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Journal of Virology, June 2007, p. 5497-5507, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02233-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Functional Characterization of the Major and Minor Phosphorylation Sites of the P Protein of Borna Disease Virus{triangledown}

Sonja Schmid, Daniel Mayer, Urs Schneider, and Martin Schwemmle*

Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany

Received 11 October 2006/ Accepted 9 March 2007

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 {varepsilon} (PKC{varepsilon}) 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 PKC{varepsilon} 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 PKC{varepsilon} sites were used but not when both CKII sites were altered. PKC{varepsilon} 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.

* Corresponding author. Mailing address: Department of Virology, University of Freiburg, Hermann Herder Strasse 11, D-79104 Freiburg, Germany. Phone: 49 761 203 6526. Fax: 49 761 203 6639. E-mail: martin.schwemmle{at}uniklinik-freiburg.de

{triangledown} Published ahead of print on 21 March 2007.

Journal of Virology, June 2007, p. 5497-5507, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02233-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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