Structure-function analysis of mammalian orthoreovirus attachment protein ó1

Abstract

To efficiently initiate infection in target cells, viruses must become tethered to the plasma membrane by engaging cellular receptors. As the first step in the viral replication cycle, attachment is a key determinant of viral tropism and pathogenesis outcomes. The adhesion process is often mediated by a viral attachment protein and can involve binding to multiple receptors at the cell surface. Mammalian orthoreoviruses (reoviruses) are non-enveloped, double-stranded RNA viruses that serve as tractable models for studies of virus-receptor interactions and viral pathogenesis. Reoviruses also show potential as vectors for vaccine delivery and oncolytic therapeutics. The reovirus attachment protein, ó1, is a filamentous, trimeric molecule that extends from the icosahedral vertices of reovirus virions. Reovirus ó1 of some serotypes binds á-linked sialic acid, a carbohydrate, and ó1 of all serotypes binds junctional adhesion molecule-A (JAM-A), a dimeric protein involved in cell-cell adhesion and tight junction formation. Goals of my dissertation research have been to elucidate details of the mechanism of ó1-JAM-A engagement and understand features of ó1 that confer functionality. I characterized a novel trimerization motif, the aspartic acid sandwich, consisting of an unusual cluster of six aspartic acid residues sandwiched between residues with aromatic and hydrophobic side chains at the subunit interface of the ó1. This motif is likely to be important in mediating structural rearrangements in ó1. I systematically analyzed the contributions of individual residues in the JAM-A dimer interface to reovirus binding and JAM-A homodimer stability. Residues with charged side chains play critical roles in stabilizing both interactions. In addition, I report the structure of a complex between ó1 and JAM-A and identify sequences in ó1 required for efficient JAM-A binding and reovirus infectivity. These studies enhance an understanding of the contributions of structural features of ó1 and JAM-A to reovirus pathogenesis outcomes in vivo and provide a basis for vector retargeting as we explore the potential of reovirus for vaccine delivery and oncolysis.