Chicken Anemia Virus VP2 is a novel dual specificity protein

42 Pages·2002·0.98 MB·English

JBC Papers in Press. Published on July 31, 2002 as Manuscript M201752200
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Chicken Anemia Virus VP2 is a novel dual specificity
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protein phosphatase
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5 Michelle A. Peters , David C. Jackson , Brendan S. Crabb and
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6 Glenn F. Browning
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8 1Department of Veterinary Science, The University of Melbourne, Victoria h
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9 3010, Australia w
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11 Fax: [phone] es
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12 [email protected] pril 1
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13 2Department of Microbiology and Immunology, The University of Melbourne, 9
14 Victoria 3010, Australia
15 3Division of Infection and Immunity, The Walter and Eliza Hall Institute of
16 Medical Research, The Royal Melbourne Hospital, Victoria 3050, Australia
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18 running title: CAV VP2 is a novel protein tyrosine phosphatase
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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
1 SUMMARY
2 The function of viral protein 2 (VP2) of the immunosuppressive circovirus
3 Chicken Anemia Virus (CAV) has not yet been established. We show that the
4 CAV VP2 amino acid sequence has some similarity to a number of eukaryotic,
5 receptor, protein-tyrosine phosphatase (PTPase) alpha proteins, as well as to
6 a cluster of human TT viruses within the Sanban group. To investigate if CAV
7 VP2 functions as a PTPase, purified glutathione S-transferase (GST) -VP2
8 fusion protein was assayed for PTPase activity using the generalized peptide
9 substrates END(pY)INASL and DADE(pY)LIPQQG, with free phosphate
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10 detected using the malachite green colorimetric assay. CAV GST-VP2 was nlo
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11 shown to catalyse dephosphorylation of both substrates. CAV GST-VP2 fro
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12 PTPase activity for the END(pY)INASL substrate had a V max of 14 925 ://w
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13 U/mg.min and a K of 18.88 µM. Optimal activity was observed between pH 6 .jb
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14 and 7 and activity was specifically inhibited by 0.01 mM orthovanadate. We by/
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15 also show that the ORF2 sequence of the CAV-related human virus TT-like t o
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16 minivirus (TLMV) possessed PTPase activity and steady state kinetics ril 1
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17 equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To 9
18 establish whether these viral proteins were dual specificity protein
19 phosphatases the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were
20 also assayed for serine/threonine phosphatase (S/T PPase) activity using the
21 generalised peptide substrate RRA(pT)VA, with free phosphate detected
22 using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV
23 GST-ORF2 fusion proteins possessed S/T PPase activity, which was
24 specifically inhibited by 50 mM sodium fluoride. CAV GST-VP2 exhibited S/T
25 PPase activity with a V of 28 600 U/mg.min and a K of 76 m M.
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1 Mutagenesis of residue C95 to serine in CAV GST-VP2 abrogated both
2 PTPase and S/T PPase activity, identifying it as the catalytic cysteine within
3 the proposed signature motif. These studies thus show that the circoviruses
4 CAV and TLMV encode a dual specificity protein phosphatases (DSP) with an
5 unusual signature motif that may play a role in intracellular signalling during
6 viral replication. This is the first DSP gene to be identified in a small viral
7 genome (2.3 kb, 3 genes), the only other two viral DSP genes occurring in the
8 complex poxvirus and baculovirus genomes.
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10 INTRODUCTION nlo
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11 Chicken anemia virus (CAV), a member of the family Circoviridae, causes h
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12 severe immunosuppression, anemia and thrombocytopenia (1).The w
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13 Circoviridae include a number of very small plant and animal viruses that are .org
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14 characterized by the possession of a single stranded, negative-sense, circular u
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15 DNA genome. As there is minimal similarity between the genomic sequence A
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16 and organization of CAV and the other characterized animal circoviruses, , 2
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17 Psittacine Beak and Feather Disease Virus (PBFDV), Pigeon Circovirus and
18 Porcine Circoviruses (PCV) 1 and 2, CAV has been reclassified within the
19 floating genus Gyrovirus. TT viruses (TTV) have recently been identified in
20 human hosts and other species as a heterogeneous cluster of single
21 stranded, negative-sense, circular DNA viruses, but have not yet been
22 cultured in vitro. Sequence analysis of this group of viruses has demonstrated
23 greatest overall homology to CAV and Takahashi et al have recently proposed
24 the classification of the TTV, Sanban viruses, Yonban viruses, TT-like
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1 minivirus (TLMV) and CAV as the Paracircoviridae. However the phylogeny of
