Evolution of TRIM5α B30.2 (SPRY) domain in New World primates

Infection, Genetics and Evolution 10 (2010) 246–253

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Evolution of TRIM5a B30.2 (SPRY) domain in New World primates Esmeralda A. Soares a, Albert N. Menezes a, Carlos G. Schrago b, Miguel A.M. Moreira a, Cibele R. Bonvicino a,c, Marcelo A. Soares a,b, Hector N. Seua´nez a,b,* a

Programa de Gene´tica – CPQ, Instituto Nacional de Caˆncer, Rua Andre´ Cavalcanti, 37 – 4o andar, 20231-050 Rio de Janeiro, RJ, Brazil Departamento de Gene´tica, Universidade Federal do Rio de Janeiro, CCS, Bloco A, sala A2-120, Cidade Universita´ria – Ilha do Funda˜o, 21949-570 Rio de Janeiro, RJ, Brazil c Laborato´rio de Biologia e Parasitologia de Mamı´feros Reservato´rios Silvestres, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, RJ, Brazil b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 August 2009 Received in revised form 14 October 2009 Accepted 16 November 2009 Available online 1 December 2009

The tripartite motif 5 protein (TRIM5) has been extensively studied in view of its ability to restrict retroviruses in mammalian hosts. The B30.2 domain, encoded by exon 8 of TRIM5, contains the major restriction determinants. We have analyzed the genetic diversity of the TRIM5 B30.2 domain in a wide range of New World primates (NWP). The TRIM5 region encoding the B30.2 domain of 35 animals, representing all NWP families and 10 genera, was PCR-amplified, sequenced and analyzed at the amino acid level. Comparisons were carried out with available GenBank data; analyses were carried out with a dataset of 44 representative sequences of 32 NWP species and 15 genera, with a human B30.2 sequence as outgroup. A high genetic diversity was observed, both with respect to length and amino acid substitutions, mainly at the three variable regions of this domain associated with the restriction phenotype. Phylogenetic reconstructions based on B30.2 DNA differed from the consensus NWP topology due to positive selection along different lineages and definite codon positions, with robust evidence either with a complete or a pruned dataset. This was especially evident in codons 406 and 496, consistently demonstrated with all methods. Positive selection was virtually absent in all NWP species when analyzing intra-specific polymorphisms except for Saguinus labiatus. Our findings indicated that NWP TRIM5 proteins have been subjected to selection, probably by retroviruses and/or retroelements. We anticipate that the diversity of NWP TRIM5 is indicative of disparate retroviral restriction phenotypes representing a plentiful source of factors countering HIV infection. ß 2009 Elsevier B.V. All rights reserved.

Keywords: TRIM5a New World primates B30.2 domain Molecular evolution Platyrrhini

1. Introduction The new class of cellular factors that restrict the early intracellular steps of retroviral replication has recently become a subject of intensive research. This is the case of the family of tripartite motif (TRIM) proteins, encoded by more than 70 different genes in humans and other mammals (Nisole et al., 2005). Extensive analysis of one member of this family, TRIM5, showed that its alpha isoform (TRIM5a) was frequently associated with restriction of several retroviruses, including HIV and SIV lentiviruses, the ethiological agents of acquired immunodeficiency in humans and other primates (Baumann, 2006; Nisole et al., 2005; Towers, 2005, 2007; Yap et al., 2004). The spectrum of retroviral restriction has been found to be highly variable among different

* Corresponding author at: Instituto Nacional de Caˆncer, Rua Andre´ Cavalcanti, 37 – 4o andar, 20231-050 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 3233 1458; fax: +55 21 3233 1423. E-mail address: [email protected] (H.N. Seua´nez). 1567-1348/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2009.11.012

primate species as well as among different retroviruses within a given species (Kratovac et al., 2008; Stremlau et al., 2005). The tripartite nature of TRIM5 proteins results from their three classical domains, namely RING, B-box and coiled-coil (Reymond et al., 2001). In addition to these domains, TRIM5a contains a B30.2 domain, encoded by the exon 8 of the TRIM5 gene and generated by alternative splicing (Perez-Caballero et al., 2005). Although there is good evidence that some of the first three domains might contribute to retroviral restriction (Diaz-Griffero et al., 2007a,b; Li et al., 2007), B30.2 is widely recognized as a major player in this process, since it harbours the retroviral capsid-binding motifs required for restriction (Perez-Caballero et al., 2005; Stremlau et al., 2005; Yap et al., 2005). TRIM5 genes have been extensively characterized in Old World primates (OWP) of Africa and Asia because these primates represent natural hosts and animal models of lentiviral infection, respectively. However, recent attention has been brought to TRIM genes from New World primates (NWP) following the description of the TRIM-cyclophilin A (TRIM-Cyp) fusion gene in owl monkeys (Nisole et al., 2004; Sayah et al., 2004). Among NWP, TRIM-Cyp has

