Background:. Peptide-based fusion inhibitors like T-20 interfere with conformational changes that normally promote HIV-1 membrane fusion by competitively binding to targets within the heptad repeat regions (HR1 an HR2) of gp41. Amino acid residues 36 - 38 within HR1 (GIV) define the level of sensitivity to T-20, but do not affect sensitivity to an overlapping peptide, T-649. When we determined the levels of T-20 and T-649 sensitivity for 29 CCR5- and 26 CXCR4-utilizing patient-derived virus that were naïve to both inhibitors, differential sensitivity (discordance) was one of the interesting phenotypes we observed. 4 discordant viruses (R11, R14, R16, and X10) were 0.8 - 1.0 log₁₀ less sensitive to T-20 than T-649, while 1 virus (X23) was unusual because it was less sensitive (by about 0.3 log₁₀) to T-649 than T-20.

Methods:. For each virus, we amplified multiple functional env clones via PCR and T/A cloned them into an expression vector for functional analysis. Next, we targeted the gp41 heptad repeat regions for sequence analysis, expecting to find substitutions in or near the GIV motif.

Results: Remarkably, all T-20-resistant clones contained the conserved GIV sequence and lacked any other features that could be clearly associated with T-20 resistance in HR1. Moreover, inspection of the X23 sequences did not reveal any features that could be immediately associated with a decreased sensitivity to T-649. Although HR1 was highly conserved among the discordant viruses and between related clones, non-conservative amino acid substitutions were observed in HR2 that could potentially affect molecular interactions between the heptad repeats.

Conclusions: These studies suggest that novel mechanisms for decreased sensitivity to T-20 and T-649 inhibition are operative in these naturally occurring viruses. Phenotypic studies are currently underway using individual clones with genotypic differences in HR2 to identify the molecular bases for the observed resistance. These studies will provide critical information about the potential for development of viral resistance to fusion inhibitors like T-20 as well as detailing the molecular interactions that drive fusion and entry using a representative and well-characterized panel of naturally occurring viruses.