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

Abstract

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...

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

“…Specific regions within the NS protein that are required for recruitment of the viral core and six other MRV proteins to VFs and for forming VFs have previously been identified (6,7,9,10,12) (Fig. 1A).…”

“…We separated the TC-NS mutants based on previously identified functions of NS to include the N-terminal third (aa 1 to 221), the central third (aa 222 to 470), and the C-terminal third (aa 471 to 721). Previous studies have found that the NS N-terminal third is both necessary and sufficient to bind virus proteins NS, 2, 1, 2, and 2, the C-terminal third is necessary and sufficient to bind 3, form VFs, and bind cellular clathrin, and the central third has been implicated in recruiting cellular protein Hsc70 to VFs (6,7,9,10,12,26,27). To examine the impact of the introduced TC-NS mutations, we cotransfected cells with each pTC-NS mutant or wild-type pT7-M3 along with plasmids expressing each of the other six virus proteins individually.…”

“…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).…”

“…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). Deletion of aa 471 to 561 disrupts VFL formation, suggesting that this region also plays a role in the assembly of these structures (12).…”

“…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). Deletion of aa 471 to 561 disrupts VFL formation, suggesting that this region also plays a role in the assembly of these structures (12).…”