E.A. Soares et al. / Infection, Genetics and Evolution 10 (2010) 246–253

been initially found in species of the genus Aotus (Ribeiro et al., 2005), and to potently restrict HIV-1, even when expressed in human susceptible cells (Sayah et al., 2004). Of interest, other events of independent TRIM-Cyp retrotransposition were later found among Asian primates like rhesus, cynomolgus and pigtailed macaques (Brennan et al., 2008; Newman et al., 2008; Virgen et al., 2008; Wilson et al., 2008), suggesting an adaptive convergence leading to retroviral restriction. With respect to retroviral restriction, a few TRIM5 genes of NWP previously characterized were shown to have evolved under positive selection, presumably by retroelements and against infections by some retroviruses (Sawyer et al., 2005), although extensive analyses of the genetic variability of the B30.2 domain have not been carried out. The very rapid rate of molecular evolution of TRIM5 in mammals, and especially in primates, has been a matter of interest to several investigators (Liu et al., 2005; Sawyer et al., 2006, 2007), as well as the strong evidence of positive selection documented by a high frequency of non-synonymous with respect to synonymous substitutions (Liu et al., 2005; Sawyer et al., 2005). In fact, the dN/dS ratios estimated for TRIM5 are among the highest ever reported for primate genes. Such uncommon mode of mammalian gene evolution probably results from virus/host interactions. In this study, we analyze the genetic diversity of the B30.2 domain across a wide range of NWP taxa. Our results showed a substantial variation of this domain, both in length and nonsynonymous substitutions, pointing to a multitude of potential retroviral restriction phenotypes. Moreover, we have evaluated events of positive selection in TRIM5a B30.2 domains across NWP lineages and codons.

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domain (exon 8) of TRIM5. Reactions were run at 94 8C for 2 min, followed by 20 cycles of 94 8C for 30 s, 55 8C for 30 s and 72 8C for 1 min. Additional 20 cycles consisted of 94 8C for 30 s, 64 8C for 30 s and 72 8C for 1 min followed by a final extension step of 72 8C for 10 min. PCR products were subsequently purified with GFX PCR DNA and Gel Band Purification kit (GE Healthcare, Brazil), quantified with DNA Mass Ladder (Invitrogen), labeled with Dynamic ET Terminator Cycle Sequencing kit (GE Healthcare) following the manufacturer’s specifications and sequenced in an ABI 377 DNA Genetic Analyzer (Applied Biosystems, Foster City, USA). Outside primers used for DNA sequencing were the same as in PCR amplifications, and two additional internal primers were used, TRIM5INTF (50 -cctttgatcgtgcccctc-30 ) and TRIM5INTR (50 grtccambcmcttttattg-30 ). 2.3. Sequence editing, alignment and phylogenetic analyses

2. Materials and methods

DNA sequences were assembled with SeqMan (DNAStar Inc., Madison, USA) and manually inspected. Sequences were subsequently aligned using BioEdit (Hall, 1999) with 21 NWP sequences retrieved from GenBank (Table 1). Deduced amino acid sequences were visualized and compared. Altogether, analyses were carried out with a dataset of 44 representative sequences of 32 NWP species and 15 genera. Additionally, a human B30.2 sequence (GenBank accession number NM033034) was used as an outgroup (Fig. 1). Phylogenetic analyses were carried out with nucleotide data. Maximum likelihood (ML) analysis was conducted in GARLI (Zwickl, 2006). The nucleotide substitution model used was GTR+G, selected by Modeltest v. 3.6 (Posada and Crandall, 1998) using both the hierarchical likelihood ratio tests and the Akaike information criterion. Bootstrap supporting values were calculated based on 1000 replicates.

2.1. Animal sources and genomic DNA

2.4. Genetic distance analyses

Whole blood (1–3 ml) was collected from 35 animals belonging to 20 species, 3 NWP families (Atelidae, Cebidae and Pitheciidae), and 10 different genera (Aotus, Alouatta, Brachyteles, Callimico, Callithrix, Leonthopithecus, Mico, Saguinus, Saimiri and Pithecia). Nomenclature and taxonomy of NWP followed the criterion of Rylands et al. (2000). Animals were held at the Centro Nacional de Primatas (Ananindeua, State of Para´) and at the Centro de Primatologia do Rio de Janeiro (Mage´, State of Rio de Janeiro), both located in Brazil. Blood collections were conducted according to the guidelines of the Sociedade Brasileira em Cieˆncia de Animais de Laborato´rio (SBCAL/COBEA). Samples were further processed at the Universidade Federal do Rio de Janeiro or at the Instituto Nacional de Caˆncer, both located in Rio de Janeiro (State of Rio de Janeiro, Brazil), where peripheral blood mononuclear cells (PBMC) were separated in Ficoll gradients.

