Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein NS 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 NS. We found that all three of these proteins-1, 2, and 2-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 NS, on the other hand, each core surface protein colocalized with NS in globular inclusions, supporting the initial hypothesis. We also found that 1, 2, and 2 each localized to filamentous inclusions formed upon the coexpression of NS and 2, a structurally minor core protein that associates with microtubules. The first 40 residues of NS, which are required for association with 2 and the RNA-binding nonstructural protein NS, were not required for association with any of the three core surface proteins. When coexpressed with 2 in the absence of NS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with 2 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 NS that had been preformed in those cells, providing evidence that NS 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 NS and 2 in this process.

Infection with many mammalian orthoreovirus (MRV) strains results in shutoff of host, but not viral, protein synthesis via protein kinase R (PKR) activation and phosphorylation of translation initiation factor eIF2␣. Following inhibition of protein synthesis, cellular mRNAs localize to discrete structures in the cytoplasm called stress granules (SGs), where they are held in a translationally inactive state. We examined MRV-infected cells to characterize SG formation in response to MRV infection. We found that SGs formed at early times following infection (2 to 6 h postinfection) in a manner dependent on phosphorylation of eIF2␣. MRV induced SG formation in all four eIF2␣ kinase knockout cell lines, suggesting that at least two kinases are involved in induction of SGs. Inhibitors of MRV disassembly prevented MRV-induced SG formation, indicating that viral uncoating is a required step for SG formation. Neither inactivation of MRV virions by UV light nor treatment of MRV-infected cells with the translational inhibitor puromycin prevented SG formation, suggesting that viral transcription and translation are not required for SG formation. Viral cores were found to colocalize with SGs; however, cores from UV-inactivated virions did not associate with SGs, suggesting that viral core particles are recruited into SGs in a process that requires the synthesis of viral mRNA. These results demonstrate that MRV particles induce SGs in a step following viral disassembly but preceding viral mRNA transcription and that core particles are themselves recruited to SGs, suggesting that the cellular stress response may play a role in the MRV replication cycle.

Cells infected with mammalian reoviruses contain phase-dense inclusions, called viral factories, in which viral replication and assembly are thought to occur. The major reovirus nonstructural protein NS forms morphologically similar phase-dense inclusions when expressed in the absence of other viral proteins, suggesting it is a primary determinant of factory formation. In this study we examined the localization of the other major reovirus nonstructural protein, NS. Although NS colocalized with NS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of NS. When coexpressed with NS, NS was redistributed and colocalized with NS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of NS to viral factories in infected cells. We have previously shown that NS residues 1 to 40 or 41 are both necessary and sufficient for NS association with the viral microtubule-associated protein 2. In the present study we found that this same region of NS is required for its association with NS. We further dissected this region, identifying residues 1 to 13 of NS as necessary for association with NS, but not with 2. Deletion of NS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of NS with NS. Furthermore, when treated with RNase, a large portion of NS was released from NS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that NS plays a key role in forming the reovirus factories and recruiting other components to them.

Mammalian orthoreoviruses are believed to replicate in distinctive, cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. The viral nonstructural protein NS has been implicated in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly. In this study, we sought to identify the regions of NS that are involved in forming factory-like inclusions in transfected cells in the absence of infection or other viral proteins. Sequences in the carboxyl-terminal one-third of the 721-residue NS protein were linked to this activity. Deletion of as few as eight residues from the carboxyl terminus of NS resulted in loss of inclusion formation, suggesting that some portion of these residues is required for the phenotype. A region spanning residues 471 to 721 of NS was the smallest one shown to be sufficient for forming factory-like inclusions. The region from positions 471 to 721 (471-721 region) includes both of two previously predicted coiled-coil segments in NS, suggesting that one or both of these segments may also be required for inclusion formation. Deletion of the more amino-terminal one of the two predicted coiled-coil segments from the 471-721 region resulted in loss of the phenotype, although replacement of this segment with Aequorea victoria green fluorescent protein, which is known to weakly dimerize, largely restored inclusion formation. Sequences between the two predicted coiled-coil segments were also required for forming factory-like inclusions, and mutation of either one His residue (His570) or one Cys residue (Cys572) within these sequences disrupted the phenotype. The His and Cys residues are part of a small consensus motif that is conserved across NS homologs from avian orthoreoviruses and aquareoviruses, suggesting this motif may have a common function in these related viruses. The inclusion-forming 471-721 region of NS was shown to provide a useful platform for the presentation of peptides for studies of proteinprotein association through colocalization to factory-like inclusions in transfected cells.Viruses with ten-segmented, double-stranded RNA genomes from the family Reoviridae, genus Orthoreovirus, are believed to replicate in distinctive, cytoplasmic inclusion bodies (7, 10, 11, 13, 23, 24, 30, 33, 37-39, 41, 44, 45 (32,36), and flock house virus (ssRNA genome, family Nodaviridae) on the cytoplasmic face of mitochondria (27). The basis and consequences of such specific localizations are the subjects of active investigations in many laboratories. We anticipate that studies of mammalian orthoreovirus (reovirus) factories in our own laboratory will provide new insights into the still poorly characterized mechanisms for RNA packaging and replication by these and other viruses in the family Reoviridae, as well as on the general significance and mechanism of concentrating viral replication at particular sites within cells.In early studies, reovirus factories were determined to contain full...

Virally induced structures called viral factories form throughout the cytoplasm of cells infected with mammalian orthoreoviruses (MRV). When expressed alone in cells, MRV nonstructural protein NS forms factory-like structures very similar in appearance to viral factories, suggesting that it is involved in forming the structural matrix of these structures. NS also associates with MRV core particles; the core proteins 2, 1, 2, 3, and 2; and the RNA-binding nonstructural protein NS. These multiple associations result in the recruitment or retention of these viral proteins or particles at factory-like structures. In this study, we identified the regions of NS necessary and sufficient for these associations and additionally examined the localization of viral RNA synthesis in infected cells. We found that short regions within the amino-terminal 220 residues of NS are necessary for associations with core particles and necessary and sufficient for associations with the proteins 2, 1, 2, 2, and NS. We also found that only the 3 protein associates with the carboxyl-terminal one-third of NS and that viral RNA is synthesized within viral factories. These results suggest that NS may act as a cytoplasmic scaffolding protein involved in localizing and coordinating viral replication or assembly intermediates for the efficient production of progeny core particles during MRV infection.