Functional characterization of the major and minor phosphorylation sites of the P protein of Borna disease virus.
The phosphoprotein P of Borna disease virus (BDV) is an essential cofactor of the viral RNA-dependent RNA polymerase. It is preferentially phosphorylated at serine residues 26 and 28 by protein kinase C epsilon (PKCepsilon) and, to a lesser extent, at serine residues 70 and 86 by casein kinase II (CKII). To determine whether P phosphorylation is required for viral polymerase activity, we generated P mutants lacking either the PKCepsilon or the CKII phosphate acceptor sites by replacing the corresponding serine residues with alanine (A). Alternatively, these sites were replaced by aspartic acid (D) to mimic phosphorylation. Functional characterization of the various mutants in the BDV minireplicon assay revealed that D substitutions at the CKII sites inhibited the polymerase-supporting activity of P, while A substitutions maintained wild-type activity. Likewise, D substitutions at the PKC sites did not impair the cofactor function of BDV-P, whereas A substitutions at these sites led to increased activity. Interestingly, recombinant viruses could be rescued only when P mutants with modified PKCepsilon sites were used but not when both CKII sites were altered. PKCepsilon mutant viruses showed a reduced capacity to spread in cell culture, while viral RNA and protein expression levels in persistently infected cells were almost normal. Further mutational analyses revealed that substitutions at individual CKII sites were, with the exception of a substitution of A for S86, detrimental for viral rescue. These data demonstrate that, in contrast to other viral P proteins, the cofactor activity of BDV-P is negatively regulated by phosphorylation.
Schmid
Sonja
S
Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Hermann Herder Strasse 11, D-79104 Freiburg, Germany.
Mayer
Daniel
D
Schneider
Urs
U
Schwemmle
Martin
M
D019943:Amino Acid Substitution / Q000235:genetics; D001891:Borna disease virus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism; D002460:Cell Line; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D003067:Coenzymes; C496537:P protein, Borna disease virus; D010750:Phosphoproteins; D015678:Viral Structural Proteins; D047390:Casein Kinase II; D051744:Protein Kinase C-epsilon; D012324:RNA-Dependent RNA Polymerase