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

Abstract

Genome replication of mammalian orthoreovirus (MRV) occurs in cytoplasmic inclusion bodies called viral factories. Nonstructural protein mu NS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, mu NS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for mu NS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (lambda 1, lambda 2, lambda 3, mu 2, and sigma 2) and nonstructural protein sigma NS, can associate with mu NS 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 mu NS expression as well as, among other effects, a reduction in infectious yields by as much as 4 log(10) 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 mu NS expression and to rescue infectious yields by similar to 100-fold. We next used mu NS mutants specifically defective at forming factory-matrix structures to show that this function of mu NS is important for MRV growth; point mutations in a C-proximal, putative zinc-book motif as well as small deletions at the extreme C terminus of mu NS prevented rescue of viral growth while causing mu NS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of mu NS, designed to represent mu NSC 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 mu NS known to be involved in both mu 2 and sigma NS association. Thus, factory-matrix formation is an important, though not a sufficient function of mu NS during MRV infection; mu NS is multifunctional in the course of viral growth.