Biophysical Investigation of the Intermediate Steps of Flavivirus Membane Fusion

• Grant number: 08/01636-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2008
• End date: June 30, 2010
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Paolo Marinho de Andrade Zanotto
• Grantee: Ana Vitória Botelho
• Host Institution: Associação Brasileira de Tecnologia de Luz Síncrotron (ABTLuS). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

Dengue virus is a member of the Flaviviridae family, genus flavivirus, and constitutes one of the most serious emerging viruses. Protein E is the major surface protein of the viral envelope and is responsible for the entry of the virus in the host cell. Virus entry occurs through the binding of protein E (envelope) to specific receptors. In this step, protein E is found as dimer positioned flat in an anti-parallel arrangement on the viral surface. After binding, the virus enters cells by receptor-mediated endocytosis inducing conformational changes in the protein, going from dimer to monomers. In the monomer form, protein E exposes its fusion loops, inserting them into the cellular membrane promoting a subsequent trimer formation, which catalyzes membrane fusion of the virus with the host cell. This fusion process creates a fusion pore, through which the virus genome is delivered into the cytoplasm of the host cell, leading to subsequent infection. The present project aims to investigate the mechanisms involved in the conformational changes of the fusion protein E, to better understand the mechanistic details associated with the fusion steps of flaviviruses. This project is based on examining the conformational changes and the oligomeric states of the E protein by thermodynamic and kinetic analysis, using as model systems the ectodomains sE, as well as the integral protein E. In addition, full-length E will be expressed and inserted into liposomes to correlate the kinetics of membrane fusion with lipid identity. This work presents experiments to deepen our understanding in the functions of the Type II fusion proteins, with repercussions for basic science as well as eventual assay development for drug discovery against Dengue and related flaviviruses.