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 between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb–CP interaction but not the NIb–NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb–NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa 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 conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells.