Reovirus Nonstructural Protein μNS Recruits Viral Core Surface Proteins and Entering Core Particles to Factory-Like Inclusions

Abstract

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.

Recovery of Infectious Bluetongue Virus from RNA

“…A second possibility involves the poorly understood processes of genome packaging and genome replication, which in the Reoviridae occur within the viral inclusion bodies. The introduction of viral ssRNA into cells by transfection omits the presence of a core particle, which has been shown to be targeted to the viral inclusion bodies in the case of mammalian orthoreoviruses (2). It has been suggested that the core produces ssRNA within the inclusion body, thus supplying viral ssRNA for packaging and replication at the site of assembly of progeny cores.…”

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.…”

“…When expressed in transfected cells from a plasmid, NS forms inclusions similar to VFs that are termed viral factory-like (VFL) structures (9). 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).…”