Cdc34p ubiquitin-conjugating enzyme is a component of the tombusvirus replicase complex and ubiquitinates p33 replication protein

Department of Plant Pathology, University of Kentucky, Lexington, KY 40546

^* To whom correspondence should be addressed. Email: pdnagy2{at}uky.edu.

	Abstract

To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used the yeast proteome microarray carrying 4088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA modifying enzymes and other proteins with yet unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Down-regulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon (rep)RNA, reduced repRNA accumulation and the activity of the tombusvirus replicase by up to 5-fold. Over-expression of wt, but not an E2 defective mutant of Cdc34p, increased repRNA accumulation, suggesting significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication.