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 and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.
Héricourt
F
F
Laboratoire de Virologie Moléculaire, Institut Jacques Monod, UMR 7592, CNRS-Universités Paris 6-Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France.
Blanc
S
S
Redeker
V
V
Jupin
I
I
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D007313:Insecta; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D017806:Tymovirus / Q000201:enzymology*; D014452:Ubiquitins / Q000737:chemistry
D011994:Recombinant Proteins; D014452:Ubiquitins; D013912:Threonine; D012694:Serine; D012324:RNA-Dependent RNA Polymerase