Expression of the Bunyamwera Virus M Genome Segment and Intracellular Localization of NSm

Abstract

Bunyamwera (BUN) virus is the prototype of the family Bunyaviridae and contains a trisegmented, single-stranded RNA genome of negative polarity. The medium (M) RNA segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, in the form of a polyprotein precursor which is cotranslationally cleaved. The gene order of the M segment is 5′ G2-NSm-G1 3′. We have raised a monospecific antiserum in rabbits to a branched chain synthetic peptide to a region of the NSm protein which specifically immunoprecipitates NSm from BUN-infected cells. Indirect immunofluorescence experiments on BUN-infected cells using this antiserum gave a perinuclear staining pattern, suggesting that like the viral structural proteins, NSm localizes to the Golgi complex. An essentially full-length M segment cDNA was cloned into a recombinant vaccinia virus under control of bacteriophage T7 promoter and terminator sequences and expressed in cells co-infected with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. G1, G2, and NSm were detected in cells dually infected with the recombinant vaccina viruses, indicating that processing of the M segment-encoded precursor does not require other BUN proteins. Immunofluorescence experiments showed that the BUN glycoproteins expressed from this recombinant vaccinia virus system localized to the Golgi complex like authentic BUN proteins.