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. 2010 Aug 24;8(8):e1000462.
doi: 10.1371/journal.pbio.1000462.

TRIM5 suppresses cross-species transmission of a primate immunodeficiency virus and selects for emergence of resistant variants in the new species

Andrea Kirmaier  1 , Fan Wu, Ruchi M Newman, Laura R Hall, Jennifer S Morgan, Shelby O'Connor, Preston A Marx, Mareike Meythaler, Simoy Goldstein, Alicia Buckler-White, Amitinder Kaur, Vanessa M Hirsch, Welkin E Johnson
Affiliations

TRIM5 suppresses cross-species transmission of a primate immunodeficiency virus and selects for emergence of resistant variants in the new species

Andrea Kirmaier et al. PLoS Biol. .

Abstract

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 TRIM5alpha, 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The rhesus macaque TRIM5 coding sequence is highly polymorphic.
Rhesus macaque TRIM5 proteins are distinguished by polymorphic variation in the B30.2/SPRY domain (encoded by TRIM5TFP and TRIM5Q alleles), or by complete replacement of the B30.2/SPRY domain with a CypA domain (encoded by the TRIM5CypA allele). Shown are polymorphic sites that differentiate six common rhesus TRIM5 alleles, including three TRIM5TFP alleles (Mamu-1, Mamu-2, and Mamu-3), two TRIM5Q alleles (Mamu-4, Mamu-5), and the TRIM5CypA allele. For more details, see references ,. Accession numbers for the depicted alleles are also available in GenBank as follows: Mamu-1 through Mamu-5 (EF113914–EF113918); TRIM5CypA (EU359036).
Figure 2
Figure 2. Differential restriction of SIVmac and SIVsm strains by multiple alleles of rhesus macaque TRIM5.
Single-cycle infectivity was measured on a panel of cell lines stably expressing six common alleles of rhesus TRIM5 ,. Alleles tested included three TRIM5TFP (Mamu-1, Mamu-2, Mamu-3, dark blue bars), two TRIM5Q (Mamu-4 and Mamu-5, light blue bars), and TRIM5CypA (orange bars). Control cells stably expressing the empty vector served as a negative control (black bars). Of the four closely related SIVs, only the rhesus macaque isolate SIVmac239 is resistant to multiple alleles. Infectivity (% GFP-positive cells) was measured by flow-cytometry (error bars indicate ± SEM). Virus stocks were first titered by serial dilution on parental cells and normalized (Figure S1). Stable cell lines were generated from CRFK cells as described in the Materials and Methods section. HIV-1NL4-3 was used as a positive control to confirm expression and function of the TRIM5Q/Q alleles. (A) SIVmac239; (B) SIVsmE041; (C) SIVsmE543-3; (D) SIVstm37/13; (E) HIV-1NL4-3; (F) Immunoblot confirming expression of HA-tagged TRIM5 proteins in cell lysates. upper panel, TRIM5 proteins; lower panel, β-actin loading control. Lanes: (1) Mamu-1; (2) Mamu-2; (3) Mamu-3; (4) Mamu-4; (5) Mamu-5; M, protein standard; TC, TRIM5CypA; V, control cells expressing vector-only. GenBank accession number for the SIVsmE041 clone: HM059825.
Figure 3
Figure 3. Attenuated replication of the sooty mangabey virus SIVsmE041 upon experimental cross-species transmission to rhesus macaques.
The cohort was described previously ; briefly, four Indian-origin rhesus macaques were inoculated intravenously with 25 ng p27 equivalent of an SIVsmE041 virus stock derived by co-culture of cells from SIV-infected sooty mangabey #E041 on PBMC from a second, SIV-negative sooty mangabey. Viral RNA levels in plasma (y-axis) were determined by quantitative RT-PCR at different time-points post-infection (x-axis). All four rhesus macaques displayed post-acute reduction of viral replication by several orders of magnitude. Genotypes were TRIM5TFP/TFP (3 animals, green lines) and TRIM5TFP/CypA (one animal, black line). The three homozygotes had acute viral RNA levels peaking between 105 and 106 (RNA copy equivalents/ml of plasma), whereas the heterozygote had the lowest acute viral RNA levels (2.3×104 RNA copy eqs/ml). Viral replication rebounded in one of the TRIM5TFP/TFP animals (black arrow), suggestive of adaptation and escape. (A) Viral RNA levels in plasma. (B) Partial sequencing of the region encoding the N-terminal domain of the viral capsid from the indicated animal (black arrow), using samples collected during acute infection. (C) Partial sequencing of the region encoding the N-terminal domain of the viral capsid from the indicated animal (black arrow) using samples collected during week 89 post-infection. Comparison of (B) and (C) revealed potential adaptive changes at amino-acid positions 97 and 108 (SIVmac239 numbering).
