateTRIM multigene family and should therefore be intensively investigated for antiviral function (fig. 6).

Based on previous studies with APOBEC andTRIM5 restriction genes, it is informative to identify antiviral restriction factors even if they are not currently active against modern viral pathogens. Restriction factors honed against evolutionarily “recent” viral infections might protect us against future viruses or viral variants or might be artificially enhanced to be active against current forms. Genes with partial activity might vary in potency within the human population. Furthermore, such genes serve as barriers to animal models of viral infection (Hatziioannou et al. 2006;Kirmaier et al. 2010). To this end, our evolutionary approach to identify potential restriction factors in theTRIM family has revealed ten canonical and noncanonical members of this gene family that bear previously unrecognized signatures of recent positive selection. These primateTRIM genes are therefore primate candidates to be investigated as novel restriction factors against viruses.

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