Studies of HCV Infection, Vaccine Development and HCV-Host interactions

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral life cycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we identified many previously unrecognized host factors required for productive HCV infection. Despite the development of highly effective HCV treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins E1 and E2 in the entry process with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by the X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here we analyze the structure of recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies as well as a small molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. 3D models were generated after extensive 2D classification analysis with negative-stain single particles datasets. We modeled the available crystal structures of the E2c and Fabs into the 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body presumably depicting the E1 and E2 stem/transmembrane domain and a protruding structure representing the E2c based on anti-E2 Fab binding. At low resolution, model generated from negatively stained analysis reveals the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of double Fab complexes resulting in a refined structural model of the E1E2 heterodimer is presented.