investigation of the nonstructural proteins of mammalian reovirus serotype 3: Mu NS and sigma NS

The reovirus nonstructural proteins muNS and sigmaNS are thought to play a role in assortment. To produce large quantities of the reovirus nonstructural proteins, two recombinant vaccinia virus systems were used, one for expression of muNS and the other for expression of muNS. The reovirus gene is under the control of the phage T7 RNA polymerase promoter. A recombinant vaccinia virus containing the gene for T7 RNA polymerase was added and protein expression was determined. Protein expression was confirmed by infecting mouse fibroblast L929 cells, harvesting the cells and running an SDS-PAGE gel, followed by western blot, followed by Western-immunoperoxidase assay using a polyclonal antibody for the reovirus proteins, or by radioimmunoprecipitation followed by an SDS-PAGE gel. Purification of the recombinant proteins was accomplished by ammonium sulfate fractionation and column chromatography. The purified proteins will be utilized to further investigate the role of the reovirus nonstructural proteins in assortment.