Background:  While HIV-1 viruses resistant to enfuvirtide (ENF) usually contain mutations in the HR1 region of the gp41 env glycoprotein, determinants of variation in ENF sensitivity of viruses not exposed to ENF remain unknown. We explored the relative contributions of the gp41 and gp120 env glycoproteins to ENF susceptibility in ENF naïve subjects by phenotypic testing of HIV-1 pseudovirions expressing chimeric gp41-gp120 env complexes.

Methods:  Env expression vectors were constructed that contained: the entire patient-derived env gene (gp160); only the patient-derived gp41; only the patient-derived gp120; or chimeras between the gp41 and gp120 from different patient viruses. Susceptibility to entry inhibitors and monoclonal antibodies was measured using a single cycle assay. Membrane fusion was assessed using CEM target cells that express CD4, CXCR4, and CCR5, and produce GFP and luciferase reporters upon fusion with env-expressing transfected effector cells.

Results:  A panel of env clones lacking any known HR1 resistance mutations was derived from ENF-naïve patient samples that exhibited a 100-fold range in ENF IC₅₀. Variation in ENF IC₅₀ for chimeras between gp41 from patient samples and gp120 from either HXB2 or JRCSF was approximately 40-fold, while ENF IC₅₀ for chimeras between gp120 from patient samples and gp41 from HXB2 or JRCSF varied by 5-fold. Chimeras generated by exchanging gp120 and gp41 from clones isolated from within patient samples with identical HR1 sequences but different ENF susceptibilities, also suggested that gp41 and gp120 were each partially responsible for reduced ENF susceptibility. There was no obvious effect of these chimeras on susceptibility to X4 and R5 inhibitors. To further delineate the influence of gp120 on the function of gp41, we evaluated neutralization of these chimeras by anti-gp41 monoclonal antibodies. Exchanging gp120 affected the sensitivity to both 2F5 and 4E10 to a much greater degree than ENF, confirming that gp120 sequence variation affects conformational changes in the gp120-gp41 complex. Membrane fusion activity was mapped dominantly to gp120.

Conclusions:  The diversity of HIV-1 env contributes to the natural variation of ENF sensitivity, with reduced ENF susceptibility in treatment-naive samples associated with genetic variability in both gp41 and gp120 regions. Both gp41 and gp120 affect fusion inhibitor susceptibility, and their interactions are also important to the env-mediated fusion process.