To ask whether TRIM5-mediated restriction plays a role in cross-species transmission and emergence of primate lentiviruses, we tested six representative alleles of rhesus macaque TRIM5 for restriction activity against four closely related viruses of old-world monkeys, SIVmac239, SIVsmE543, SIVsmE041, and SIVstm/37.16 (Table 1). SIVmac239 is a molecular clone of a highly adapted, emergent virus of rhesus macaques [25], generated in the 1980s by experimental passage of SIV-positive plasma through a series of five monkeys [26]. In all likelihood, SIVmac239 is descended from a cross-species transmission event that took place in the 1960s in captive colonies of rhesus macaques [5], probably as the unintended consequence of experiments involving transfer of biological material from SIVsm-positive sooty mangabeys to rhesus macaques [27]. Regardless of origin, as a result of this long association with macaques, experimental infection with SIVmac239 reproducibly results in high levels of persistent viral replication [28]. In contrast, SIVsmE543-3 is a molecular clone derived by intentional inoculation of a rhesus macaque with plasma from an SIVsm-infected sooty mangabey, followed by passage through one additional rhesus macaque [29]; thus, opportunity for SIVsmE543-3 to adapt to macaques was limited to only two animals. As a result, SIVsmE543-3 replication in macaques is highly variable, with acute viral loads ranging from 10³ to 10⁸ viral RNA copies/ml plasma, and set-point values from <100 to 10⁸. Variation in SIVsmE543-3 infected animals is consistent with an influence of genetic variation in a host gene or genes [30]. SIVsmE041 is a biological isolate cultured directly from an SIV-positive sooty mangabey [31] and has therefore not experienced any prior adaption to rhesus macaques. SIVstm/37.16 is an SIV isolate from a different species, the stump-tailed macaque (M. arctoides), and represents an independent cross-species transmission event involving transmission of SIVsm directly to M. arctoides animals [3],[27],[32],[33]. The relevant properties of these four viruses are summarized in Table 1.

Infectivity of all four viruses was measured on cell lines stably expressing six common alleles of rhesus macaque TRIM5, including three TRIM5^TFP alleles, two TRIM5^Q alleles, and the TRIM5^CypA allele (Figure 2). SIVmac239 was resistant to all six. SIVsmE041, SIVsmE543, and SIVstm were also resistant to TRIM5^Q but unlike SIVmac239 were sensitive to both TRIM5^CypA alleles and TRIM5^TFP alleles (Figure 2B,C,D). Of the four SIV strains tested, only SIVmac239 is the product of decades of replication and spread in rhesus macaques. Thus, the comparison suggests that sensitivity to TRIM5^TFP and TRIM5^CypA alleles represents the ancestral phenotype and that emergence of SIVsm (as SIVmac) in rhesus macaques required acquisition of adaptive changes to overcome those particular types of alleles. In contrast, the SIVsm variants that first invaded rhesus macaques were probably inherently resistant to TRIM5^Q alleles.

To analyze the impact of TRIM5 variation on cross-species transmission directly, we acquired samples from two independent SIVsm/macaque cohorts. The smaller cohort consisted of four sooty mangabeys and four rhesus macaques that had been experimentally inoculated with SIVsmE041 [34]. Infection of sooty mangabeys (natural hosts of SIVsm) resulted in ready take of virus and persistent infection (unpublished data), whereas infection of macaques resulted in a transient infection followed by a decrease in viral replication to a point near or below the detection limit (Figure 3). Using archived DNA, we determined that the four macaques included three TRIM5^TFP/TFP homozygotes and one TRIM5^TFP/CypA heterozygote. Thus, the alleles present in these four animals were all of the same types that restricted SIVsmE041 in tissue culture (Figure 2B). In one animal, replication resurged during the first year, reaching ~10⁴ RNA copies/ml plasma (Figure 3). Capsid sequences recovered from this animal revealed the appearance of two fixed changes, R97S and V108A (SIVmac239 numbering) at the late time point (Figure 3C). In contrast, amplification and sequencing from acute infection, from the SIVsmE041 inoculum, and from an infected sooty mangabey, revealed only the ancestral states at both positions (R97 and V108) (Figure 3B).

