Primates have evolved a variety of restriction factors that prevent retroviral replication. One such factor, TRIM5␣, mediates a postentry restriction in many Old World primates. Among New World primates, Aotus trivirgatus exerts a similar early restriction mediated by TRIMCyp, a TRIM5-cyclophilin A (CypA) chimera resulting from a CypA retrotransposition between exons 7 and 8 of the TRIM5 gene. convergent evolution ͉ cyclophilin A ͉ HIV ͉ retroviruses ͉ TRIM5␣ T he current HIV pandemic resulting from cross-species transmissions of simian immunodeficiency viruses SIVcpz or SIVsmm to humans has been well documented (1). However, the mechanisms enabling such transmissions are not yet fully understood. Mammals have evolved several restriction factors capable of inhibiting the replication of certain retroviruses in a species-specific manner. One of the best described primate host-restriction factors is TRIM5␣, which is expressed in most Old World primates.Macaca mulatta TRIM5␣ exerts an early, postentry block to HIV-1 replication (2). TRIM5␣ is a member of the tripartite motif family of proteins, characterized by the ordered Nterminal to C-terminal expression of a RING domain, B-Box, and coiled coil, also known as an RBCC domain (3). The TRIM5␣ isoform also expresses a B30.2 domain at the C terminus, which is required for recognition of the incoming retroviral capsid (4-7). Changes to the B30.2 domain have been shown to dramatically affect the breadth and potency of TRIM5␣-mediated anti-retroviral activity. For instance, the ability to restrict HIV-1 replication may be conferred to Homo sapiens TRIM5␣ by changing a single residue to the amino acid found in M. mulatta TRIM5␣, R332P (8). Similarly, site-directed mutagenesis studies have demonstrated that mutations around the cyclophilin A (CypA) binding loop of HIV-1 capsid effect the potency of TRIM5␣-mediated restriction, suggesting that the B30.2 domain interacts with or near the CypA binding loop (5, 9, 10).New World primates Aotus trivirgatus exert a postentry restriction to HIV-1 mediated by a TRIM5-CypA chimera. Sequencing the A. trivirgatus TRIM5 gene identified a LINE-1-mediated retrotransposition of CypA into intron 7, resulting in the expression of a fusion protein called TRIMCyp, which is unique to the Aotus genus (11-13). TRIMCyp retains the N-terminal tripartite motif of all TRIM family members, but the B30.2 domain of TRIM5␣ is replaced by the CypA domain. Functionally, TRIM5␣ and TRIMCyp are similar, preventing reverse transcription of incoming viruses at an early postentry stage. However, TRIMCyp exerts a more potent restriction to incoming retroviruses than TRIM5␣, and unlike TRIM5␣, TRIMCyp-mediated restriction is sensitive to cyclosporin A. A. trivirgatus has been shown to express only TRIMCyp,.We recently demonstrated that the Old World primates Macaca nemestrina do not express TRIM5␣. Instead, they transcribe novel isoforms TRIM5 and TRIM5 (14). These isoforms likely arise because of a single-nucleotide polymorphism (SNP) at the intron 6 splice acc...

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed by many different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted to changes in PKR. As a result of this "arms race," modern TRS1 alleles in CMVs may function differently in cells derived from alternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replication of a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not have these activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfills these functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNA and, in the cell types in which they can rescue VV⌬E3L replication, they also bind to PKR and prevent phosphorylation of the ␣-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophosphorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations in this critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanism of PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of the factors that contributes to species specificity of cytomegalovirus replication.

A growing number of studies indicate that host-species-specific and virus-strain-specific interactions of viral molecules with the host innate immune system play a pivotal role in determining virus host range and virulence. Because interacting proteins are likely constrained in their evolution, mutations that are selected for to improve virus replication in one species, may stochastically alter the ability of a viral antagonist to inhibit immune responses in hosts the virus has not yet encountered. Based on recent findings of host-species interactions of poxvirus, herpesvirus and influenza virus proteins, we propose a model for viral fitness and host range, which considers the full interactome between a specific host-species and a virus, resulting from the combination of all interactions, positive and negative, that influence whether a virus can productively infect a cell and cause disease in different hosts.

The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5␣ isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5␣-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (⌬2) in the transcript at the 5 terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5. This deletion is likely due to a single nucleotide polymorphism that alters the 3 splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (⌬exon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5. There are 18 amino acid differences between M. nemestrina TRIM5 and Macaca mulatta TRIM5␣, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5␣-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5 or TRIM5 show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo. The human immunodeficiency virus (HIV) pandemic is the result of cross-species transmissions of simian immunodeficiency virus (SIV) from non-human primates to humans (28).The recent discovery of human T-cell leukemia virus types 3 and 4 suggests that cross-species transmission of retroviruses is not an infrequent occurrence (33). However, to establish a productive infection in a new species, it is necessary for retroviruses to evade host-specific restriction factors. A broad range of antiretroviral host restriction factors has been identified in mammalian species. The best-described primate host restriction factors are APOBEC3F/G and TRIM5␣. APOBEC3F/G are cytidine deaminases which exert a late block to retroviral replication, inhibiting the virus in target cells rather than producer cells (29), whereas TRIM5␣ exerts a dominant block to infection immediately after viral entry into the cell through the inhibition of reverse transcription (30).TRIM5␣ is a member of the tripartite motif family of proteins, also called RBCC proteins, because of the presence of a RING domain (C3HC4 type), one or two B boxes, and a coiled-coil region in an ordered arrangement from N terminus to C terminus (23). Six isoforms of TRIM5 have been identified in mammals (26). These proteins have been shown to form homo-and heteromultimers, although only homomultimers of TRIM5␣ have been shown to restrict lentiviruses (3,8,15,...