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Originally published In Press as doi:10.1074/jbc.M201752200 on July 31, 2002

J. Biol. Chem., Vol. 277, Issue 42, 39566-39573, October 18, 2002
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Chicken Anemia Virus VP2 Is a Novel Dual Specificity Protein Phosphatase*

Michelle A. PetersDagger §, David C. Jackson, Brendan S. Crabb||, and Glenn F. BrowningDagger **

From the Departments of Dagger  Veterinary Science and  Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia and the || Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Victoria 3050, Australia

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV) has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) alpha  proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a Vmax of 14,925 units/mg·min and a Km of 18.88 µM. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mM orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mM sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a Vmax of 28,600 units/mg·min and a Km of 76 µM. Mutagenesis of residue Cys95 to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.

* This study was supported by the Egg Industry Committee of the Rural Industries Research and Development Corp.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of an Australian Postgraduate Award and a scholarship supplied by the Rural Industries Research and Development Corp.

** To whom correspondence should be addressed: Dept. of Veterinary Science, University of Melbourne, Victoria 3010, Australia. Tel.: 613-8344-7342; Fax: 613-8344-7374; E-mail: glenfb@unimelb.edu.au.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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