Background:  Hypervariable loops V1/V2 and V3 on HIV gp120 are immunodominant, contain type-specific neutralizing epitopes, and protect conserved core domains from humoral immune responses. The V3 loop also contributes to interactions with chemokine receptors and largely determines X4 or R5 tropism. We reported that a CD4-independent variant of HIV-2/NIHz, termed VCP, could tolerate a partial deletion of its V3 loop while remaining replication competent. With a goal of deriving functional HIV Env lacking these domains as novel immunogens, we determined whether this Env could be used to derive a virus that could replicate without V1/V2 and V3 in combination.

Methods:  HIV-2/VCP Env were constructed that contained V1/V2 and/or V3 deletions and evaluated in cell–cell fusion and viral pseudotype assays. An iterative process of viral adaptation, Env cloning and further mutagenesis was used to derive Env and viruses that could tolerate additional loop deletions while remaining functional.

Results: An Env with only the first and last 6 amino acids in V3 was able to mediate fusion and entry, and viruses with this Env were infectious, albeit with slower kinetics and reduced cytopathicity. Serial passaging yielded viruses with improved growth capacity, and an Env clone was derived from which all but the first and last amino acids of V3 were deleted. After another round of adaptation, Env cloning and mutagenesis, a fusion-competent Env and a replication-competent virus were obtained that lacked V1/V2 and V3 in combination. Adaptive changes included in gp120 a loss of conserved glycosylation sites and in gp41 mutations in HR-1. This “gp120 core” Env exhibited CD4-dependent tropism for both X4- and R5-expressing cells and could utilize additional co-receptors from CXC and CC chemokine receptor families. Interestingly, all V3-deleted viruses became completely or partially resistant to small molecule inhibitors of CXCR4 (AMD3100, T140) and CCR5 (TAK779, Compound167) suggesting that these inhibitors act by disrupting a V3 loop-co-receptor interaction.

Conclusions:  Our findings represent a proof of concept that replication-competent HIV lacking V1/V2 and V3 can be derived. Because variable loops are implicated in protecting gp120 core domains from antibody responses, loop-deleted HIV may be useful for producing novel immunogens that can focus humoral responses to conserved and/or cryptic epitopes on the Env trimer. Efforts to extend this work to HIV-1 are underway.

Keywords: hypervariable loops; hiv gp120; chemokine receptor inhibitors