The second and larger cohort consisted of historical samples from 44 SIVsmE543-3-infected rhesus macaques. Genotype frequencies in this cohort were 30% TRIM5^TFP/TFP, 23% TRIM5^TFP/Q, 26% TRIM5^TFP/CypA, 12% TRIM5^Q/Q, 9% TRIM5^Q/CypA, and 0% TRIM5^CypA/CypA. Animals with two restrictive alleles (TRIM5^TFP/TFP and TRIM5^TFP/CypA) had dramatically diminished viral replication compared to TRIM5^Q/Q homozygotes, with mean (geometric) differences of 830-fold and 1,728-fold, respectively, by 8 wk post-infection (Figure 4). Animals with one restrictive allele (TRIM5^TFP/Q and TRIM5^CypA/Q heterozygotes) displayed intermediate levels of viral replication. Taken in conjunction with the clear differences in restriction of SIVsmE543-3 by TRIM5^TFP, TRIM5^CypA, and TRIM5^Q in vitro (Figure 2C), these results are consistent with allelic variation in TRIM5 having a significant impact on SIVsmE543 replication kinetics in rhesus macaques. We also obtained archived DNA from animal #E543, the source of the original SIVsmE543-3 clone [29], and determined that this animal had been a TRIM5^Q/Q homozygote. The fact that E543 bore a non-restrictive genotype may well have facilitated isolation of the original SIVsmE543-3 clone.

Several reports describe a correlation between specific alleles of class I MHC and enhanced control of SIVmac239/SIVmac251 infection in rhesus macaques [35]–[37]. Specifically, MHC class-I Mamu-B*08 and Mamu-B*17 alleles have been associated with lower levels of chronic phase viral replication in SIVmac239-infected animals [35],[37]. Several observations argue against the dramatic differences in viral replication of SIVsmE543-3 in rhesus macaques being due to Mhc class I rather than TRIM5. First, the TRIM5 and class I MHC loci are on different chromosomes, reducing the probability of a chance association between suppressive alleles of TRIM5 and a specific allele or alleles of class I MHC with activity against SIVsmE543-3. More importantly, the effects of class I Mhc on SIVmac239 replication do not manifest during the acute stage of infection [38], whereas the correlation with TRIM5 genotype is already apparent during acute infection (Figure 4D). Finally, Goldstein et al. demonstrated that variation in susceptibility of cells taken from naïve animals (prior to infection) and tested ex vivo correlated with viral replication levels in vivo, when the same animals were subsequently infected with SIVsmE543; such results argue in favor of an inherent, genetic cause of variation in susceptibility and against an effect of virus-specific adaptive immune responses induced by infection [30]. Finally, we typed all 43 animals in the SIVsm543-3 cohort for the presence of the Mamu-B*08 and Mamu-B*17 alleles and found no association between these alleles and the observed differences in Figure 4. Most importantly, none of the infected animals with TRIM5^Q/Q or TRIM5^TFP/CypA genotypes were Mamu-B*08 or Mamu-B*17 positive, so the statistically significant differences between those two groups cannot be attributed to Mamu-B*08 or Mamu-B*17 associated control of SIVsmE543. Thus, the observed correlation between TRIM5 genotype and SIVsmE543-3 replication levels is not due to a spurious association between suppressive alleles of TRIM5 and class I MHC alleles previously associated with control of SIVmac239. It is important to note, however, that this result does not rule out a general influence of class I Mhc on viral replication levels in rhesus macaques, only that MHC genotype does not explain the correlation depicted in Figure 4. Among other things, allelic variation in MHC class I may well contribute to the significant variation observed within groups (Figure 4C,D,E).

Three macaques in the SIVsmE543-3 cohort also had patterns of resurgent viral replication consistent with escape from suppression (Figure 5A). All three animals had two restrictive alleles and included one TRIM5^TFP/TFP homozygote and two TRIM5^TFP/CypA heterozygotes. We amplified gag sequences encoding the NTD of CA from all three animals, and from a TRIM5^Q/Q animal, and compared these to the original SIVsmE543-3 clone (Figure 5B). Strikingly, an R97S change was present in every clone from all three animals, typically due to an AGA->AGC substitution, although 3/15 clones in one animal were AGA->AGT. No changes were found in the corresponding region of virus from the non-restrictive TRIM5^Q/Q animal. Thus, an identical R97S change appeared independently in four animals with suppressive TRIM5 genotypes, including three SIVsmE543-infected animals (Figure 5B) and one SIVsmE041-infected animal (Figure 3).

Phylogenetic analyses are consistent with a minimum of two historical transmissions of SIVsm into macaques, one into stump-tailed macaques (SIVstm), and the other into rhesus macaques (SIVmac); both transmissions are thought to have occurred in captive macaques sometime prior to the 1970s [3]. If TRIM5-mediated restriction influences cross-species transmission of primate lentiviruses, we predict the existence of adaptive changes in SIV isolates corresponding to such events. Indeed, alignment of the NTD of several lentiviruses in the SIVsm/SIVmac/HIV-2 lineage revealed multiple potential adaptations in SIVmac (Figure 6). Most striking is an inferred R97S change, identical to the change that appeared in the SIVsmE041 and SIVsmE543 experimental cohorts described above (Figures 3 and ​and5B).5B). Based on phylogeny of the SIVsm/SIVmac/HIV-2 lineage [3], the R97S change was probably selected twice, once coinciding with emergence of SIVmac in rhesus macaques and once coinciding with emergence of SIVstm in stump-tailed macaques. Consistent with this interpretation, the underlying nucleotide substitutions are different in the two viruses (AGA->AGC in SIVstm, and AGA->TCA in SIVmac). However, S97 is also found in a small percentage of HIV-2 and SIVsm isolates; thus, it is possible that one or both historical transmissions were initiated by an SIVsm with serine at position 97, rather than de novo mutation as seen in the experimental cohorts. In either case, these combined observations strongly suggest that S97 is selectively advantageous in macaques.

The second putative adaptation is a highly unusual LPA/QQ substitution at the tip of the CA 4–5 loop (QQ^89,90 in SIVmac239). How this change was generated is unclear but must have involved multiple point mutations and a net loss of three nucleotides. While QQ^89,90 is very common among SIVmac isolates, P⁹⁰ (the proline in LPA^89–91) is extremely well conserved in SIVsm and HIV-2 isolates (Figure 6). The highly unusual nature of the LPA->QQ substitution, together with its location in a stretch of residues known to affect TRIM5-mediated restriction [14],[39], mark it as a potential adaptation that arose to circumvent restriction by rhesus macaque TRIM5 proteins.

To ask whether the R97S and LPA/QQ changes in SIVmac strains arose as adaptations to overcome rhesus TRIM5, we reverted these sites to the ancestral sequence in the context of the macaque-adapted strain SIVmac239. We then tested SIVmac239^S97R and SIVmac239^QQ->LPA for gain-of-sensitivity to different rhesus TRIM5 alleles. Indeed, the S97R reversion resulted in increased sensitivity to all three TRIM5^TFP alleles but had no effect on resistance to the TRIM5^Q or TRIM5CypA alleles. In contrast, the QQ^89,90-to-LPA^89-91 reversion resulted in sensitivity to both TRIM5^TFP and TRIM5CypA alleles. This closely resembles the pattern displayed by SIVsm isolates (see Figure 2B and C), confirming that these represent bona fide adaptations to overcome restriction by rhesus TRIM5 alleles. Because QQ^89,90 is in the 4–5 loop of capsid, the adaptation probably functions by altering the TRIM5CypA binding site. The structural basis for the interaction between the TRIM5 B30.2/SPRY domain and viral CA is not yet well defined, and it is not clear why QQ^89,90 also affects resistance to TRIM5^TFP alleles. However, this result is consistent with studies showing that site-directed mutations in the HIV-1 CypA-binding domain influence binding and restriction by TRIM5α proteins [39]. A better understanding of the influence of CA positions 89–91 on restriction awaits more detailed, structural understanding of the interaction between CA and TRIM5α.

