Formation of the factory matrix is an important, though not a sufficient function of nonstructural protein μNS during reovirus infection

Genome replication of mammalian orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein μNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, μNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for μNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (λ1, λ2, λ3, μ2, and σ2) and nonstructural protein σNS, can associate with μNS in these structures. In the current study, small interfering RNA targeting M3 was transfected in association with MRV infection and shown to cause a substantial reduction in μNS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log10 values. By also transfecting in vitro-transcribed M3 plus-strand RNA containing silent mutations that render it resistant to the small interfering RNA, we were able to complement μNS expression and to rescue infectious yields by ~ 100-fold. We next used μNS mutants specifically defective at forming factory-matrix structures to show that this function of μNS is important for MRV growth; point mutations in a C-proximal, putative zinc-hook motif as well as small deletions at the extreme C terminus of μNS prevented rescue of viral growth while causing μNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of μNS, designed to represent μNSC and still able to form factory-matrix structures, is unable to rescue MRV growth, localizing one or more other important functions to an N-terminal region of μNS known to be involved in both μ2 and σNS association. Thus, factory-matrix formation is an important, though not a sufficient function of μNS during MRV infection; μNS is multifunctional in the course of viral growth.