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Reovirus NS Protein Localizes to Inclusions through an Association Requiring the µNS Amino Terminus

Department of Microbiology and Molecular Genetics,¹ Virology Training Program, Harvard Medical School, Boston, Massachusetts 02115²

Received 11 October 2002/ Accepted 23 January 2003

Cells infected with mammalian reoviruses contain phase-dense inclusions, called viral factories, in which viral replication and assembly are thought to occur. The major reovirus nonstructural protein µNS forms morphologically similar phase-dense inclusions when expressed in the absence of other viral proteins, suggesting it is a primary determinant of factory formation. In this study we examined the localization of the other major reovirus nonstructural protein, NS. Although NS colocalized with µNS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of µNS. When coexpressed with µNS, NS was redistributed and colocalized with µNS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of NS to viral factories in infected cells. We have previously shown that µNS residues 1 to 40 or 41 are both necessary and sufficient for µNS association with the viral microtubule-associated protein µ2. In the present study we found that this same region of µNS is required for its association with NS. We further dissected this region, identifying residues 1 to 13 of µNS as necessary for association with NS, but not with µ2. Deletion of NS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of NS with µNS. Furthermore, when treated with RNase, a large portion of NS was released from µNS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that µNS plays a key role in forming the reovirus factories and recruiting other components to them.

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