Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection.
We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The alpha-catalytic subunit of CK2 (CK2alpha) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatography-tandem mass spectrometry. The tobacco CK2alpha gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2alpha in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and long-distance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.
Ivanov
Konstantin I
KI
Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland. konstantin.ivanov@helsinki.fi
Puustinen
Pietri
P
Gabrenaite
Rasa
R
Vihinen
Helena
H
Rönnstrand
Lars
L
Valmu
Leena
L
Kalkkinen
Nisse
N
Mäkinen
Kristiina
K
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D047390:Casein Kinase II; D002384:Catalysis; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D017800:Potyvirus / Q000254:growth & development / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism / Q000821:virology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000821:virology
D036022:Capsid Proteins; D010940:Plant Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D013912:Threonine; D004004:Dichlororibofuranosylbenzimidazole; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases