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

Abstract

Mammalian orthoreoviruses replicate and assemble in the cytosol of infected cells. A viral nonstructural protein, NS, forms large inclusion-like structures called viral factories (VFs) in which assembling viral particles can be identified. Here we examined the localization of the cellular chaperone Hsc70 and found that it colocalizes with VFs in infected cells and also with viral factory-like structures (VFLs) formed by ectopically expressed NS. Small interfering RNA (siRNA)-mediated knockdown of Hsc70 did not affect the formation or maintenance of VFLs. We further showed that dominant negative mutants of Hsc70 were also recruited to VFLs, indicating that Hsc70 recruitment to VFLs is independent of the chaperone function. In support of this finding, NS was immunoprecipitated with wild-type Hsc70, with a dominant negative mutant of Hsc70, and with the minimal substrate-binding site of Hsc70 (amino acids 395 to 540). We identified a minimal region of NS between amino acids 222 and 271 that was sufficient for the interaction with Hsc70. This region of NS has not been assigned any function previously. However, neither point mutants with alterations in this region nor the complete deletion of this domain abrogated the NS-Hsc70 interaction, indicating that a second portion of NS also interacts with Hsc70. Taken together, these findings suggest a specific chaperone function for Hsc70 within viral factories, the sites of reovirus replication and assembly in cells. Mammalian orthoreoviruses have a genome of 10 doublestranded RNA (dsRNA) segments that are encased in a double-layered, nonenveloped capsid. The replication and assembly of reoviruses are thought to take place in distinct cytoplasmic inclusion bodies called viral factories (VFs) (33). The matrix of these structures is formed by the nonstructural viral protein NS (5). The factories are not static elements but can fuse with other viral factories in the same infected cell (J. S. L. Parker, unpublished findings). During the course of infection, other viral proteins are recruited to the viral factories at distinct times (3,8,28). By thinsection electron microscopy, the matrix of viral factories appears to consist of fibrils that have a distinct kink (9, 10, 35). However, no atomic resolution structure of NS is available. If expressed alone, without other viral proteins, the 80-kDa NS protein forms viral factory-like structures (VFLs) that resemble VFs in infected cells (5). The carboxyl-terminal (C-terminal) one-third of NS, comprising amino acids (aa) 471 to 721, is sufficient for VFL formation (2). 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 ...

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