Characterization of the biological activities of the tomato bushy stunt virusp19 gene.

摘要

The objectives of this study were to identify and characterize RNA elements of the Tomato bushy stunt virus (TBSV) p19 gene and the activities of its protein. For the first objective, deletion analyses identified two regions within the p19 gene involved in efficient TBSV RNA accumulation. One of these is a 79 nucleotide (nt) region termed IE (Inhibitory Element) residing in the 5′ half of the 3′-proximal overlapping p19 and p22 genes. The other RNA element, encompassing the 3 ′ end of the p19 and p22 genes, is divided into two regions that are termed SUR-III (Sequence Upstream Region III) and III-A (5 ′ portion of region III). Computer-aided RNA structure prediction identified two possible hairpins in each region that can potentially interact with each other due to the presence of complementary nucleotide sequences. The deletion of either of these two hairpins prevents efficient viral RNA accumulation, indicating that the co-existence of these secondary structures is required for TBSV replication. Deletion analysis indicates that the inhibitory activity of IE exerts its influence only in the absence of SUR-III Computer-aided sequence and structure prediction showed that the regions corresponding to SUR-III and III-A are conserved among members in the Tombusvirus genus.; For the second objective, immunocytological studies were performed which showed that TBSV is localized in the xylem in Nicotiana benthamiana , pepper and spinach in addition to its accumulation in the phloem. Girdling assays to investigate xylem usage for systemic infection was inconclusive. However, six of twelve and four of nine girdled-N. benthamiana infected with TBSV and Potato virus X showed systemic infection, respectively.; For the third objective, it was shown with a yeast two-hybrid system and a viral protein copurification strategy that no other viral proteins interact with P19. However, the yeast two-hybrid system confirmed P19 self-interaction, for which the arginine at position 72 was critical. Comparisons of the biochemical and infectivity tests indicated that the P19 self-interaction may be responsible for both symptom severity and TBSV movement. These assays confirmed that the self-interaction of P19 is biologically significant.