2 this group of viruses remains an area of active revision (2). The highest
3 sequence similarity between CAV and TTV is seen in the non-coding region
4 and between ORF2 of TTV and VP2 of CAV. The high level of sequence
5 conservation between CAV and TTV within these coding regions suggests
6 VP2 may play a critical role in viral replication.
7 CAV encodes only three proteins, with overlapping ORFs in three frames.
8 ORF3 encodes the 45-52 kDa capsid protein, VP1, ORF2 encodes the 11-13
9 kDa VP3 that has been shown to have apoptotic activity in transformed cell
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10 lines (1) and ORF1 encodes a 28 kDa non-structural protein, VP2, of nlo
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11 unknown function. VP2 is expressed at barely detectable levels during fro
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12 infection (3) and the low level of expression is consistent with a non-structural, ://w
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13 regulatory protein involved in viral replication and infection. .jb
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14 Preliminary comparisons of the CAV VP2 sequence to sequences available in by/
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15 the Genbank database identified similarity to a number of eukaryotic receptor t o
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16 protein tyrosine phosphatases (R-PTPases), particularly the human placental, ril 1
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17 rat, mouse and chicken R-PTPase alpha precursors. Reversible protein 9
18 phosphorylation plays a crucial role in the regulation of cellular processes
19 such as metabolism, gene regulation, cell cycle control, cytoskeletal
20 organization and cell adhesion. The PTPase family is highly diverse and
21 includes the eukaryotic receptor-like transmembrane proteins and soluble
22 cytosolic proteins, as well as bacterial PTPases, such as the YopH PTPase
23 from pathogenic Yersinia species and a viral PTPase, VH1, found in Vaccinia
24 virus (4) .
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1 The aim of this study was to investigate CAV VP2 and the homologous ORF2
2 of TT virus as novel viral PTPases and as serine/threonine protein
3 phosphatases (S/T PPase). This investigation primarily stemmed from the
4 finding of some sequence similarity between CAV VP2 and a number of
5 PTPases. In order to establish the catalytic function of VP2 the protein was
6 expressed as a fusion with glutathione S-transferase (GST) and the activity of
7 the fusion protein determined using universal oligopeptide PTPase substrates.
8 The classification of the VP2 PTPase was assessed by examination of the
9 kinetic parameters, the optimal pH for activity and the sensitivity of the
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10 enzyme to orthovanadate. S/T PPase activity was similarly assessed using a o
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11 universal oligopeptide substrate. Within the proposed signature motif of VP2 e
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12 the cysteine 95 residue was mutagenised to serine and protein phosphatase h
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13 activity was assessed. ww
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15 EXPERIMENTAL PROCEDURES A
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16 Sequence analysis 9
17 Protein sequences with homology to CAV VP2 were identified by searches of
18 the Genbank database using BLASTX (Basic Local Alignment Search Tool)
19 via the NCBI interface (www.ncbi.nlm.nih.gov). Sequences identified by this
20 method were then aligned to the CAV VP2 sequence using Macboxshade
21 v3.2 (R. Fusch and Glaxo Wellcome). A separate alignment was produced for
22 both the R-PTPases and the TTV sequences.
