Identification of host proteins interacting with potyviral RNA-dependent RNA polymerase and investigation of the roles of the carboxy terminus of cucumber poly-(A) binding protein 1 in potyviral replication and cellular translation

Viruses rely on host factors to complete their replication and life cycles. The Potyviridae, whose members have a positive sense, single-stranded RNA genome with a viral-encoded, 3' poly-(A) tail, is the largest and most economically important plant virus family. However, host factors involved in potyviral replication have not been identified. In this work, I sought to identify such host proteins and examine how they are involved in potyviral replication and in host cellular processes. I reported here a cucumber poly-(A) binding protein, CS-PABP1, as a promising candidate that could be involved in potyviral replication. CS-PABP 1 interacted with RNA-dependent RNA polymerase (RdRp, viral replicase) of zucchini yellow mosaic potyvirus (ZYMV) in the yeast two-hybrid and in vitro binding assays. Since the 3' poly-(A) of potyviral genome is the site where the minus strand synthesis starts and ZYMV RdRp itself does not bind to poly-(A), we propose that one of the consequences of the interaction is to recruit RdRp or partially assembled replication complexes onto the poly-(A) of viral genome.;Deletion analysis indicated that the carboxy terminus of PABP (PABP-CT), which has not been previously well studied, was essential for the interaction. I provided evidence in this work that PABP-CT and its partners were involved in translational regulation. Cucumber PCI6 (P&barbelow;ABP-C&barbelow;T i&barbelow;nteracting), PCI243 and Arabidopsis ERD15 (e&barbelow;arly r&barbelow;esponsive to d&barbelow;ehydration) interacted with PABP-CT and contain a 12-amino-acid motif that is present in human PABP-CT interactors. Deletion and point mutation analyses of PCI6 indicated that the motif was necessary for the interaction. PCI6 inhibited translation in wheat germ and mouse ascites Krebs2 translation systems. A non-PABP interacting PCI6 mutant did not inhibit translation in wheat germ, and caused reduced translation in the Krebs2 system. PCI6 is a wound- and jasmonic acid-inducible protein. The above results suggested a possible translational regulation in gene expression upon stresses or other stimuli.;Like other higher eukaryotes, cucumber PABPs exist as a gene family. A second member of the cucumber PABP family, CS-PABP2, which shares 86% identity to CS-PABP1 and is expressed constitutively in different tissues, was amplified. While CS-PABP2 interacted with host PABP-CT interactors as well as that of CS-PABP1, it did not interact with ZYMV RdRp. Mutational analysis on a ZYMV RdRp region, which shares similarity to the 12-amino-acid motif, suggested that RdRp might interact with CS-PABP1 using a different motif than the host interactors.