Genetic distances within New World and catarrhine primates were calculated using the GTR+R model with HyPhy (Pond et al., 2005). The catarrhine dataset comprised the following sequences retrieved from GenBank: EU260465 (Homo sapiens), DQ437595 (Pan troglodytes), AY710300 (Gorilla gorilla), DQ437601 (Pongo pygmaeus), AY710299 (Hylobates lar), AY710298 (Colobus guereza) and DQ437602 (Macaca nemestrina).

2.2. PCR amplification and sequencing of the TRIM5 B30.2 domain PBMC genomic DNA was extracted with the QIAGEN genomic DNA extraction kit (QIAGEN, Chatsworth, CA), and 5 ml (approximately 1 mg) of each DNA were used in a single round of PCR amplification reaction containing 5 ml of Taq DNA polymerase buffer (10), 2.5 ml of MgCl2 at 50 mM, 0.4 ml of dNTP mix (at 25 mM of each dNTP), 0.5 ml of each primer (at 25 pmol/ml), and 0.25 ml Platinum Taq DNA polymerase at 5 U/ml (Invitrogen, CA). Forward and reverse primers TRIM5Ex8SeqF (50 -gtaaaacgacggccagttcccttagctgacctgttaattt-30 ) and TRIM5Ex8SeqR (50 -ggaaacagctatgaccatggctgtacagaaggggctgag-30 ) (Song et al., 2005a) amplified a DNA fragment of approximately 700 bp containing the B30.2

2.5. Analysis of selective pressure in the B30.2 domain of NWP To assess whether the B30.2 domain of NWP was subjected to positive selection, we performed a conservative analysis of average dN and dS values and ML tests. Average dN and dS values were estimated with MEGA v.4.1 (Kumar et al., 2008) using the Pamillo– Bianchi method (Pamillo and Bianchi, 1993), which considers the transition/transversion ratio. The significance of differences between dN and dS was estimated with the Z statistics, with standard errors based on 1000 bootstrap replicates using MEGA v.4.1. ML tests for detecting selective pressure were performed with the Codeml program of the PAML 4 package (Yang, 2007) and HyPhy. We inputted the currently accepted phylogenetic topology of NWP genera (Schneider et al., 2001), with a further inclusion of one representative of the genus Mico as sister branch of Cebuella, an association supported by mtDNA control region data (Tagliaro et al., 1997) and comparative karyology (Canavez et al., 1996). Intra-generic diversity was excluded from ML analyses because phylogenetic relationships within genera are not well resolved (Schneider et al., 2001) and because ML tests are not suited for analyzing sequences with low genetic divergence (Anisimova et al.,

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Table 1 New World primate specimens analyzed in this study. Specimen

Species name

GenBank accession #

1a.A.belzebul_1b 1.A.belzebul_2 1.A.belzebul_3 1.A.sara_GBc 1.A.geoffroyi_GB1 1.A.geoffroyi_GB2 1.B.arachnoides_1 1.B.arachnoides_2 1.B.arachnoides_3 1.L.lagotricha_GB1 1.A.azarae 1.A.trivirgartus_GB 2.C.goeldii 2.C.goeldii_GB 2.M.argentata_GB 2.M.argentata 2.M.emiliae 2.C.geoffroyi_1 2.C.geoffroyi_2 2.M.humeralifera 2.C.jacchus_1 2.C.jacchus_2 2.C.jacchus_3 2.C.kuhlii_1 2.C.kuhlii_2 2.C.penicillata 2.C.pygmaea_GB 2.C.albifrons_GB 2.L.chrysomelas_1 2.L.chrysomelas_2 2.L.chrysopygus_1 2.L.chrysopygus_2 2.L.chrysopygus_3 2.L.rosalia_1 2.L.rosalia_2 2.S.b.martinsi 2.S.bicolor bicolor 2.S.imperator 2.S.imperator_GB 3.S.labiatus_GB1 2.S.labiatus_GB2 2.S.mystax 2.S.oedipus_GB1 2.S.oedipus_GB2 2.S.boliviensis_GB1 2.S.boliviensis_GB2 2.S.sciureus 2.S.sciureus_GB1 2.S.sciureus_GB2 2.S.ustus 3.C.moloch_GB 3.C.donacophilus_GB 3.P.irrorata_1 3.P.irrorata_2 3.P.pithecia 3.P.pithecia_GB

