Reovirus σNS and μNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection

Abstract

Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, NS and NS, and structural protein 3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed NS, NS, and 3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed NS is functional as it complements the defect in temperature-sensitive, NS-mutant virus tsE320. In both transfected and infected cells, NS is found in punctate cytoplasmic structures and 3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of NS and 3 is not altered when the proteins are expressed together or with NS. However, when expressed with NS, NS colocalizes with NS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both NS and NS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, NS interacts with NS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that NS and NS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.

Carboxyl-Proximal Regions of Reovirus Nonstructural Protein μNS Necessary and Sufficient for Forming Factory-Like Inclusions

“…The inclusions formed by NS in such experiments are notably similar in appearance to globular reovirus factories formed in infected cells, as visualized by either phase-contrast or immunofluorescence (IF) microscopy (4,7,30), suggesting that NS forms the matrix of the factories (7). Moreover, NS can associate with several other reovirus proteins and recruit them to these inclusions in transfected cells.…”

“…Results from our laboratory and others have recently shown that a single reovirus protein, NS, is sufficient for forming phase-dense globular inclusions in the cytoplasm of transfected cells (4,7). The inclusions formed by NS in such experiments are notably similar in appearance to globular reovirus factories formed in infected cells, as visualized by either phase-contrast or immunofluorescence (IF) microscopy (4,7,30), suggesting that NS forms the matrix of the factories (7).…”

“…Moreover, NS can associate with several other reovirus proteins and recruit them to these inclusions in transfected cells. To date, the viral proteins that have been reported to be recruited to inclusions formed by reovirus NS are microtubule-binding core protein 2 (7); nonstructural and ssRNA-binding protein NS (4,26); and the core surface proteins 1, 2, and 2 (6). Whole core particles released into the cytoplasm during cell entry are also recruited to NS inclusions (6).…”

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

“…Moreover, the stack images suggested that viroplasms are well-defined spherical structures. In reoviruses, it has also been reported that sNS, the non-structural protein homologous to rotavirus NSP2 with ssRNA binding activity and capacity to form higher-order homo-oligomeric structures (Huismans & Joklik, 1976;Richardson & Furuichi, 1985;Gillian & Nibert, 1998;Gillian et al, 2000), can also form inclusionbody-like structures when co-expressed with reovirus protein mNS (Becker et al, 2003). NS2 of bluetongue virus and rotavirus NSP2 have been considered to be proteins of similar function, since they share NTPase activity, non-specific ssRNA binding and localization to inclusions bodies Fillmore et al, 2002).…”

Structure and function of S9 segment of grass carp reovirus Anhui strain

“…The capsid shells are built by virus protein-protein interaction and vital to impede host cell immune clearance mechanism. The Y2H system has been become a powerful genetic approach to identify protein-protein interaction in Reoviridae [4,7,30,34]. For application in GCRV protein-protein interaction identification, GCRV873 (GCRV genotype I) NS80 is verified to interact with proteins NS38, VP4, and VP6 as well as itself, whereas no interactions involving the four protein pairs NS38-VP4, NS38-VP6, VP4-VP4, and VP4-VP6 are observed [6].…”