Association of reovirus proteins with the structural matrix of infected cells

The interaction of reovirus with the cytoskeleton was investigated. The soluble components of infected cells were extracted with the nonionic detergent NP-40 in a physiological buffer, and a cytoskeletal extract was prepared from the detergent-insoluble fraction. We observed a selective association of viral-specified products with the cytoskeleton that was temporally controlled. Viral dsRNA appeared first on the framework but after several hours was found also in the soluble phase, encapsidated in mature virions. The initial viral translation products were associated exclusively with the soluble fraction, but concomitant with the appearance of dsRNA, viral proteins microNS and sigma 3 were detected on the cytoskeleton. Several hours later, all viral proteins were detected on the framework. Viral polypeptide microNS exhibited unique spatial distribution patterns that correlated with viral assembly: Before dsRNA replication, it appeared as diffusely distributed protein; a few hours later, it was detected in punctate foci interconnected by tiny filaments; several hours later, it appeared as an extensive fiber network that traversed the foci. The other viral proteins were detected only within viral foci. MicroNS remained bound to the matrix fraction after treatment with DNase, Mg2+, and high salt, treatments that released other viral proteins. This distribution pattern was virus-directed because passage of virus at high multiplicity of infection induced mutations that prevented assembly of the microNS-coated filament organization. A small fraction of the viral-specified products that included polypeptide microNS, but not viral dsRNA, was coprecipitated from cytoskeletal extracts with proteins of mol wt approximately 55K by monoclonal antibodies that recognized tubulin and vimentin. Disruption of this interaction by long exposure to colchicine did not prevent association of viral proteins or RNA with the matrix, indicating that viral products were not transported through these interactions. The results indicate that reovirus morphogenesis includes temporal and spatial controls not described previously.