Background:  It is generally thought that CCR5 and CXCR4 are the only HIV-1 co-receptors of relevance in vivo.

Methods:  We determined the co-receptor use of HIV-1 envelopes (Env) cloned directly from plasma from infected patients taken sequentially over time. The Env surface unit (gp120) was cloned from 8 men at both seroconversion (6 to 28 days after onset of acute symptoms) and >1 year after infection. The gp120 sequences were inserted into pHxB2-MCS-D-env and viruses generated in 293T cells. Co-receptor use was assessed on the NP2 and the U87 indicator cell lines.

Results:  All patients harbored CCR5-using (non-CXCR4) viruses. However, 7 of the 8 patients harbored Env (25 of 51 clones) that could use CCR3 almost as efficiently as CCR5. Infection by CCR3 could be blocked by the CCR3 antagonist UCB35625, verifying the specificity of the interaction. We also isolated viruses from the same cohort by peripheral blood mononuclear cell (PBMC) co-culture. In contrast to the cloned Env, the PMBC isolates had a CCR5-only phenotype. One patient progressed toward disease development (CD4 <200/mL, viral load increasing). Env amplified at this time point differed markedly from the earlier R5R3 phenotype, being either R3R5X4- or R5-only-using.

Conclusions:  Virus isolation by PBMC co-culture favors the selection of CCR5-only-using viruses. Our results suggest a selective pressure in vivo to maintain CCR3 use.