Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5-and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by sequential interaction of the envelope glycoprotein with CD4 and one of two chemokine receptors, CCR5 or CXCR4 (1, 5, 10, 11). Most primary infections involve transmission of viruses using CCR5 as the preferred coreceptor (8,25). Evolution of coreceptor use by HIV-1 from CCR5 to CXCR4 is known to be associated with poorer clinical prognosis (3,8) and can be assumed to be one pathway leading to resistance to CCR5 inhibitors currently in clinical trials (28,30,34). Moreover, treatment with CCR5 inhibitors may select for minor populations of viruses with the ability to utilize CXCR4. Understanding the evolution of coreceptor switching in terms of the fitness costs to the virus is thus important. Although the sequence of the V3 variable loop of HIV-1 gp120 envelope is known to contribute to coreceptor use (6,14,18,42,51), sequence variation in or near the V1/V2 loop is also an important influence on coreceptor choice (13,22,23,31,39,47,48,52,53). We have previously characterized coreceptor switch mutants selected by rapid substitution of U87-CD4-CXCR4 cells for U87-CD4-CCR5 cells in vitro (32). Mutations confined to the V3 region were sufficient to alter coreceptor use for some virus envelopes, but other viruses required additional mutations in or adjacent to the V1/V2 region for successful coreceptor switching. These prior studies allowed analysis of the starting virus, an occasional intermediate, and the final succ...