Alouatta belzebul Alouatta belzebul Alouatta belzebul Alouatta sara Ateles geoffroyi Ateles geoffroyi Brachyteles arachnoides Brachyteles arachnoides Brachyteles arachnoides Lagothrix lagotricha Aotus azarae Aotus trivirgatus Callimico goeldii Callimico goeldii Mico argentatad Mico argentata Mico emiliae Callithrix geoffroyi Callithrix geoffroyi Mico humeralifera Callithrix jaccus Callithrix jacchus Callithrix jacchus Callithrix kuhli Callithrix kuhli Callithrix penicillata Cebuella pygmaea Cebus albifrons Leontopithecus chrysomelas Leontopithecus chrysomelas Leontopithecus chrysopygus Leontopithecus chrysopygus Leontopithecus chrysopygus Leontopithecus rosalia Leontopithecus rosalia Saguinus bicolor martinsi Saguinus bicolor bicolor Saguinus imperator Saguinus imperator Saguinus labiatus Saguinus labiatus Saguinus mystax Saguinus oedipus Saguinus oedipus Saimiri boliviensis Saimiri boliviensis Saimiri sciureus Saimiri sciureus Saimiri sciureus Saimiri ustus Callicebus moloch Callicebus donacophilus Pithecia irrorata Pithecia irrorata Pithecia pithecia Pithecia pithecia

Pending Pending Pending AY843511 AY843516 AY740616 Pending Pending Pending AY843520 Pending AY684993 Pending DQ437599 DQ437603 Pending Pending Pending Pending Pending Pending Pending Pending Pending Pending Pending AY843512 DQ437596 Pending Pending Pending Pending Pending Pending Pending Pending Pending Pending DQ437598 AY740615 AY843518 Pending DQ437597 DQ229285 AY928202 AY740614 Pending DQ437605 AY843517 Pending EU124690 AY843519 Pending Pending Pending AY843518

Sequence data from highlighted specimens were used for inferring positive selection shown in Fig. 2B. a Numbers preceding sample name refer to NWP families (1.Atelidae; 2.Cebidae; 3.Pitheciidae). b Sequence data from specimens in bold were generated in this study. c Sequence data from specimens followed by ‘‘GB’’ were retrieved from GenBank. d Formerly classified as Callithrix species Callithrix argentata, Callithrix emiliae and Callithrix humeralifera, the Amazon basin callithrichides are now classified as Mico.

2001). The M1a/M2a and M7/M8 tests of PAML were carried out (Wong et al., 2004). Models M1a and M7 differ in the discretization of v values between 0 and 1, while M2a and M8 display alternative hypotheses allowing the inclusion of a positive selection category. The PARRIS test of HyPhy (Scheffler et al., 2006), which considers recombination and reduces the rate of false positives, was also used. When using HyPhy, synonymous rates were allowed to vary

over codons by using a dual general discrete distribution (GDD) with three bins each for synonymous and non-synoymous rates (Pond and Muse, 2005). With Codeml, codon sites were considered to be positively selected when assigned to the v > 1 category with posterior probability higher than 95% using the Bayes Empirical Bayes approach (Yang et al., 2005). We used three different HyPhy methods to identify positively selected codons: the single likelihood ancestor counting (SLAC), the fixed effects likelihood (FEL), and the random effects likelihood (REL) (Pond and Frost, 2005a). All methods were run allowing heterogeneity of synonymous rates, which was reported to control the rate of false positives (Pond and Muse, 2005). Analysis of lineages (i.e., branches) under positive selection was carried out with the genetic algorithm of Pond and Frost (Pond and Frost, 2005b) available online, in the GABranch application of the DataMonkey.org server. The MG94xHKY85 model of codon evolution was used. 3. Results 3.1. Genetic diversity of TRIM5 B30.2 in NWP An amino acid alignment of B30.2 sequences (Fig. 1) showed extensive variation in length and amino acid substitutions at all taxonomic levels. Variations in length were present in all three B30.2 variable regions (V1–V3). In V1, in addition to the 9-amino acid indel present in all NWP respective to humans (Song et al., 2005a), Alouatta sara (Bolivian red howler monkey) showed an additional histidine residue at position 336 when compared to other NWP. In V2, a 2-amino acid indel including a cysteine residue, potentially involved in disulfide bonds, was observed in all atelids (group 2; Fig. 1) with respect to the other two families. The highest variation in length was observed in V3. One variation consisted of a 9-amino acid indel due to an incomplete V3 duplication previously described in six cebid species comprising marmosets, tamarins, capuchins, and squirrel monkeys (Ohkura et al., 2006), which was further found in all NWP herein analyzed (Fig. 1). Most strikingly, however, was the finding of multiple, additional duplications in atelids. The majority of atelid genera (Ateles, Brachyteles and Lagothrix in Fig. 1) showed a 55-amino acid indel resulting from an incomplete V3 triplication, as previously reported in Ateles (Song et al., 2005a). Moreover, Alouatta, the most basal genus of this family, showed additional indels with respect to the remaining atelids (Fig. 1). Several amino acid replacements were also observed. Multiple family-, subfamily-, genus- and species-specific substitutions were observed (Fig. 1), some of which involving placement or replacement of cysteine residues potentially involved in disulfide bonds (e.g., positions 345, 347 and 517 in Fig. 1). Finally, several species-specific substitutions were also observed. We also analyzed B30.2 regions recently found to be involved in retrovirus restriction independently from capsid binding (Sebastian et al., 2009). The first three regions, referred as 358, 362 and 367 by Sebastian et al. (2009), and corresponding to the amino acid triads KHY (359–361), EVD (363–365) and NKS (368–370) in NWP, were found to be strongly conserved in the species herein analyzed. While the first two were absolutely invariable, the third region showed serine at position 370 in the Pitheciidae and Atelidae and arginine in all Cebidae; while one spider monkey specimen carried aspartic acid at position 368. The fourth region (480) reported in (Sebastian et al., 2009), corresponding to amino acids 553–555 in our alignment, was more variable among NWP. While the consensus triad was MTC, MIC was found in Ateles, TTC in Mico and some Callithrix, and VTC in some Saguinus. Average genetic distances within New World and catarrhine primates, using the GTR+G model, were of 0.069  0.006 and

