Analysis of Putative Interactions Between Potyviral Replication Proteins and Plant Retinoblastoma Proteins

Sequence comparisons suggest that the RNA-dependent RNA polymerase (NIb) of potyviruses and bymoviruses, as well as the viral polymerase of potexviruses may contain a putative retinoblastoma protein (pRb) binding motif. The possibility that the potyviral NIb may function in the nucleus through interactions with plant pRb-related (RBR) proteins, and the modifications of the cell cycle was investigated by a combination of mutagenesis of the NIb and yeast two-hybrid system (YTHS). Mutation of a highly conserved glutamic acid residue in the putative pRb-binding motif of the NIb had no detectable phenotypic effect on replication of Potato virus A (PVA). Furthermore, the NIb proteins from Potato virus V and PVA failed to interact with maize or tobacco RBR proteins in yeast. Although the conservation of the motif for pRb interaction in plant RNA viruses is intriguing, these proteins from plant RNA viruses appear not to interact with plant RBR proteins.     

Tyrosine 66 of Pepper vein banding virus genome-linked protein is uridylylated by RNA-dependent RNA polymerase

Pepper vein banding virus (PVBV), a member of the genus potyvirus, is a single-stranded positive-sense RNA virus and it primarily infects plants of the family Solanaceae. Genome organization and gene expression strategy of the potyviruses are similar to the picornaviruses, although they infect widely different hosts and have distinctly different morphologies. The genomic RNA of PVBV has a viral genome-linked protein (VPg) at the 5'-terminus and a poly(A) tail at the 3'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV NIb and VPg were over-expressed in Escherichia coli and purified under non-denaturing conditions. PVBV NIb was found to be active as polymerase and it could uridylylate the VPg in a template independent manner. N- and C-terminal deletion analysis of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation. The amino acid residue uridylylated by PVBV NIb was identified to be Tyr 66 by site-directed mutagenesis. It is possible that in potyviruses, replication begins with uridylylation of VPg which acts as primer for progeny RNA synthesis.