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TRIM5α polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of human immunodeficiency virus (HIV) vaccine strategies in rhesus macaques. We previously reported that the TRIM5α-sensitive SIV from sooty mangabeys (SIVsm) clone SIVsmE543-3 acquired amino acid substitutions in the capsid that overcame TRIM5α restriction when it was passaged in rhesus macaques expressing restrictive TRIM5α alleles. Here we generated TRIM5α-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5α alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5α genotypes as efficiently as in macaques with permissive TRIM5α genotypes. These results demonstrated that mutations in the SIV capsid can confer SIV resistance to TRIM5α restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies. IMPORTANCE Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5α alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques, and the introduction of two amino acid changes in the capsid transfers this improved replication to the parent clone. In the present study, we introduced these mutations into a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved the replication of this strain in macaques with the restrictive TRIM5α genotype and thus will eliminate the confounding effects of TRIM5α in vaccine studies.

Rhesus monkey rhadinovirus (RRV), a simian gamma-2 herpesvirus closely related to the Kaposi sarcoma-associated herpesvirus, replicates lytically in cultured rhesus monkey fibroblasts and establishes persistence in B cells. Overlapping cosmid clones were generated that encompass the entire 130-kilobase-pair genome of RRV strain 26-95, including the terminal repeat regions required for its replication. Cloned RRV that was produced by cotransfection of overlapping cosmids spanning the entire RRV26-95 genome replicated with growth kinetics and to titers similar to those of the parental, uncloned, wild-type RRV26-95. Expression cassettes for secreted-engineered alkaline phosphatase (SEAP) and green fluorescent protein (GFP) were inserted upstream of the R1 gene, and the cosmid-based system for RRV genome reconstitution was used to generate replication-competent, recombinant RRV that expressed either the SEAP or GFP reporter gene. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. Heat-inactivated sera from rhesus monkeys that were naturally or experimentally infected with RRV were assayed for their ability to neutralize RRV-SEAP and RRV-GFP infectivity using rhesus monkey fibroblasts. Sera from RRV-positive monkeys, but not RRV-negative monkeys, were consistently able to neutralize RRV infectivity when assayed by the production of SEAP activity or by the ability to express GFP. The neutralizing activity was present in the immunoglobulin fraction. Of the 17 rhesus monkeys tested, sera from rhesus monkey 26-95, i.e., the monkey that yielded the RRV 26-95 isolate, had the highest titer of neutralizing activity against RRV26-95. This cosmid-based genetic system and the reporter virus neutralization assay will facilitate study of the contribution of individual RRV glycoproteins to entry into different cell types, particularly fibroblasts and B cells.

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5TFP/TFP or TRIM5Cyp/TFP. Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5Cyp. Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIMTFP alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates., Author Summary Human immunodeficiency virus (HIV) resulted from the transmission of simian immunodeficiency viruses (SIV) from nonhuman primates followed by adaptation and expansion as a pandemic in humans. This required the virus to overcome a variety of intrinsic host restriction factors in humans in order to replicate efficiently. Similarly, SIV encounters restriction factors upon cross-species transmission between nonhuman primates, specifically from a natural host species such as sooty mangabey monkeys to rhesus macaques. Previously we observed significant differences in the levels of virus replication of SIV among rhesus macaques due to subtle differences in one of these restriction factors, TRIM5 among individual macaques. Although a restrictive version of TRIM5 resulted in lower viremia, we also observed that the virus spontaneously mutated in the viral capsid gene and that these mutations were associated with escape from TRIM5 restriction. In the present study, we found that introduction of these escape mutations into the parental virus confers resistance to TRIM5 both in tissue culture and in macaques. These studies provide direct evidence that TRIM5 is a critical factor influencing the cross-species transmission of SIV in primates.

Infections following repeated, low-dose (RLD), mucal S(H)IV exposures of macaques are used to model sexual HIV exposures for biomedical prevention testing. Different susceptibilities among animals can complicate study designs. In rhesus macaques, TRIM5 alleles Q, CypA, and TFP are resistance factors for infection with some S(H)IV strains, but not for SIVmac239 due to its capsid properties. SIVmac239-derived SHIVSF162P3 has been demonstrated to reproducibly infect mucosally in vaginal and rectal RLD models. To further test the suitability of SHIVSF162P3 for RLD models, we studied the influence of the TRIM5 genotype on susceptibility to rectal RLD infection and on plasma viremia by analyzing 43 male Indian rhesus macaques from control arms of completed studies. The median number of exposures required for infection was three (Q/Q, n=4) (TRIM5 alleles, number of macaques, respectively), four (Q/CypA, n=7), three (TFP/Q, n=15), three (TFP/TFP, n=15), and two (TFP/CypA, n=2); TRIM5(CypA/CypA) was not represented in our study. Median peak viremia (log10 viral copies/ml) in infected animals was 7.4 (Q/Q, n=4), 7.2 (Q/CypA, n=6), 7.3 (TFP/Q, n=13), 7.1 (TFP/TFP, n=15), and 6.5 (TFP/CypA; n=2). Neither susceptibility nor peak viremia was significantly different (log rank test, Kruskal-Wallis test, respectively). Rhesus macaques' susceptibility to RLD SHIVSF162P3 is independent of the TRIM5 TFP, CypA, and Q alleles, with the limitation that the power to detect any impact of CypA/CypA and TFP/CypA genotypes was nonexistent or low, due to absence or infrequency, respectively. The finding that TRIM5 alleles do not restrict mucosal infection or ensuing replication rates suggests that SHIVSF162P3 is indeed suitable for RLD experimentation.

Here, we describe the evolution of antigenic escape variants in a rhesus macaque that developed unusually high neutralizing antibody titers to SIVmac239. By 42 weeks postinfection, 50% neutralization of SIVmac239 was achieved with plasma dilutions of 1:1,000. Testing of purified immunoglobulin confirmed that the neutralizing activity was antibody mediated. Despite the potency of the neutralizing antibody response, the animal displayed a typical viral load profile and progressed to terminal AIDS with a normal time course. Viral envelope sequences from week 16 and week 42 plasma contained an excess of nonsynonymous substitutions, predominantly in V1 and V4, including individual sites with ratios of nonsynonymous to synonymous substitution rates (dN/dS) highly suggestive of strong positive selection. Recombinant viruses encoding envelope sequences isolated from these time points remained resistant to neutralization by all longitudinal plasma samples, revealing the failure of the animal to mount secondary responses to the escaped variants. Substitutions at two sites with significantdN/dSvalues, one in V1 and one in V4, were independently sufficient to confer nearly complete resistance to neutralization. Substitutions at three additional sites, one in V4 and two in gp41, conferred moderate to high levels of resistance when tested individually. All the amino acid changes leading to escape resulted from single nucleotide substitutions. The observation that antigenic escape resulted from individual, single amino acid replacements at sites well separated in current structural models of Env indicates that the virus can utilize multiple independent pathways to rapidly achieve similar levels of resistance.

Standard vectors for high-level expression elicited undetectable levels of the gH and gL glycoproteins of rhesus monkey rhadinovirus (RRV) following transient-transfection assays under a variety of conditions. These same vectors and conditions yielded high levels of RRV gB expression. Unlike other genes of RRV, both the gH and gL genes were noted to have a highly aberrant, suboptimal codon usage. High levels of RRV gH and gL expression were achieved by two alternative means: codon optimization or coexpression of RRV ORF57. The failure of gH and gL to be expressed in the absence of ORF57 and in the absence of codon optimization could not be explained by the failure of RNA to egress from the nucleus. Rather, the defect in gH and gL expression appeared to be cytoplasmic in nature. It is not clear at the present time whether the aberrant codon usage for gH and gL of RRV is an intentional regulatory strategy used by the virus or whether it is driven by some external force, such as intrinsic immunity. In any event, our results indicate that the need of ORF57 for gH and gL expression can be circumvented by codon optimization, that RRV ORF57 acts principally to allow translation of gH and gL RNA in the cytoplasm, and that this activity of ORF57 is related in some way to the aberrant codon usage of the gH and gL RNAs.

Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary “arms-race” between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary “arms-race” hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations., Author Summary APOBEC3G is a host factor that can inhibit replication of primate lentiviruses, including HIV-1, HIV-2, and the related simian immunodeficiency viruses (SIVs) of African primates. As a consequence, primate lentiviruses encode a protein, called Vif, which can induce degradation of APOBEC3G. Given its antiviral role, APOBEC3G may be an important genetic barrier to interspecies jumping of primate lentiviruses. To study this possibility, we asked whether APOBEC3G affected transmission of SIV from sooty mangabeys (SIVsm) to rhesus macaques and subsequent emergence of pathogenic SIVmac in the 1970s. We found that APOBEC3G of sooty mangabeys and rhesus macaques have divergent protein sequences, and that the Vif proteins of SIVsm (Vif-SIVsm) cannot counteract rhesus macaque APOBEC3G. We mapped Vif-SIVsm resistance to a specific substitution in the N-terminal domain of rhesus APOBEC3G, in which a highly conserved tyrosine is replaced by leucine-arginine (Y→LR). We also identified a viral counter-adaptation, found in the Vif proteins of all SIVmac strains, which specifically confers the ability to antagonize APOBEC3G of rhesus macaques. This change was most likely selected during adaptation of SIV to its new host. Together, these results demonstrate that APOBEC3G can serve as a critical genetic determinant of interspecies transmission of primate immunodeficiency viruses.

Cross-species transmission of simian immunodeficiency virus from sooty mangabeys (SIVsm) into rhesus macaques, and subsequent emergence of pathogenic SIVmac, required adaptation to overcome restriction encoded by the macaque TRIM5 gene., Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5α, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission., Author Summary The human immunodeficiency viruses HIV-1 and HIV-2 originated from cross-species transmission of simian immunodeficiency viruses (SIVs) from chimpanzees (SIVcpz) and sooty mangabeys (SIVsm), respectively. A related virus, SIVmac, causes AIDS-like pathogenesis in rhesus macaques; like HIV-2, SIVmac is the product of a cross-species jump of SIVsm from sooty mangabeys. The primate TRIM5 gene encodes a factor with potent antiviral activity when tested in the laboratory, and TRIM5 proteins are thought to play a role in restricting the movement of viruses between species in nature. In this study, we show that genetic variation in the TRIM5 gene of rhesus macaques heavily influences the outcome of cross-species transmission of SIVsm and that emergence of SIVmac in rhesus macaques in the 1970s required adaptations to circumvent the genetic barrier imposed by the rhesus macaque TRIM5 gene. Our results confirm the hypothesis that TRIM5 can influence the process of cross-species transmission and emergence of viruses related to HIV-1 and HIV-2 and serve as a striking illustration of how host genes can influence virus evolution.