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Fig. 1. Alignment of the deduced amino acid sequence of TRIM5 B30.2 (exon 8) domain in NWP. Numbers preceding species names refer to the three NWP families to which they belong: 1. Atelidae; 2. Cebidae; 3. Pitheciidae (Schneider et al., 2001). Numbers following species names depict different amino acid sequences. Species names followed by ‘‘GB’’ correspond to sequences retrieved from GenBank. The three variable regions (V1–V3) are depicted with horizontal bars above the alignment, as previously described (Sawyer et al., 2005; Song et al., 2005b). Amino acid residue numbering at top of the alignment was defined as in Ohkura et al. (2006), representing positions of the entire TRIM5a protein. The human B30.2 domain was placed at the bottom of the alignment. Dashes represent gaps.

0.045  0.006, respectively. The standard errors of these estimates were small and, consequently, without overlap of 95% confidence intervals (0.05724–0.08076 and 0.03324–0.05676, respectively), indicating that the number of substitutions per site were higher in New World primates. 3.2. Phylogenetic analysis and differential selection at the TRIM5 B30.2 domain The major phylogenetic clusters currently recognized (Schneider et al., 2001) were maintained, as was the case of the Atelidae

(Fig. 2A). Pithecids were also grouped although the internal arrangement of this family could not be estimated due to lack of representative specimens of Chiropotes and Cacajao. A few exceptions to consensus topologies were also observed; of note, Aotus appeared as the most basal NWP lineage instead of grouping with the Cebidae. Cebus and Saimiri grouped with the Pitheciidae rather than with the Aotinae and Callitrichinae; and Callimico appeared as a sister branch of the Leontopithecus/Saguinus lineage rather than a sister branch of Callithrix. Average dN and dS estimates indicated that non-synonymous substitutions had accumulated more frequently than synonymous

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substitutions along the evolution of the B30.2 domain (d¯ N ¼ 0:102  0:010 and d¯ S ¼ 0:047  0:007), the average difference (d¯ N  d¯ S ) equaling 0:055  0:011, which was >0 (neutral expectation). This difference was significant (Z-test  5; p < 0.01), strongly suggesting that this domain had gone through a process of adaptive evolution. All ML tests reinforced the finding of positive selection. The M1a/M2a model comparison was significant (p < 0.01), and the v parameter estimate of the positive selection class equaled 6.41 (Table 2). The M7/M8 test also rejected the model without a category of positively selected sites, and the v estimate was also very high (6.86). Finally, the PARRIS test, accounting for the rate of

heterogeneity of synonymous sites, was also significant, with v = 3.96. The BEB approach applied to the parametric values of models M2a and M8 revealed several codon sites under positive selection across the B30.2 domain. Both models assigned the same sites to the v > 1 class (Table 3), with posterior probability >99%. The SLAC method of HyPhy, which relies on the reconstruction of codon states along the phylogeny, found two sites, namely 406 and 496; these sites were also identified with Codeml models. The FEL and REL methods also estimated positively selected sites (Table 3). As expected, the number of codons inferred at v > 1 was smaller in SLAC and FEL. The REL method is similar to Codeml’s strategy, and

