Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Computational and Molecular Biophysics

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Daved Fremont

Abstract

Flaviviridae are a family of enveloped, positive-stranded RNA viruses responsible for a variety of diseases including encephalitis, hemorrhagic fever and hepatocellular carcinoma. The envelope: E) proteins that coat the outer surface of these viruses provide the molecular machinery that drives receptor interaction and membrane fusion. The assignment of biological functions to specific structural elements of these E proteins has proven crucial to the understanding of viral entry into host cells. Clearance is dependent upon the presence of neutralizing antibodies that are able to disrupt several stages of this process. Given their fundamental role in the viral life cycle, we sought to determine the structural basis for envelope protein interaction with antibodies and receptors for human pathogens of the Flaviviridae family Japanese Encephalitis Virus, Hepatitis C Virus and St. Louis Encephalitis Virus. Viruses of the Flavivirus genus within Flaviviridae are grouped into serocomplexes with similar clinical manifestations that are defined by cross-neutralization tests with polysera from heterologous infections. Japanese Encephalitis Virus: JEV) is the leading cause of viral encephalitis and prototypical member of the JEV serocomplex. We determined the 2.1Ã… resolution crystal structure of the JEV E protein ectodomain to investigate whether structural features could contribute to our understanding of serocomplex-specific pathogenesis. JEV E possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies revealed residues localized to the domain I lateral ridge, fusion loop, domain III lateral ridge and domain I-II hinge. The dimer interface, however, is remarkably small and lacks several contacts present in other flavivirus E homodimers. Uniquely conserved histidines of the JEV serocomplex suggest that pH-mediated structural transitions may be assisted by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation of dimer structure and stability may influence the assembly, receptor interaction and uncoating of virions. St. Louis Encephalitis Virus: SLEV) is another member of the JEV serocomplex with similar pathogenesis to JEV. We determined the 4.0 Ã… structure of the SLEV E protein in the post-fusion trimer conformation to compare it with E trimer structures from other flavivirus serocomplexes. SLEV E crystallized as a trimer in the absence of lipids or detergents, requiring only low pH. However, its domain arrangement was nearly identical to other post-fusion structures. This suggests that viruses can alter dimer assembly but the structure of the activated, fusogenic conformation may be more strictly conserved. The only member of Flaviviridae known to chronically infect humans is Hepatitits C Virus: HCV). HCV is blood borne and carried by roughly 3 percent of the world's population. Clinical manifestations include hepatitis, cirrhosis and hepatocellular carcinoma. HCV envelope protein E2 mediates interaction with host receptors CD81 and scavenger receptor BI: SR-BI) and is the primary target of neutralizing antibodies. To elucidate detailed biochemical roles for these receptors' interactions with E2, we determined that the E2 ectodomain: sE2) interacts with soluble CD81 large extracellular loop: CD81-LEL) with 2:2 stoichiometry, and that this interaction inhibits subsequent engagement of SR-BI. We then evaluated the affinity and kinetics of sE2:CD81-LEL binding. Interaction between these proteins was enhanced by deletion of hypervariable region 1: HVR1) of E2 and modulated by the genotype from which sE2 was generated. Furthermore, neutralization of HVR1-deleted HCV by a cross-reactive antibody was enhanced in a genotype-specific manner that correlated with sE2:CD81-LEL affinity measurements. Our results suggest that E2 cannot engage CD81 and SR-BI simultaneously, that HVR1 obscures conserved CD81 and antibody binding sites, and that genotypic variation influences HCV host receptor preference.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7DR2SG3

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