Novel roles for the flavivirus envelope protein in the virus life cycle

Elisa M La Bauve, Purdue University

Abstract

Flaviviruses are responsible for serious human diseases, including dengue fever, yellow fever and West Nile. They are enveloped viruses and the envelope protein (E) is thought to play an important role in the virus life cycle, the nature of which is poorly understood. The flavivirus E protein undergoes a series of conformational changes during its life cycle, starting with the immature prM-E heterodimer. In the final stage of maturation, E rearranges into sets of antiparallel homodimers arranged into sets of trimers known as E protein "rafts". This exposes a furin cleavage site on the pr portion of prM, which is then cleaved, resulting in the mature particles, which are then released. Infection of new host cells involves receptor-mediated endocytosis. Once inside the host cell endosome, the subsequent drop in pH brings about a third rearrangement of E into fusogenic parallel trimers, initiating fusion of host and viral membranes, releasing the viral genome into the host cell cytoplasm. The objective of this work was to elucidate the roles that E plays in the virus life cycle. Three aspects of E were examined. Firstly, the role of two conserved helices found within the stem anchor region of the envelope protein were examined. A series of deletions were introduced into highly conserved regions of the helices. All these deletions proved lethal. It was determined that any mutations introduced into the helices resulted in a defect in virus fusion, disrupting entry. This suggests a role for the helices in formation and stability of the fusogenic trimer during the fusion process in entry. Secondly, stabilizing residues between E protein rafts in the mature particle were examined. Regions of potential contact between rafts were identified and conserved residues from within these regions were selected for mutagenesis. Though these were all single point mutations, all proved lethal. It was determined that all these mutants were defective in maturation. This suggests the existence of a stabilizing lattice of contact points required for proper formation of the mature particle. Lastly, an in-silico screen of an NIH small molecule library identified compounds, which docked into two binding pockets between E monomers in the E dimer interface. Four compounds from this screen exhibited significant antiviral activity by disrupting the maturation process.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Molecular biology|Microbiology|Virology

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