615 Analysis of Patient-derived HIV-1 Isolates Suggests a Novel Mechanism for Decreased Sensitivity to Inhibition by Enfuvirtide and T-649 M. Heil*1, J. Decker1,2, D. T-. Chen1, J. Sfakianos1, G. Shaw1,2, E. Hunter1, C. Derdeyn1 1Univ of Alabama at Birmingham and 2Howard Hughes Med Inst
Background: Peptide fusion inhibitors (PFI) are a promising class of anti-retroviral drugs that are proposed to prevent molecular interactions between 2 helical domains of gp41 (HR1 and HR2) necessary for virus entry. Enfuvirtide, previously T-20, is a potent inhibitor of HIV-1 replication in vivo and has successfully completed phase III trials. Enfuvirtide-resistant variants selected in vitro or in sub-optimally dosed patients (pts) frequently contain changes in amino acid residues 36-< /en>38 (glycine-isoleucine-valine, GIV) of HR1. Our previous studies showed that some PFI naïve patient (pt) isolates of HIV-1 exhibited reduced sensitivity to these inhibitors. Our hypothesis is that regions outside the HR1 domain of gp41 can modulate sensitivity to PFI.
Methods: We have determined the Env sequence of 6 pt isolates and have tested both native as well as chimeric env genes encoding the gp41 protein from PFI sensitive or resistant Envs for sensitivity to PFI and an HR1 mimicking inhibitor 5-helix.
Results: Five (5) of the 6 pt isolates showed reduced sensitivity to T20 and/or T-649 (a T-1249 analogue), but lacked changes in residues 36-< /en>38 or relevant changes in HR1. Sequence analysis of gp41 revealed that the Envs that were less sensitive to both inhibitors (R14 and X23) exhibited multiple amino acid substitutions in the N-terminus of HR2, whereas Envs with substitutions in the C-terminus of HR2 (X10) were less sensitive only to Enfuvirtide. Chimeric env genes showed that it is sequences in gp41 that modulate sensitivity to PFI. In virus infection-inhibition assays R14 was significantly less sensitive to the 5-helix inhibitor than R23, X10, or R21.
Conclusions: PFI insensitive Env variants exist in the PFI naïve population that lack changes in the GIV motif of HR1 but which have multiple changes in HR2. The results of sequencing and 5-helix inhibition support the concept that in some of these the kinetics of 6-helix formation allows escape from drug, while for other insensitive Envs a more stable HR1-HR2 interaction is likely the basis for resistance. These studies provide critical information about the potential for development of viral resistance to fusion inhibitors, as well as detailing the molecular interactions that drive fusion and entry.
