Adaptive Mutations in a Human Immunodeficiency Virus Type 1 Envelope Protein with a Truncated V3 Loop Restore Function by Improving Interactions with CD4

Abstract

We previously reported that a human immunodeficiency virus type 1 (HIV-1) clade B envelope protein with a severely truncated V3 loop regained function after passage in tissue culture. The adapted virus, termed TA1, retained the V3 truncation, was exquisitely sensitive to neutralization by the CD4 binding site monoclonal antibody b12 and by HIV-positive human sera, used CCR5 to enter cells, and was completely resistant to small molecule CCR5 antagonists. To examine the mechanistic basis for these properties, we singly and in combination introduced each of the 5 mutations from the adapted clone TA1 into the unadapted envelope. We found that single amino acid changes in the C3 region, the V3 loop, and in the fusion peptide were responsible for imparting near-normal levels of envelope function to TA1. T342A, which resulted in the loss of a highly conserved glycosylation site in C3, played the primary role. The adaptive amino acid changes had no impact on CCR5 antagonist resistance but made virus more sensitive to neutralization by antibodies to the CD4 binding site, modestly enhanced affinity for CD4, and made TA1 more responsive to CD4 binding. Specifically, TA1 was triggered by soluble CD4 more readily than the parental Env and, unlike the parental Env, could mediate entry on cells that express low levels of CD4. In contrast, TA1 interacted with CCR5 less efficiently and was highly sensitive to antibodies that bind to the CCR5 N terminus and ECL2. Therefore, enhanced utilization of CD4 is one mechanism by which HIV-1 can overcome mutations in the V3 region that negatively affect CCR5 interactions.The human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) mediates sequential binding to CD4 and a coreceptor, with these interactions triggering conformational changes in Env that result in fusion between the viral and cellular membranes (2,12,66). The V3 loop in the gp120 subunit of the Env protein is thought to interact with the extracellular loops (ECLs) of the seven-transmembrane domain HIV-1 coreceptors, CCR5 and CXCR4 (9,10,28,45,51), while the base of the V3 loop and the bridging sheet region of gp120 are thought to engage the amino-terminal domains of the coreceptors (23). In addition, the V3 loop plays a major role in determining whether a given virus strain utilizes CCR5, CXCR4, or both coreceptors subsequent to CD4 binding (6, 7, 57). Perhaps because of its role in coreceptor engagement, the overall length of the V3 loop is highly conserved, as are specific residues that may play key roles in receptor binding (11,33,70). However, the V3 loop is also a target for neutralizing antibodies, making it subject to immune selection (20,25,26,44,47). In addition, the V3 loop as well as the highly variable V1/V2 region shield more conserved regions of Env that are also involved in receptor binding (16,20,33,58,59).The importance of the V3 loop for Env function is shown by the fact that genetic deletion of residues in V3 typically results in a nonfunctional Env protein (5,19,67). While V3 loopdeleted Envs ap...

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