Neither reversion affected resistance of SIVmac239 to TRIM5^Q, consistent with our observation that SIVsmE041 and SIVsmE543, which retain ancestral states at these sites, were also resistant to TRIM5^Q (Figure 2). We conclude that TRIM5^Q alleles did not significantly hamper SIVsm colonization of rhesus macaques or its emergence as SIVmac in the 1970s. In fact, TRIM5^Q/Q animals may have facilitated initial transmission of SIVsm among macaques, permitting higher levels of replication and increasing the probability of adaptation and spread.

In this study, we specifically demonstrated that the RF gene TRIM5 can suppress replication of a primate immunodeficiency virus in vivo at the time of cross-species transmission, and that TRIM5-mediated suppression of viral replication selects for acquisition of adaptive changes in the viral capsid protein. The data included retrospective genotyping of two independent, cross-species transmission cohorts, identification of at least two adaptations selected for resistance to TRIM5 during emergence of the SIVmac lineage, and functional assessment of allele-specific restriction in cell-culture. While the adaptations in SIVmac239 capsid identified here (R⁹⁷S and LPA^89–91/QQ^89,90) have a direct effect on restriction sensitivity, the modification of the otherwise highly conserved residues R97 and P90 may well have required additional adaptive adjustments elsewhere in the CA or the Gag polyprotein. Identifying such changes, if they exist, will require systematic testing of combinations of residues that vary between the SIVmac and SIVsm lineages for impact on relative infectivity in the presence and absence of TRIM5 expression.

The transmission event(s) leading to spread and emergence of SIVsm in the U.S. macaque colonies have not been identified, although there is indirect evidence suggesting that transmission may have resulted from experimental procedures involving the transfusion of material from one species into another [27]. Regardless of the mechanism(s) of initial transmission, our data strongly suggest that adaptation to overcome restriction by a specific subset of rhesus macaque TRIM5 alleles played a key role in spread of SIVsm and its emergence as SIVmac in captive colonies of rhesus macaques. Based on cell-culture assays, analysis of experimental cohorts, and functional identification of adaptive changes in the SIVmac239 capsid, we propose a simple but likely scenario for the influence of variation in the rhesus macaque TRIM5 gene on emergence of SIVmac in the U.S. National Primate Research Center macaque colonies (Figure 7). Briefly, at the time of initial cross-species exposure, we believe that the SIVsm source was likely to be sensitive to restriction by a subset of rhesus macaque TRIM5 alleles, including those with a TFP sequence at residues 339–341 in the B30.2/SPRY domain (TRIM5^TFP) and those that produce a TRIM5CypA chimera by alternative splicing (TRIM5^CypA). In contrast, we have yet to identify an SIV strain that is sensitive to alleles with a Q at position 339 (TRIM5^Q); thus, it seems likely that animals bearing TRIM5^Q alleles (particularly TRIM5^Q/Q homozygotes) permitted initial spread and adaptation of SIVsm to rhesus macaques, potentially facilitating acquisition of adaptations to overcome other, as yet unidentified genetic barriers encountered in the newly invaded host species. The appearance of the R-to-S and the LPA-to-QQ changes in CA would have opened the way for the virus to spread to a larger percentage of the macaque population, ultimately leading to emergence of pathogenic SIVmac.

None of the viruses tested in this study except HIV-1 were sensitive to the rhesus macaque TRIM5^Q alleles. It is noteworthy that all known human alleles of TRIM5 have a Q at the homologous position, suggesting that human TRIM5 may not pose a critical barrier to transmission of SIVsm into human populations; in this regard, it would be interesting to assess the ability of human TRIM5 variants to restrict divergent primary isolates of SIVsm found in regions of endemic infection among African nonhuman primates and to look for correlations between TRIM5 and susceptibility to HIV-2 in human AIDS cohorts.

