Year of Publication
Doctor of Philosophy (PhD)
Dr. Peter D. Nagy
Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In my thesis I describe (i) the effect of phosphorylation of p33, (ii) the RNA chaperone-like activity of p33, and (iii) the role of HSP70s a host proteins in the viral replication. To test the effect of phosphorylation on p33 function, I used in vitro phosphorylated p33. I found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. Phosphorylation-mimicking mutations rendered p33 nonfunctional in plant protoplasts and in yeast.
Based on these results, I propose that the primary function of phosphorylation of p33 is to regulate its RNA binding capacity, which could affect the assembly of new viral replicase complexes, recruitment of the viral RNA template into replication and/or release of viral RNA from replication. Thus, phosphorylation of p33 might help in switching the role of the viral RNA from replication to other processes, such as viral RNA encapsidation and cell-to-cell movement. Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, small RNA viruses might use RNA chaperones for replication as shown here for the p33 replication protein. In vitro experiments demonstrated that the purified recombinant p33 facilitated RNA synthesis on plusstranded and double-stranded (ds)RNA templates up to 5-fold. In addition, p33 rendered dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNA. Altogether, the RNA chaperone activity of p33 might perform similar biological functions to the helicases. SSa a yeast HSP70 found in the viral replication complex and shown to facilitate viral replication (Serva and Nagy, 2006)To dissect the mode of action of SSA in the viral replication I used temperature sensitive and deletion mutants. Both showed miss localization of p33 compared to the wild type. Purified SSA rendered non functional bacterial expressed p92 functional in an in vitro replication assay. SSa might play a role in the transportation and assembly of viral replication proteins.
Stork, Jozsef, "ROLE OF P33 IN TOMBUSVIRUS REPLICATION" (2009). University of Kentucky Doctoral Dissertations. 684.