Reovirus σ1 conformational flexibility modulates the efficiency of host cell attachment

Abstract

Reovirus attachment protein {sigma}1 is a trimeric molecule containing tail, body, and head domains. During infection, {sigma}1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows {sigma}1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in {sigma}1 from a compact form on virions to an extended form on ISVPs. To determine the importance of {sigma}1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can crosslink {sigma}1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that crosslink the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride. Together, these data suggest that {sigma}1 conformational flexibility modulates the efficiency of reovirus host cell attachment. IMPORTANCENonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, {sigma}1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that crosslink {sigma}1 monomers in non-reducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of {sigma}1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that crosslinking the {sigma}1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that {sigma}1 conformational rearrangements mediate the efficiency of reovirus host cell attachment.