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 microNS, encoded by genome segment M3, is a major constituent of these structures. When expressed without other viral proteins, microNS forms cytoplasmic inclusions morphologically similar to factories, suggesting a role for microNS as the factory framework or matrix. In addition, most other MRV proteins, including all five core proteins (lambda1, lambda2, lambda3, micro2, and sigma2) and nonstructural protein sigmaNS, can associate with microNS 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 microNS 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 microNS expression and to rescue infectious yields by ~100-fold. We next used microNS mutants specifically defective at forming factory-matrix structures to show that this function of microNS 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 microNS prevented rescue of viral growth while causing microNS to be diffusely distributed in cells. We furthermore confirmed that an N-terminally truncated form of microNS, designed to represent microNSC 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 microNS known to be involved in both micro2 and sigmaNS association. Thus, factory-matrix formation is an important, though not a sufficient function of microNS during MRV infection; microNS is multifunctional in the course of viral growth.

Identification of Functional Domains in Reovirus Replication Proteins μNS and μ2

“…Our findings and those previously reported by Arnold et al (2) show that NSC, though capable of selfassembly into inclusion-like structures, cannot support viral replication. Such aspects of NS structure-function relationships point to compulsory heterointeractions between NS and 2 and NS proteins at one or more steps in the viral RNA life cycle, perhaps recruitment or retention of (ϩ)-strand RNAs at sites of viral replication, dsRNA synthesis, or genome packaging.…”

“…Minimum sequences required for autoassembly of NS protein into inclusion-like structures are contained within the 161 to 251 C-terminal amino acids (9). The C-terminal two to eight amino acid residues and a putative metal-chelating motif (perhaps selective for Zn 2ϩ ) involving His 570 and Cys 572 appear to play critical roles in this process (2,9). These sequences may contribute to a dimerization domain perhaps involved in NS homotypic or heterotypic interactions required to nucleate inclusions (9).…”

“…Viral inclusions contain dsRNA (56), viral proteins (19), and both complete and incomplete virion particles (19). The viral nonstructural proteins NS and NS and minor core protein 2 are collectively required for the genesis and maturation of viral inclusions in reovirus-infected cells (2,28). Expression of these proteins from cloned cDNAs in the absence of infection results in spontaneous assembly of intracytoplasmic structures exhibiting inclusion morphology (6,39).…”

“…The N-terminal 40 residues of NS contain interacting domains for 2 and NS (12,39), neither of which associates with NSC (12,39). Using RNA interference (RNAi)-based trans-complementation approaches, NSC was found to be incapable of supporting viral growth in NSsilenced cells (2,28), pointing to the significance of interactions between NS, 2, and NS for viral replication. However, the concerted activities of NS and other replication proteins within inclusions have not been defined in structural or functional terms.…”

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

“…Moreover, NS formation of VFLs results in specific VFL localization of each of the five viral structural proteins that make up the core particle ( 1, 2, 3, 2, and 2), the nonstructural NS protein that is involved in virus translation and replication, as well as the intact core particle itself (6,(9)(10)(11). The mechanism of VFL formation by NS is not fully understood; however, several regions of the protein, including the carboxyl (C)-terminal 7 amino acids (aa) and a putative metal chelating structure formed by amino acids His570 and Cys572, have been shown to be necessary for VFL formation in transfected cells and replication in infected cells (12)(13)(14). Additionally, the C-terminal 250 amino acids (aa 471 to 721) of NS, which comprise a predicted coiled-coil domain, are sufficient to form VFL structures (12,15).…”