Background: CD4-independent entry via CCR5 and/or CXCR4 by HIV, SIV, and FIV isolates suggests that chemokine receptors may represent the primordial receptor for these retroviruses with usage of CD4 evolving later facilitating the entry process. HIV Env, which consists of variable loops, constant domains, and extensive glycosylation, may also have gone through an evolutionary process optimizing entry and immune evading mechanisms in the context of host immune responses. To further examine the role of variable loops and conserved regions in Env function, we generated Env proteins with deletions of V1/V2 and/or V3 loops.

Methods: Using a variant of HIV-2/NIHz termed HIV-2/vcp that exhibits high affinity binding to CXCR4 and infects CD4^- cells using CXCR4, Envs were constructed containing a deletion of V1/V2 (DV1/V2), a deletion of all but the first and last 6 amino acids of V3 (DV3 [6,6]), and both deletions (DV1/V2:V3 [6,6]) and analyzed in co-receptor binding and cell-cell fusion assays. Viruses were generated with these Envs and assessed in viral replication assays.

Results: The DV1/V2 deletion alone had no effect on gp120-CXCR4 binding or CD4-independent CXCR4 fusion, and DV1/V2 virus was fully replication competent on CD4-positive SupT1 and CD4-negative BC7 cells. Remarkably, DV3 (6,6) and DV1/V2:V3 (6,6) Envs were still able to mediate fusion and viruses with these Envs could replicate on SupT1 cells although with decreased kinetics and cytopathicity compared to wild-type HIV-2/vcp. Sequential passage of these variable loop-deleted viruses generated variants displaying increased replication kinetics and cytopathicity, and envs cloned from these cultures displayed increased cell-cell fusion and enhanced replication and cytopathicity when incorporated into viruses. Interestingly, compensatory mutations associated with this phenotype were located in both gp120 and gp41. Remarkably, viruses with DV3 (6,6) were refractory to CXCR4 antagonist AMD3100 despite continued CXCR4 utilization suggesting an essential role for V3 in the mechanism of AMD3100 inhibition.

Conclusions: These results demonstrate that functional HIV Env variants can be generated lacking V1/V2 and the majority of V3 and may represent a primordial Env core. Variable loops may have evolved to evade humoral immune responses. Thus, functional Env cores with variable loop deletions may serve as better immunogens in eliciting novel immune responses against conserved regions on the gp120 core.