Background:  The HIV-1 V3 loop is an immunodominant target for neutralizing antibodies and determines tropism through poorly defined interactions with CXCR4 or CCR5. Unlike other variable loops, V3 is highly conserved in length and is typically 34 to 35 amino acids. Our lab has derived replication competent variants of HIV-2 lacking V3 and is exploring their function, immunogenicity, and susceptibility to co-receptor antagonists. In this study we determined if isolates of HIV-1 could be derived with V3 deletions.

Methods: Various V3 deletions were introduced in the dual tropic HIV-1/R3A Env and evaluated in cell fusion assays and in viruses. Viruses that could replicate with truncated V3 loops were serially passaged to improve fitness and Env clones were sequenced and characterized.

Results:  Although several deletion mutants were nonfunctional, an Env with only the first and last 9 amino acids in V3 mediated fusion and generated an infectious virus, albeit one with slower kinetics. Serial passaging yielded a virus with improved replication capacity, although it infected only CCR5⁺ cell lines. Cloned Env exhibited enhanced fusion on CCR5, but in contrast to parental R3A were unable to utilize CXCR4. Adaptive changes included a loss of 2 glycosylation sites in gp120 and an Ala to Val change at the gp41 amino terminus. Interestingly, an R3A Env with a deletion of 2 amino acids at the base of V3 was exclusively X4 tropic and unable to utilize CCR5. All Env with the ∆V3(9,9) mutation were completely resistant to small-molecule inhibitors of CCR5 (TAK779, CMPD-167, and Schering D), consistent with the model that these inhibitors act by disrupting an interaction of a distal domain of V3 with CCR5.

Conclusions:  HIV-1 can be derived with partial deletions of the V3 loop. The location of these deletions can have profound effects on X4 vs R5 tropism. These findings suggest that subdomains within V3 can be identified that contribute to the differential use of X4 and R5. Our findings also show that a deletion of the V3 crown results in resistance to small-molecule inhibitors of CCR5. These studies reveal a novel approach for analyzing Env structure and function and possibly for developing new classes of antiviral drugs that target CCR5.