Background:  Hypervariable loops V1/V2 and V3 on the HIV gp120 envelope protein (Env) facilitate interactions with chemokine receptors and contain neutralizing epitopes. They also may protect conserved domains on the gp120 core from humoral immune responses. The V3 loop determines tropism and participates directly in chemokine receptor binding. Although some viruses with V1/V2 deletions are replication competent, Env lacking V3 has not been functional. We determined whether a variant of HIV-2/NIHz, termed VCP, remarkable for its CD4-independent, high-affinity binding to CXCR4, could tolerate partial or complete deletions of V3. 

Methods:  Env was constructed that contained deletions in V3 described below and evaluated in cell/cell fusion and viral pseudotype assays and on viruses. 

Results:  Env containing only the first and last 6 amino acids in V3, designated ∆V3(6,6), mediated fusion and entry, and viruses generated were infectious, albeit with slower kinetics and reduced cytopathicity. Following serial passaging, variants emerged that replicated with accelerated kinetics; cloned Env from these cultures showed increased fusogenicity. Compensatory changes included, in gp120 the loss of conserved glycosylation sites, and in gp41 mutations in heptad repeat region-1. Env clones with these mutations remained functional when V3 was fully deleted by a ∆V3(1,1) mutation leaving only the first and last amino acid, and again, infectious viruses could be generated and serially passaged. Cloned Env from these cultures also became more fusogenic and this time acquired positively charged residues in the gp120 core. Compared with the CD4-independent parental VCP, Env with V3 deletions  became strictly CD4-dependent, exhibited broader co-receptor usage, and, although remaining CXCR4 tropic, became completely resistant to AMD3100. Using a panel of CXCR4 and CXCR2 chimeras, these Env also showed increased dependence on the CXCR4 amino terminus.

Conclusions:  Thus, replication-competent HIV can be generated in the absence of V3. Because variable loops are implicated in protecting gp120 core domains from humoral immune responses, loop-deleted viruses may be useful in eliciting novel immune responses to cryptic epitopes on the Env trimer.  Further analyses of the effect of these deletions on HIV-1 and SIV isolates are underway.

Keywords: envelope glycoproteins; variable loops; viral entry