Mutation of chicken anemia virus VP2 differentially
affects serine/threonine and tyrosine protein
Michelle A. Peters,13 David C. Jackson,2Brendan S. Crabb3
and Glenn F. Browning1
Glenn F. Browning
1,2Department of Veterinary Science1and Department of Microbiology and Immunology2, The
University of Melbourne, Victoria 3010, Australia
3Division of Infection and Immunity, The Walter and Eliza Hall Institute of Medical Research,
The Royal Melbourne Hospital, Victoria 3050, Australia
Received 16 April 2004
Accepted 29 October 2004
Novel dual-specificity protein phosphatases (DSPs), which catalyse the removal of phosphate
from both phosphotyrosine and phosphoserine/phosphothreonine substrates, have recently been
identified in two viruses within the family Circoviridae. Viral protein 2 (VP2) of chicken anemia
virus (CAV) and ORF2 of TT virus have been shown to possess DSP activity in vitro. CAV VP2 is
unusual in possessing two vicinal cysteines within the protein phosphatase signature motif. The
first cysteine residue (C95) within the motif has been identified by mutagenesis as the essential
catalytic cysteine. In this study, it was shown that virus mutated at this residue displayed a
marked inhibition of growth, with titres reduced 104-fold, and reduced cytopathogenic effect in
cell culture, indicating that viral DSP activity may be significant during infection. As with virus
mutated at the first cysteine residue, mutation of the second cysteine (C97) within the motif
resulted in a marked reduction in viral growth and attenuation of cytopathogenicity in infected
cell cultures. However, mutagenesis of this second cysteine only reduced phosphotyrosine
phosphatase activity to 70% of that of wild-type VP2, but increased phosphoserine/
phosphothreonine phosphatase activity by as much as 700%. The differential effect of the
C97S mutation on VP2 activity does not appear to have parallels in other DSPs and suggests a
unique role for the second cysteine in the function of these viral proteins, particularly in vivo.
Novel dual-specificity protein phosphatases (DSPs) have
recently been identified in two viruses within the family
Circoviridae (Peters et al., 2002): TT virus (TTV), which is
detected commonly in adult humans, but is of unknown
significance (Takahashi et al., 2000), and chicken anemia
virus (CAV), which causes severe immunosuppression in
chickens (Noteborn et al., 1991). Reversible protein phos-
phorylation plays a crucial role in the regulation of cellular
processes and the activation of immune effector cells
(Chui et al., 1994; Dutz et al., 1995; Kanda & Hirai, 2001;
Poovorawan et al., 2001; Safadi et al., 2001; Schievella et al.,
1993; Shibayama et al., 2001; Vasconcelos et al., 2001) and
(VP2) may contribute to virus-induced immunosuppres-
sion and that better understanding of the functions of these
proteins may improve our understanding of the patho-
genesis of both of these viruses.
The family of DSPs possesses the highly conserved signature
motif CXXXXXR. Protein tyrosine phosphatases (PTPases)
catalyse the removal of phosphate from phosphotyrosine
via a cysteinyl phosphate intermediate formed with the
active cysteine in the signature motif. The signature motif
ICNCGQFRKH, from residues 94 to 103, has been identi-
fied in CAV VP2, and mutation of C95 has been shown to
abrogate both PTPase and serine/threonine protein phos-
phatase (S/TPPase) activities (Peters et al., 2002), confirm-
ing that this residue is essential to the catalytic mechanism
and that this viral DSP has features analogous to those of
Numerous detailed studies of protein phosphatases have
identified structural features that contribute to the activa-
tion states and substrate specificities of protein phospha-
tases within the cell (Chiarugi et al., 2001; Li & Dixon,
2000; Schumacher et al., 2002). In general, the structural
motifs surrounding the catalytic cleft contribute to the
3Present address: Department of Veterinary Pathobiology, Purdue
University, 406 Sth University Dve, ADDL-47907, West Lafayette, IN,
0008-0197 G 2005 SGM Printed in Great Britain623
Journal of General Virology (2005), 86, 623–630
the United Kingdom for antibody to chicken anaemia agent. Avian
Pathol 17, 315–324.
Noteborn, M. H. M., de Boer, G. F., van Roozelaar, D. J. & 9 other
authors (1991). Characterization of cloned chicken anemia virus
DNA that contains all elements for the infectious replication cycle.
J Virol 65, 3131–3139.
Peters, M. A., Jackson, D. C., Crabb, B. S. & Browning, G. F. (2002).
Chicken anemia virus VP2 is a novel dual specificity protein
phosphatase. J Biol Chem 277, 39566–39573.
Phenix, K. V., Meehan, B. M., Todd, D. & McNulty, M. S. (1994).
Transcriptional analysis and genome expression of chicken anaemia
virus. J Gen Virol 75, 905–909.
Poovorawan, Y., Tangkijvanich, P., Theamboonlers, A. & Hirsch, P.
(2001). Transfusion transmissible virus TTV and its putative role in
the etiology of liver disease. Hepatogastroenterology 48, 256–260.
Safadi, R., Or, R., Ilan, Y. & 8 other authors (2001). Lack of known
hepatitis virus in hepatitis-associated aplastic anemia and outcome
after bone marrow transplantation. Bone Marrow Transplant 27,
Schievella, A. R., Paige, L. A., Johnson, K. A., Hill, D. E. &
Erikson, R. L. (1993). Protein tyrosine phosphatase 1B undergoes
mitosis-specific phosphorylation on serine. Cell Growth Differ 4,
Schumacher, M. A., Todd, J. L., Rice, A. E., Tanner, K. G. & Denu,
J. M. (2002). Structural basis for the recognition of a bisphos-
phorylated MAP kinase peptide by human VHR protein phospha-
tase. Biochemistry 41, 3009–3017.
Nishizawa, T., Tsuda, F. & Okamoto, H. (2001). Inverse relationship
between the titre of TT virus DNA and the CD4 cell count in
patients infected with HIV. AIDS 15, 563–570.
T., Masuda, G.,Ajisawa, A.,Takahashi, M.,
Takahashi, K., Hijikata, M., Samokhvalov, E. I. & Mishiro, S. (2000).
Full or near full length nucleotide sequences of TT virus variants
(types SANBAN and YONBAN) and the TT virus-like mini virus.
Intervirology 43, 119–123.
Tonks, N. K., Diltz, C. D. & Fischer, E. H. (1991). Purification and
assay of CD45: an integral membrane protein-tyrosine phosphatase.
Methods Enzymol 201, 442–451.
Vasconcelos, H. C. F., Menezes, M. E. & Niel, C. (2001). TT virus
infection in children and adults who visited a general hospital in the
south of Brazil for routine procedure. Mem Inst Oswaldo Cruz 96,
630 Journal of General Virology 86
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