Interaction between Tomato spotted wilt virus N protein monomers involves nonelectrostatic forces governed by multiple distinct regions in the primary structure

The ambisense RNA genome of Tomato spotted wilt virus (TSWV) is packaged by interaction with numerous copies of the virus encoded nucleocapsid (N) protein to form a subvirion structure called a ribonucleoprotein (RNP). RNPs are central to the viral replication cycle because they, and not free viral RNA, serve as templates for viral gene expression and genome replication. N protein monomers bind to viral RNA molecules in a cooperative manner. We have examined regions of the N protein that are involved in the N-N interactions that likely contribute to the cooperative binding of N to viral RNA. We created random and alanine scanning mutants of N and then screened the mutants for defects in N-N interaction using reverse and forward yeast two-hybrid assays. Our experiments identified residues in three distinct regions of the primary structure of the protein, residues 42 to 56, 132 to 152, and in the C-terminal 26 amino acids, that contribute to N-N dimerization or multimerization. The interactions between N monomers mediated by the residues we identified are of a nonelectrostatic nature.