Figure 4
Figure 4. TRIM5 genotype and replication of SIVsmE543-3 in rhesus macaques.
Archived samples were obtained from 43 Indian origin rhesus macaques that had been infected intravenously (n = 35) or intrarectally (n = 9) with SIVsmE543-3 (TCID 50% from 1 to 1,000). None of the animals were treated or vaccinated prior to infection. Genomic DNA extracted from stored PBMC was used to determine TRIM5 genotype. Data are color-coded by genotype, as follows: red, TRIM5Q/Q; orange, TRIM5Q/CypA; blue, TRIM5TFP/Q; green, TRIM5TFP/TFP; and black, TRIM5TFP/CypA. (A) Viral replication (RNA copy equivalents per milliliter of plasma) through 12 wk post-infection; (B) Same data presented as geometric mean values for each genotype; (C) area under the curve; (D) acute infection (defined as peak viremia for each animal during the first 4 wk post-infection); (E) 8 wk post-infection. Viral replication levels were compared by non-parametric one-way ANOVA (Kruskal-Wallis test) with Dunn's post-test. Pairs of groups that differed significantly are indicated (p<0.01 or p<0.001).
Figure 5
Figure 5. Emergence of Trim5-resistant SIV in vivo.
(A) Viral replication suggestive of suppression followed by adaptation in three animals, as indicated by a green line (TRIM5TFP/TFP homozygote) and two black lines (TRIM5TFP/CypA heterozygotes). (B) Sequences encoding a portion of the N-terminal region of CA were amplified, cloned, and sequenced from all three animals, as well as from a TRIM5Q/Q homozygote with typical, high levels of persistent viral replication. Also shown is the sequence of the SIVsmE543-3 clone. An R97S change was present in every clone from the three animals with restrictive genotypes, but not in the non-restrictive TRIM5Q/Q control animal. At the nucleotide level, both Arg(AGA)-to-Ser(AGC) and Arg(AGA)-to-Ser(AGT) were seen. The aligned amino-acid sequences correspond to residues 89–113 in the N-terminal domain of the SIVmac239 capsid; however, SIVsmE543-3 has one additional amino-acid in this region compared to SIVmac239, so that R97S actually appears at position 98 in the depicted alignment.
Figure 6
Figure 6. Adaptations in the CA of SIVmac strains confer resistance to a subset of rhesus TRIM5 alleles.
(A) Partial alignment of the NTD of CA from multiple primate lentiviruses highlights the unusual QQ89,90 sequence at the tip of the CypA-binding loop, which is unique to the SIVmac lineage. Also shown is an inferred change at position 97 at the base of the loop (in helix 5), identical to the R97S change that arose in the SIVsm experimental cohorts shown in Figures 2 and 3. (B) Location of LPA89–91 and R97 on the HIV-2 CA crystal structure, highlighted in red (structure is from reference [23]). (C) Infectivity of parental SIVmac239; bars are color-coded as described in the legend of Figure 2. (D) Infectivity of SIVmac239S97R, in which S97 has been reverted to the ancestral R97, reveals a gain of sensitivity to TRIM5TFP alleles (dark blue bars). (E) Infectivity of SIVmac239QQ/LPA (in which QQ89,90 has been reverted to the ancestral LPA89–91) reveals a gain of sensitivity to TRIM5TFP alleles (dark blue bars) and TRIM5CypA (orange bars). (F) Changing the ancestral IPA to QQ in SIVsmE041 (SIVsmE041IPA/QQ) results in a gain of resistance to TRIM5CypA.
Figure 7
Figure 7. Scenario depicting the inferred role of rhesus macaque TRIM5 in emergence of SIV in captive Asian macaque colonies in the late 20th century.
Colored circles represent hypothetical populations of sooty mangabey monkeys (red circle) and rhesus macaques (blue circle). In this scenario, at the time of transmission, SIVsm was initially resistant to rhesus TRIM5Q alleles but sensitive to alleles of the TRIM5TFP and TRIM5CypA types. Initially (1), Infection of animals bearing TRIM5Q alleles (particularly TRIM5Q/Q animals) permitted high-titer, persistent infection, and the greatest potential for further transmission in the new host species. Replication in these animals also provided opportunity for adaptation to the new host. Subsequently (2), the appearance of adaptive changes in the N-terminal domain of the viral capsid protein (including S97 and QQ89,90) allowed viral replication in a larger percentage of the population and ultimately facilitated emergence of pathogenic SIVmac.
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