Localization of Mammalian Orthoreovirus Proteins to Cytoplasmic Factory-Like Structures via Nonoverlapping Regions of μNS

Abstract

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.

Analysis and application of the SRBSDV P9-1 octamer crystal structure for the target of α-amino phosphonate derivatives

“…Inmammalian genera of the family reoviridae, μNS is sufficient to form VFs (viral factories) [24], and it recruits σNS to VFs [27]. The μNS protein acts as a scaffold to recruit viral core surface proteins [25], while σNS binds preferentially to ss-RNA and ss-DNA [26,27]. In members of the genus fijivirus such as RBSDV, nonstructural protein RBSDV P9-1 is important for the functioning of the relative proteins in Vps during viral morphogenesis [28], RBSDV P9-1 self-associates into aggregate formations and preferentially binds ss-RNA molecules [29,30].…”

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.…”

“…During MRV infection, inclusion bodies called viral factories (VFs) form, which were first identified in 1962 and later described as small particles that coalesced to form a reticulum-like structure around the nucleus (2,3). VFs have historically been hypothesized to be the location of viral replication and assembly of viral core particles (4,5), and recent data showing core particle recruitment and positive-strand RNA synthesis at VFs (6,7) and translation of viral RNA within and around VFs (8) suggest that they are also the site of viral transcription and translation.…”

The Cellular Chaperone Hsc70 Is Specifically Recruited to Reovirus Viral Factories Independently of Its Chaperone Function

“…This minimal factory-forming region has two predicted coiled-coil domains linked by a putative zinc hook and followed by a short C-terminal tail (26). The first one-third of NS (aa 1 to 221) has been identified as a scaffold for the recruitment of the viral proteins 1, 2, 2, 2, and NS; in contrast, the RNA-dependent RNA polymerase (RdRp), 3, interacts with the C-terminal minimal factory-forming region (5,27,28). So far, no function has been elucidated for the middle portion of NS (aa 222 to 470).…”