To examine mutational pathways that lead to CXCR4 use of HIV-1, we analyzed the genotypic and phenotypic characteristics of envelope sequences from a large panel of patient virus populations and individual clones containing different V3 mutations. Basic amino acid substitutions at position 11 were strong determinants of CXCR4-mediated entry but required multiple compensatory mutations to overcome associated reductions in infectivity. In contrast, basic amino acid substitutions at position 25, or substitutions at positions 6-8 resulting in the loss of a potential N-linked glycosylation site, contributed to CXCR4-mediated entry but required additional substitutions acting cooperatively to confer efficient CXCR4 use. Our assumptions, based upon examination of patient viruses, were largely confirmed by characterizing the coreceptor utilization of five distinct panels of isogenic envelope sequences containing V3 amino acid substitutions introduced by site-directed mutagenesis. These results further define the mutational pathways leading to CXCR4 use and their associated genetic barriers.
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