Fig. 2. (A) Maximum likelihood (ML) topology of NWP based on B30.2 nucleotide sequences using he human B30.2 sequence as outgroup. Numbers above branches show ML bootstrap estimates. Only values above 70% are shown. Numbers following species names depict different sequences. Species names followed by ‘‘GB’’ correspond to sequences retrieved from GenBank. (B) Positive selection analysis across generic lineages of NWP. Tree topology was forced according to the currently accepted NWP phylogeny as determined by Schneider et al. (2001). Branches in bold correspond to lineages under positive (diversifying) selection determined by the GABranch algorithm of Datamonkey. Values above branches correspond to dN/dS median values (1 is used for values of infinite order, when dS = 0). Arrows and boxed text indicate major evolutionary variations in length in lineages where they have occurred (see text for details).

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Fig. 2. (Continued ).

several codons were commonly found with both approaches. However, only codons 406 and 496 were consistently identified with all algorithms. The GABranch method inferred four v classes to which tree branches were assigned. In two of these classes v was <1 (0.071 and 0.818), while the other two classes v was considerably higher than 1, including one category with v reaching infinity, without any inferred synonymous substitution. Adaptive evolution was estimated along several branches (p > 99%; Fig. 2B). 4. Discussion This study showed an unparalleled level of genetic variation at the B30.2 (SPRY) domain of TRIM5 in New World primates, which was far higher than previously estimated (Ohkura et al., 2006; Song et al., 2005a). These changes affected protein length and involved amino acid substitutions across the whole B30.2 domain, particularly at its three most variable regions. In addition to the indels, several cysteine residues were found to vary in each of the three regions (Fig. 1), a finding suggesting that TRIM5a might Table 2 Maximum likelihood-based tests of occurrence of positive selection along the TRIM5 B30.2 gene region. The v ˆ value is that inferred for the positive selection class; pˆ ½v > 1 refers to the relative probability of the v > 1 class. Test

vˆ

pˆ ½v > 1

2D ln L

M1a/M2a M7/M8 PARRIS

6.41 6.86 3.96

0.23 0.23 0.32

93.8** 93.2** 20.7**

**

Significant at p < 0.01.

Table 3 Positively selected sites estimated by maximum likelihood methods. Numbers in bold depict codons shown to have undergone positive selection with all models and algorithms. Model

Amino acid sites

M2a

311, 408, 311, 417, 406, 310, 391, 341,

M8 SLAC REL FEL

317, 417, 317, 418, 496 311, 406, 390,

324, 418, 324, 496,

325, 496, 325, 529,

340, 529, 340, 541,

341, 347, 382, 383, 389, 390, 391, 406, 541, 544 341, 347, 382, 383, 389, 390, 391, 406, 408, 544

317, 324, 324, 340, 341, 347, 371, 382, 387, 388, 389, 390, 408, 409, 417, 494, 496, 529, 541, 544 391, 406, 408, 496

show significant structural differences among NWP. As these regions contain most determinants of TRIM5a antiviral activity, their evolutionary diversity may result in a wide range of TRIM5a phenotypes directed against different retroviruses and/or retroelements. Our results may therefore widen the spectrum of restriction mechanisms of TRIM5a proteins in primates. Previous reports have shown that some definite residues were crucial for TRIM5a restriction properties and specificity. This was the case of the human TRIM5a (huTRIM5a) Y336 site (corresponding to site 337 in our alignment; Fig. 1) which was shown to be determinant for B-MLV restriction (Maillard et al., 2007). We also found a tyrosine residue at this site in Aotus while, in Saimiri, this site was occupied by arginine, and by histidine in all other NWP herein analyzed. Of note, Y336R was reported to abrogate the BMLV restriction capacity of huTRIM5a (Maillard et al., 2007) while, in Callimico goeldii, residues Y410 and D412 (at sites 383 and 385 in our alignment) were shown to contribute to the restrictive phenotype against SIVmac (Ohkura et al., 2006). In Saimiri, site 383 contained phenylalanine instead of tyrosine. The K324N substitution (asparagine for lysine) in human and rhesus TRIM5a was shown to increase HIV-1 inhibition (Stremlau et al., 2005). The same position showed lysine in Aotus, Lagothrix and Saimiri, glutamic acid in the remaining atelides, pithecides and Cebus, valine in Callimico, Callithrix, Mico and Cebuella, alanine in Leontopithecus, and phenylalanine in Saguinus. Other B30.2 residues of proven relevance in retroviral restriction like R332 (Li et al., 2006) or HIV disease progression in humans, like H419 (Goldschmidt et al., 2006), lie in regions that were apparently deleted in NWP with respect to the human. Variations were also observed in two of the four relevant regions for a capsid bindingindependent mechanism of retroviral restriction (Sebastian et al., 2009). The observed diversity around B30.2 regions of functional relevance is noteworthy and phenotypic evaluations of these variants await further investigation. B30.2 nucleotide sequences of Aotus trivirgatus and Aotus azarae were the most divergent among all NWP (Fig. 2A). Aotus is the only NWP genus showing an insertion, between TRIM5 exons 7 and 8, of a retrotransposed CypA copy which results in a chimeric TRIM-Cyp protein lacking the B30.2 domain (Ribeiro et al., 2005). Whether this domain was transcribed and subsequently spliced out remains an open question. We failed to detect B30.2 domain transcription in an A. trivirgatus cell line (data not shown), despite the presence of an evolutionary conserved open reading frame. This was