An unexpected finding from analysis of the experimental transmission cohorts was the establishment of infection even in animals with restrictive genotypes. In many cases, we found that once animals with restrictive genotypes were infected, viral replication often continued to be suppressed to low levels for many weeks (Figure 4). In nature, lower viremia could reduce the probability that the infected individual will pass the virus on to additional individuals in the new host population, thus extending the role of TRIM5 in preventing emergence beyond the initial cross-species transmission event(s). However, the retrospective analysis of SIVmac emergence does not necessarily address the impact of TRIM5 during natural exposure and transmission, and it is possible that the observed pattern is, at least in part, the consequence of experimental routes of transmission. For example, in many SIV/macaque studies, including the cohorts analyzed here, high titer stocks of virus (TCID₅₀ in the range of 1 to 1,000) are used to initiate infections by intravenous injection. Since the effect of restriction by TRIM5α is known to be saturable, it is possible that a large bolus of localized infection manages to initially swamp out restriction and permits virus to seed a large number of virus producing-cells and initiate a transient acute infection. In this regard, experimental intravenous infection of macaques may more closely resemble accidental human exposure to HIV-1 via blood-transfusion or contaminated, hypodermic syringes. It is possible that the effect of TRIM5 could be greater under conditions of natural exposure to virus, such as through sexual contact or fighting; however, we are currently unaware of any studies that address this issue. In recent years, many SIV/AIDS vaccine studies have incorporated low-dose mucosal challenges, to more closely mimic the conditions of sexual transmission. Under such conditions, it is possible that the effect of TRIM5 genotype on infection will be even more pronounced. Just as historical samples were used for this study, genotyping of appropriate archived samples from completed or ongoing SIV/vaccine studies may also prove useful for evaluating the interactions between TRIM5 genotype, viral dose, and route of transmission.

Alternatively, there are published reports indicating that TRIM5 gene expression is interferon-regulated [48],[49]; thus, it is also possible that the full impact of TRIM5 on viral replication in vivo is not manifest until after the first round of infection is well underway. Such a pattern is consistent with the observation that the impact of TRIM5 genotype in the SIVsmE543-3 infected animals appeared to persist during the weeks following acute infection (Figure 4). The TRIM5 gene is also known to encode multiple splice-isoforms [50], including mRNA molecules lacking viral specificity encoded by a B30.2/SPRY or CypA domain; however, it is not known whether these are differentially expressed in vivo in response to infection or whether patterns of isoform expression differ between tissues. In vivo studies will be required to determine how TRIM5 is regulated, whether regulation changes in response to viral infection, and to identify the patterns of TRIM5 expression in cell types responsible for initial infection and spread in vivo.

The impact of variation in rhesus macaque TRIM5 has practical implications for preclinical AIDS vaccine research. SIVsmE543 and the related SIVsmE660 serve as heterologous challenge strains for widely used SIVmac239-derived vaccine immunogens in rhesus macaques [38],[51]–[54]. The pronounced effect of TRIM5 variation on SIVsmE543 infection is likely to confound comparison of vaccinated and control groups, particularly if they are not balanced for restrictive and permissive alleles; this may be especially true for studies with typically small numbers of animals (n=4–6) in each group. This may also be true of SIV strains not examined here, and possibly extends to other SIV/AIDS model organisms including Chinese-origin rhesus macaques and other commonly studied Macaca species, such as M. nemestrina and M. fascicularis. Moreover, vaccines expressing Gag, the target of TRIM5, may be affected by TRIM5 genotype. This is potentially the case for live-attenuated SIV, where vaccine efficacy is influenced by the degree to which the vaccine strain replicates in the inoculated animal [55]. Finally, as discussed above, the potential effects of viral dose or route of transmission on restriction by TRIM5 remain to be determined.

Figure S1