Reovirus nonstructural protein μNS recruits viral core surface proteins and entering core particles to factory-like inclusions

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein muNS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with muNS. We found that all three of these proteins-lambda1, lambda2, and sigma2-localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with muNS, on the other hand, each core surface protein colocalized with muNS in globular inclusions, supporting the initial hypothesis. We also found that lambda1, lambda2, and sigma2 each localized to filamentous inclusions formed upon the coexpression of muNS and mu2, a structurally minor core protein that associates with micro-tubules. The first 40 residues of muNS, which are required for association with mu2 and the RNA-binding nonstructural protein sigmaNS, were not required for association with any of the three core surface proteins. When coexpressed with mu2 in the absence of muNS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with mu2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of muNS that had been preformed in those cells, providing evidence that muNS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins muNS and mu2 in this process.