Capsid-membrane and capsid-glycoprotein interactions in alphavirus budding

Thomas Anthony Wilkinson, Purdue University

Abstract

Alphaviruses are small enveloped viruses that have a wide variety of insect and mammalian hosts. The final assembly of these viruses within infected cells is thought to be directed by interactions between the icosahedral nucleocapsid and viral spike proteins, which are embedded in the plasma membrane. The critical determinants for viral assembly were explored initially in solution using NMR spectroscopy, but more effectively using a biochemical binding assay that employed artificial vesicles to simulate the plasma membrane. In this binding assay, constructs of Sindbis virus capsid protein were examined for binding to liposomes containing synthetic peptides corresponding to the cytoplasmic domain of the E2 envelope glycoprotein. Prior to reconstitution into these liposomes, the synthetic E2 peptides were modified with C16 alkyl groups at cysteine positions, thus mimicking the E2 glycoprotein palmitoylation found in vivo. The binding assay demonstrates a direct interaction between these reconstituted synthetic peptides and a Sindbis virus capsid construct containing residues 19-264. These binding assays also show an interaction between Sindbis capsid protein 19-264 and liposomes containing neutral or negatively-charged lipids. Shorter capsid protein constructs do not show high affinity for membranes or to reconstituted E2 peptides. The results of these experiments elucidate protein sequence requirements for the binding of Sindbis virus capsid protein to the cytoplasmic domain of the E2 glycoprotein. The membrane-binding properties of the capsid protein further suggest that the plasma membrane may play an active role in alphavirus assembly by recruiting capsid protein to sites in the cell where capsid-E2 interactions are favored. Finally, a study to evaluate methods for translating nuclear Overhauser effect measurements into distance constraints for NMR structure calculations has been initiated, and the progress of this study to date is described. The results from this study should have wide relevancy to structural problems that would be tackled by NMR, including the study of a Sindbis virus capsid-cdE2 complex.

Degree

Ph.D.

Advisors

Post, Purdue University.

Subject Area

Microbiology|Molecular biology

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