CHARACTERIZATION OF THE NUCLEIC-ACID BINDING-PROPERTIES OF TOMATO SPOTTED WILT VIRUS NUCLEOCAPSID PROTEIN

Tomato spotted wilt tospovirus (TSWV) is the type member of the plant- infecting viruses of the genus Tospovirus in the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral tr anscription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coil BL21 (DE3) cells . Full-length N, N amino and carboxy halves, and two N carboxy-termina l regions were expressed and isolated by metal chelate affinity chroma tography. The N protein, both of its halves and the extreme carboxy-te rminal region, bound cooperatively and irrespective of sequence to rad iolabeled single-stranded RNA produced by runoff transcription of clon es of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N prote in did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml , similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abi lities of Tospovirus and Hantavirus nucleocapsid proteins, the only ch aracterized nucleocapsid proteins of the family Bunyaviridae. (C) 1998 Academic Press.