284 HIV-1 Fusion Inhibitors Bind to the N-Helical Coiled-coil Domain in Receptor-activated gp41 N. Kilgore, K. Salzwedel, M. Reddick, G.Allaway, C. Wild* Panacos Pharm, Gaithersburg, MD
Background: While it has been established that peptides modeling the C-helical region of HIV-1 gp41 are potent in vivo inhibitors of virus replication a number of questions concerning their mechanism of action, including their specific molecular target, remain unanswered. It has been proposed, but never directly demonstrated that these peptides block virus entry by interacting with the N-helical coiled-coil of gp41 to disrupt the formation or function of a 6-helix bundle structure. Using a 6-helix bundle-specific monoclonal antibody with isolate restricted Env reactivity, we provide the first direct evidence that in receptor-activated viral Env, C-peptide entry inhibitors bind to the gp41 N-helical coiled-coil to form a peptide/protein hybrid structure and in doing so disrupt native 6-helix bundle formation.
Methods: Lysate immunoprecipitation assays were used to characterize both the Env and 6-helix bundle binding specificity of a murine monoclonal antibody generated against rgp140. A surface immunoprecipitation assay employing either soluble CD4 or cell-associated receptors was used to characterize Mab binding to a receptor-activated form of gp41. The C-peptide was modeled after the HIV-1 LAI envelope sequence.
Results: We demonstrated that the Mab specifically bound to a conformational epitope within the gp41 6-helix bundle in an isolate restricted manner. Using the appropriate combination of C-peptide and virus we were able to rescue binding of our restricted Mab to gp41 from a non-reactive isolate. These results demonstrate that C-peptides bind specifically to the N-helical coiled-coil domain of receptor-activated gp41 to form a hybrid 6-helix bundle.
Conclusions: In a previous study, we established that C-peptide entry inhibitors bound to an unidentified gp41 target. Here we extend that work by using a 6-helix bundle-binding Mab with isolate restricted Env reactivity to provide direct evidence for C-peptide binding to the N-helical coiled-coil domain of receptor-activated gp41. Specifically, we show that a C-peptide modeled from a Mab-reactive viral isolate can interact with receptor-activated gp41 from a non-reactive isolate to form a hybrid 6-helix bundle and rescue antibody binding. Although the specific interaction of C-peptide inhibitors with the N-helical domain of gp41 has been one of several mechanisms proposed to account for the antiviral activity of C-peptides this report provides the first direct proof of this binding.