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consistent with our previous proposition that the CypA retrotransposition had been a relatively recent event (ca. 1.5 MYBP), probably anteceding Aotus radiation, and that the retrotransposed copy resulted in very few amino acid substitutions respective to the original, genomic CypA copy (Ribeiro et al., 2005). Phylogenetic analysis based on the B30.2 domain, at the nucleotide level, revealed some discrepancies with the consensus NWP topology using concatenated structural genes (Schneider et al., 2001). These probably resulted from the disparate selective pressures to which TRIM5 had been subjected during NWP radiation. Aotus was placed as the most divergent NWP taxon, differing from the consensus arrangement of Schneider et al. (2001). Although the precise phylogenetic placement of Aotus respective to other New World primate genera is still controversial, grouping either with Cebus/Saimiri or Callithrix in Wildman et al. (2009), its location within the Cebidae is almost universally accepted. Only one report considered Aotus in the separate family Aotidae (Groves, 2005), but recent analyses of extensive datasets of nuclear sequences have ruled out this proposition (Wildman et al., 2009). Similarly, Cebus/Saimiri grouped with the pithecids in our analysis rather than with the Callitrichinae and the Aotinae, and Callimico grouped with Leontopithecus/Saguinus rather than with Callithrix/Mico (Fig. 2A). These discordances most likely result from the strong positive selection in the Trim5 B30.2 domain. Our estimates of selection across lineages were generally congruent with those reported with representatives of some NWP genera (Sawyer et al., 2005). Slight differences between their data and our findings are probably due to the fact that our analysis included representatives of 15 NWP genera. Most NWP lineages had v values reaching infinity, a consequence of the lack of observed synonymous substitutions. Although this scenario may cause overestimation of v, the concordance of multiple results obtained here for codon- and lineage-based positive selection using different algorithms warrants robustness to our conclusions. Several codon sites were found under strong positive selection in our analyses (Table 3). Two of them (406 and 496) were commonly found with all methods used and were highly variable among NWP, exhibiting lineage-specific amino acid replacements (see Fig. 1). Interestingly, both sites were located in B30.2 variable regions, 406 and 496 at the beginning and end of V3, respectively. We also conducted all analyses of differential selective forces acting on codons using the complete taxa dataset and the ML tree topology (Fig. 2A). The amino acid sites inferred to have undergone adaptive molecular evolution were essentially the same estimated with the pruned dataset (Fig. 2B). Again, codons 406 and 496 were positively selected independently of the method used, showing that our findings were robust with respect to taxon sampling. Our results showed that selective pressure on B30.2 variable regions either led to adaptive changes in response to different and evolving retroviruses and/or retroelements or imposed tight evolutionary constraints for the maintenance of binding properties. Although lentiviruses have not been to present reported in NWP, the possibility that they have infected these primates in the past still remains to be demonstrated and cannot be completely ruled out. Recent reports identified extinct lentiviruses or primitive forms of them which had infected prosimians (Gifford et al., 2008) and even non-primate species like leporids (Katzourakis et al., 2007; Keckesova et al., 2009). Moreover, other retroviruses, like Simian Foamy Viruses (SFV) and betaretroviruses (Colcher et al., 1977; Heberling et al., 1977; Schweizer and Neumann-Haefelin, 1995), as well endogenous retroviruses, like HERV-W and HERV-E (Kim et al., 2008; Yi and Kim, 2006), have been reported in NWP. Exposure to these viruses must have exerted different levels of selective pressure on TRIM5 across different NWP taxa.

