Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome, We have expressed an d purified the turnip yellow mosaic virus (TYMV) RdRD in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexah istidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified prote in in vitro. Phospho amino acid analysis and immunoblotting experiments ide ntified serine and threonine residues as being the subject of phosphorylati on. Peptide mass mapping using MS analysis of a protein digest revealed tha t phosphorylation sites are localized within a putative PEST sequence [a se quence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibo dies specific for ubiquitin conjugates, we were able to demonstrate that th e TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect c ells. These observations suggest that the TYMV RdRp may be processed select ively by the ubiquitin/proteasome degradation system upon phosphorylation o f the PEST sequence.