Background: The clinical success of the HIV entry-inhibitor DP-178 (T20) has generated considerable interest in the discovery of small molecules with similar antiviral activity. A major focus of this effort has involved studies to determine the mechanism of action of peptides such as DP-178 that model the HIV-1 gp41 C-helix. Previously we have shown that DP-178 binds to a fusion-active form of viral envelope and it has been proposed that this peptide blocks virus entry by interacting with the N-helical domain of gp41 and disrupting the formation or function of a 6-helix bundle structure. To test this theory we used antibodies specific for this gp41 structure to characterize the effect of DP-178 on 6-helix bundle formation.

Methods: Anti-gp41 antibodies were generated using peptides mimicking the N- and C-helical domains of gp41. Immunoprecipitation (IP) assays and immunofluorescent staining (IFS) were used to characterize the formation of the 6-helix bundle in cell surface-expressed envelope in both laboratory-adapted and primary HIV-1 isolates. These techniques were used to determine the triggers of 6-helix bundle formation and the effect of C-peptide entry inhibitors on the formation of this structure.

Results: A mixture of gp41 peptides was used to raise antibodies against structures found in fusion-active viral envelope. These antibodies were used to show that sCD4 is sufficient to trigger 6-helix bundle formation in gp41 from prototypic and primary virus isolates. Using IP assays and IFS we determined that at concentrations >100X the IC₉₀ for inhibition of virus entry C-peptides had no effect on the binding of antibody to the 6-helix bundle found in sCD4-triggered forms of gp41.

Conclusions: These results confirm the observation that sCD4-induced conformational changes in gp120 mediate the interaction of gp41 helical domains leading to formation of the 6-helix bundle. Importantly, they extend earlier results to include the primary virus isolate ADA suggesting that sCD4-induced changes in envelope structure mimic those that occur during natural infection. We also show that high concentrations of C-peptide inhibitors have no effect on the formation of the 6-helix bundle as measured by binding of antibodies specific for this structure. This result suggests that the antiviral activity of DP-178 and similar peptides does not result from a dominant-negative interaction with the N-helical domain gp41.