Functions of the nonstructural protein σNS in reovirus replication

Abstract

Viral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, using in vitro and cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. High-resolution optical and electron microscopy showed that reovirus replication organelles are membranous networks and that σNS appears to facilitate the recruitment of endoplasmic reticulum membranes to these structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life and facilitates construction of replication organelles. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.