Background:  Most HIV-1 transmissions result in a predominantly R5 virus infection. With time, X4 variants arise and coexist with R5 virus variants in ~50% of subtype B-infected individuals. Whether X4 viruses evolve during the course of infection or are transmitted but selected against early in infection is unclear.

Methods:  We tested the hypothesis of X4 virus evolution by infection of rhesus macaques with an isolate recovered at late stage disease of an R5 simian-human immunodeficiency virus (SHIV) -infected animal. We reasoned that because this isolate, designated SHIV_SF162P3N, is more diverse and divergent, it would allow for rapid evolution of the viral population, which may lead to a change in co-receptor preference. We monitored the level of virus replication and CD4⁺ T-cell loss, as well as the genotype and phenotype of circulating viruses in rhesus macaques infected intravenously with R5 SHIV_SF162P3N.

Results:  Of 3 macaques infected with SHIV_SF162P3N, 1 showed precipitous peripheral CD4⁺ T-cell if I drop at 16 weeks post infection. This animal failed to seroconvert and at the time of euthanasia at 24 weeks post infection with clinical signs of simian AIDS, the circulating CD4⁺ T-cell count was 1 cell/mL blood with almost complete depletion of CD4⁺ T lymphocytes in all tissue compartments examined. Sequence analysis of virus recovered at necropsy revealed an increase in the overall positive charge of the V3 loop and loss of a highly conserved glycosylation site at the N-terminus of the loop. Infection of rhesus peripheral blood mononuclear cells (PBMC) in vitro with the recovered virus was blocked by CXCR4, but not CCR5 inhibitor, while infection of rhesus macaques in vivo induced rapid and dramatic peripheral and lymphoid CD4⁺ T-cell loss, features characteristic of X4 SHIV infection.

Conclusions:  We report here the first case of X4 virus evolution in a R5 SHIV-infected rhesus macaques, demonstrating that co-receptor switch can happen in a non-human primate model of HIV/AIDS. Co-receptor switch in macaques appears to require a process of multiple mutation accumulation, and is associated with envelope sequence changes similar to those reported in humans, suggesting that the R5 to X4 evolution pathways in the 2 hosts overlap. The finding of X4 emergence in a macaque with low virus-specific immune responses lends support to a role for antiviral immunity in suppressing HIV-1 co-receptor switch.