Despite the scarce knowledge on the molecular biology of viral infections in NWP, some species are promising models for biomedical research. Callitrichines and aoutines are especially suitable for these studies due to their low cost of maintenance in captivity, small size, high reproductive index and availability in South America. With respect to HIV research, the recent discovery of TRIM-Cyp in Aotus (Nisole et al., 2004; Sayah et al., 2004) has encouraged virologists to carry out molecular studies with these primates. The high genetic variability of TRIM5 herein reported further widens the possibilities of using NWP as animal models for HIV/AIDS. It is worthwhile mentioning that this diversity has been already assayed when a limited set of NWP TRIM5a proteins was functionally tested against the Mason–Pfizer monkey virus. Tamarin (Saguinus labiatus) and squirrell monkeys (Saimiri sciureus) TRIM5a efficiently blocked viral infection unlike TRIM5a from the spider monkey (Ateles geoffroyi) (Diehl et al., 2008). The high diversity of NWP TRIM5a will also encourage the search for new restriction factors to countering HIV infection in humans. Acknowledgements We are grateful to Alcides Pissinatti (Centro de Primatologia do Rio de Janerio, FEEMA) for providing blood samples and to Ieda P. Ribeiro (Universidade Federal do Rio de Janeiro) for technical assistance. Work supported by CNPq (grants no. 473768/2006-2 and 151453/2007-1) and FAPERJ (grant no. E-26/152.839-2006). References Anisimova, M., Bielawski, J.P., Yang, Z., 2001. Accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution. Mol. Biol. Evol. 18 (8), 1585–1592. Baumann, J.G., 2006. Intracellular restriction factors in mammalian cells—an ancient defense system finds a modern foe. Curr. HIV Res. 4 (2), 141–168. Brennan, G., Kozyrev, Y., Hu, S.L., 2008. TRIMCyp expression in Old World primates Macaca nemestrina and Macaca fascicularis. Proc. Natl. Acad. Sci. U.S.A. 105 (9), 3569–3574. Canavez, F., Alves, G., Fanning, T.G., Seuanez, H.N., 1996. Comparative karyology and evolution of the Amazonian Callithrix (Platyrrhini, Primates). Chromosoma 104 (5), 348–357. Colcher, D., Heberling, R.L., Kalter, S.S., Schlom, J., 1977. Squirrel monkey retrovirus: an endogenous virus of a new world primate. J. Virol. 23 (2), 294–301. Diaz-Griffero, F., et al., 2007a. Comparative requirements for the restriction of retrovirus infection by TRIM5alpha and TRIMCyp. Virology 369 (2), 400–410. Diaz-Griffero, F., et al., 2007b. Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain. J. Virol. 81 (19), 10362–10378. Diehl, W.E., Stansell, E., Kaiser, S.M., Emerman, M., Hunter, E., 2008. Identification of postentry restrictions to Mason–Pfizer monkey virus infection in New World monkey cells. J. Virol. 82 (22), 11140–11151. Gifford, R.J., et al., 2008. A transitional endogenous lentivirus from the genome of a basal primate and implications for lentivirus evolution. Proc. Natl. Acad. Sci. U.S.A. 105 (51), 20362–20367. Goldschmidt, V., et al., 2006. Role of common human TRIM5alpha variants in HIV-1 disease progression. Retrovirology 3, 54. Groves, C., 2005. Order primates. In: Wilson, D.E. (Ed.), Mammal Species of the World. John Hopkins University Press, Baltimore, pp. 111–184. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95. Heberling, R.L., Barker, S.T., Kalter, S.S., Smith, G.C., Helmke, R.J., 1977. Oncornavirus: isolation from a squirrel monkey (Saimiri sciureus) lung culture. Science 195 (4275), 289–292. Katzourakis, A., Tristem, M., Pybus, O.G., Gifford, R.J., 2007. Discovery and analysis of the first endogenous lentivirus. Proc. Natl. Acad. Sci. U.S.A. 104 (15), 6261– 6265. Keckesova, Z., Ylinen, L.M., Towers, G.J., Gifford, R.J., Katzourakis, A., 2009. Identification of a RELIK orthologue in the European hare (Lepus europaeus) reveals a minimum age of 12 million years for the lagomorph lentiviruses. Virology 384 (1), 7–11. Kim, H.S., et al., 2008. Molecular characterization of the HERV-W env gene in humans and primates: expression, FISH, phylogeny, and evolution. Mol. Cells 26 (1), 53–60. Kratovac, Z., et al., 2008. Primate lentivirus capsid sensitivity to TRIM5 proteins. J. Virol. 82 (13), 6772–6777. Kumar, S., Nei, M., Dudley, J., Tamura, K., 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief. Bioinform. 9 (4), 299–306.

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