Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein.
The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
Massey
D M
DM
Department of Physiology and Biophysics, University of Medicine and Dentistry, New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Deans
N
N
Lenard
J
J
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D002213:Capsid / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase; D013997:Time Factors; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D014758:Viral Core Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000302:isolation & purification
D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D012367:RNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D011494:Protein Kinases; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D000241:Adenosine