A POTYVIRUS POLYMERASE INTERACTS WITH THE VIRAL COAT PROTEIN AND VPG IN YEAST-CELLS

The two-hybrid system was used to test for pairwise interactions betwe en the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA po lymerase (or Nib protein) and two other TVMV-encoded proteins: the Nla protein, which consists of genome-linked protein (VPS) and proteinase domains, and the viral coat protein (CP). Using this approach, we fin d that the Nib protein interacts with both the Nla protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the Nib protein diminishes the Nlb-CP interaction but not t he Nlb-Nla interaction. Likewise, mutations in the vicinity of the Nla protein to which the genomic RNA is covalently attached eliminate the Nlb-Nla interaction. We conclude that the Nib protein interacts with the VPS domain of the Nla protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of Viral RNA synthesis in infected cells. We also conc lude that the CP interacts with the Nlb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interac tion in the functioning of the Nib protein or the CP is unclear, but m ay involve regulation of Viral RNA synthesis in infected cells. (C) 19 95 Academic Press, Inc