TRIM5α Resistance Escape Mutations in the Capsid Are Transferable between Simian Immunodeficiency Virus Strains

Abstract

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.

FIG 1

TRIM5α polymorphism restricts replication of SIVsmE660 clones. Single-cycle infectivity of three SIVsmE660 clones was measured in triplicate on a panel of cell lines stably expressing rhesus TRIM5^TFP, TRIM5^Q, and TRIM5^CypA alleles. Infectivity was measured as the percentage of green fluorescent protein-positive (GFP⁺) cells. Black bars show the negative vector without TRIM5 expression as a control.

FIG 2

Capsid sequences of SIVsmE660 clones. The amino acid sequences of the Gag capsids of SIVsmE660 clones were aligned to the capsid sequence of the TRIM5α-sensitive clone SIVSmE543-3. Identical amino acids are shown as dots. Amino acid residues 37 and 98 and the region of the cyclophilin A binding loop, which are associated with TRIM5α sensitivity, are highlighted in yellow.

FIG 3

Introduction of amino acid substitutions into the SIVsmE660 clone confers resistance to TRIM5α restriction. Amino acid substitutions P37S and R98S were introduced into the capsid of the virus clone SIVsmE660-FL14 to generate SIVsmE660-FL14SS. (A to C) The single-cycle infectivity of SIVsmE660-FL14SS (B) was measured in triplicate on a panel of cell lines stably expressing rhesus TRIM5^TFP, TRIM5^Q, and TRIM5^CypA alleles and compared with those of wild-type SIVsmE660-FL14 (A) and SIVmac239 (C). (D) The infectivity of HIV-1 NL4-3 was measured as a negative control.

FIG 4

Acquisition and replication of SIVsmE660 and SIVsmE660-SS clones during the acute phase of infection in rhesus macaques. (A) Each macaque was inoculated i.r. with 1,000 TCID₅₀ of virus, and infection was monitored by measuring plasma viral RNA loads. Four weeks later, any of the macaques that remained uninfected were inoculated intrarectally on a weekly schedule with same amount of virus until they became infected. The acquisition of infection in each group is shown as a percentage of uninfected macaques after each inoculation, and groups were compared by a log rank test (P = 0.3193). (B) Median plasma viral RNA copy numbers during the acute phase of infection (up to 8 weeks postinfection) in each group are shown and were compared by two-way ANOVA (P > 0.05). (C) Peak plasma viral RNA copy numbers in each group are shown and were compared by a Kruskal-Wallis one-way ANOVA (P = 0.3523). (D) Plasma viral RNA copy numbers at set points are shown and were compared by a Kruskal-Wallis one-way ANOVA (P = 0.3413).

FIG 5

Viral loads and survival curves of SIVsmE660- and SIVsmE660-SS-infected rhesus macaques. Viral loads were quantified and are shown as RNA copy numbers in rhesus macaques. (A) SIVsmE660-SS-infected macaques with the TRIM5^Q/Q genotype. (B) SIVsmE660-SS-infected macaques with the TRIM5^TFP/TFP genotype. (C) SIVsmE660-SS-infected macaques with the TRIM5^TFP/CypA genotype. (D) Wild-type SIVsmE660-infected macaques with the TRIM5^TFP/Q genotype. (E) Cumulative survival of macaques in each group are shown as Kaplan-Meier curves and were compared by a log rank test (P = 0.134). Animals expressing an MHC allele known to be restrictive for SIVmac239 are identified by an asterisk.