Abstract
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 mu NS 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, sigma NS. Although sigma NS colocalized with mu NS in viral factories during infection, it was distributed diffusely throughout the cell when expressed in the absence of mu NS. When coexpressed with mu NS, sigma NS was redistributed and colocalized with mu NS inclusions, indicating that the two proteins associate in the absence of other viral proteins and suggesting that this association may mediate the localization of sigma NS to viral factories in infected cells. We have previously shown that mu NS residues 1 to 40 or 41 are both necessary and sufficient for mu NS association with the viral microtubule-associated protein mu 2. In the present study we found that this same region of micro NS is required for its association with sigma NS. We further dissected this region, identifying residues 1 to 13 of mu NS as necessary for association with sigma NS, but not with mu 2. Deletion of sigma NS residues 1 to 11, which we have previously shown to be required for RNA binding by that protein, resulted in diminished association of sigma NS with mu NS. Furthermore, when treated with RNase, a large portion of sigma NS was released from mu NS coimmunoprecipitates, suggesting that RNA contributes to their association. The results of this study provide further evidence that mu NS plays a key role in forming the reovirus factories and recruiting other components to them.
Highlights
The nonfusogenic mammalian orthoreoviruses are double-stranded RNA viruses that contain 10 genome segments encased by a multilayered protein capsid
When coexpressed with the filamentous form of 2, NS colocalizes with 2 on microtubules in a pattern very similar to that seen during infection (9). These findings have led us to hypothesize that while 2 plays an important role in anchoring the factories to microtubules, NS is the primary determinant of factory formation and may be involved in recruiting other components required for RNA assortment, minus-strand synthesis, and core assembly (9)
Recombinant NS expressed in baculovirus-infected cells is found in similar large nucleoprotein complexes, which dissociate into 7- to 9S complexes upon treatment with RNase A or high-salt washes, suggesting that NS forms small oligomers in the absence of RNA (16). NS any implications for the roles of NS (and NS), as well as the structural protein 3, have been isolated from infected cells in complexes with viral ssRNA, leading to the proposal that these proteins are involved in preparing the viral transcripts for minus-strand synthesis and packaging into progeny cores (2). NS has been recently proposed to nucleate viral factories, based on evidence that it localizes to factories throughout infection and that a viral mutant with a temperature-sensitive NS protein is defective for factory formation at restrictive temperatures (5)
Summary
Fresh DMEM was added to the cells, and they were incubated for a further 14 h at 37°C before preparing for IP For both immunostaining and IP experiments, cells in dishes or on coverslips were inoculated with T1L or T3DN reovirus at a multiplicity of 10 PFU per cell in phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 [pH 7.5]) supplemented with 2 mM MgCl2. Immunostained cells were washed a final three times with PBS, incubated for 5 min in 300 nM 4Ј,6-diamidino-2-phenylindole, and mounted on slides with Prolong reagent (Molecular Probes). Immunoprecipitating antibodies that had been incubated for 2 h with protein A-conjugated magnetic beads (Dynal Biotech, Lake Success, N.Y.) and washed six times with Raf buffer were added to the cell lysates, which were incubated, rotating, overnight at 4°C. After centrifugation at 13,000 ϫ g, supernatants were removed from the pellets and sample buffer was added to both supernatants and pellets, which were boiled and loaded on SDS-PAGE gels
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