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1 Molecular Cloning of CAV viral protein 2
2 The Australian isolate of CAV, CAU269/7, was used in all experiments
3 (Genbank Accession AF227982) (5). CAV ORF1 (encoding VP2) (Genbank
4 Accession AAF34787.1) was amplified by polymerase chain reaction (PCR)
5 from the double stranded replicative form of the CAV genome. Cellular DNA
6 was purified from MDCC-MSB1 cells 48 h after infection with CAV using
7 proteinase K and sodium dodecyl sulphate (SDS) lysis and phenol/chloroform
8 extraction (6). Oligonucleotide forward primer CAV.1 – 5’
9 CGGTCCGGATCCATGCACGGAAACGGCGGACAAC 3’ and reverse primer
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10 CAV.2 – 5’ GGTTTGGAATTCTCACACTATACGTACCGGGGC 3’ were nlo
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11 synthesized to incorporate BamHI and EcoRI restriction endonuclease fro
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12 cleavage sites within their respective 5’ ends. A 100 m l reaction mixture was ttp://w
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13 prepared containing 300 m M each of dATP, dCTP, dGTP and dTTP, 2 mM .jb
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14 MgCl , 0.2 m M of each primer, 10 m l of 10x Taq DNA polymerase buffer, 2 U by/
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15 of Taq DNA polymerase (Promega, Madison, WI), and 2 m l of template DNA. t on
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16 The PCR was incubated at 95(cid:176) C for 2 min, followed by 40 cycles of 96(cid:176) C for ril 14
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17 40 s, 60(cid:176) C for 40 s and 72(cid:176) C for 40 s, with a final incubation at 72(cid:176) C for 5 min.
18 The PCR products were analyzed by agarose (1%) gel electrophoresis and a
19 band consistent with the expected size of 677 bp was excised and purified
20 using a Qiaex II (Qiagen, Basel, Switzerland) gel extraction kit according to
21 the manufacturer’s instructions. The purified product was digested with BamHI
22 and EcoRI and ligated to appropriately digested pGEX-4T-2 (Promega). E.
23 coli strain DH5a was transformed with the ligated plasmid and cultured at
24 37(cid:176) C on Luria-Bertani agar (LA) containing ampicillin at 50 m g/ml. The cloned
25 DNA was sequenced using a Taq Dye Deoxy Terminator Cycle Sequencing
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1 kit (Perkin Elmer, Wellesley, MA) using commercial sequencing primers
2 (Promega) specific for pGEX-4T-2.
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4 Molecular Cloning of TLMV viral protein 2
5 Purified nested PCR product extending from primer M-1360 to primer M-1363
6 (258 bp-886 bp) of TLMV strain CBD231 was kindly supplied by Dr Shunji
7 Mishiro (Genbank Accession AB026930) (2). TLMV ORF2 was amplified by
8 PCR from the M-1360 to M-1363 template. Oligonucleotide forward primer
9 TLMV.1 – 5’ TTGGATCCATGAGCAGCTTTCTAACACCATC 3’ and reverse D
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10 primer TLMV.2 – 5’ GGCGAATTCTTACCCATCGTCTTCTTCGAAATC 3’ d
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11 were synthesised to incorporate BamHI and EcoRI cleavage sites within their h
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12 respective 5’ ends. A 50 m l reaction mixture was prepared containing 300 m M ://ww
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13 each of dATP, dCTP, dGTP and dTTP, 2 mM MgCl2, 0.2 m M of each primer, 5 .org
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14 m l of 10x Taq DNA polymerase buffer, 1 U of Taq DNA polymerase ue
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15 (Promega), and 1 m l of template DNA. The PCR reaction was incubated at p
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16 96(cid:176) C for 2 min, followed by 40 cycles of 96(cid:176) C for 40 s, 56(cid:176) C for 40 s, then , 20
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17 72(cid:176) C for 40 s, and a final incubation at 72(cid:176) C for 5 min. Purification, digestion
18 and cloning of the 295 bp PCR product into the pGEM-T plasmid vector
19 (Promega) were performed as described above for the CAV VP2 gene. The
20 insert was subsequently subcloned into the pGEX-4T-2 plasmid vector and
21 the sequence of the insert was verified.
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23 Mutagenesis of C95 residue in CAV-VP2
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1 Overlap extension PCR (7) was used to introduce a mutation into the CAV
2 VP2 gene sequence to change the cysteine residue at position 95 to serine
3 (C95S). The PCR template was pCAU269/7 in the plasmid vector pGEX-4Z
4 (Promega) (5). The oligonucleotide pair, positive-sense -5’
5 CGCTAAGATCAGCAACTGCG 3’ and negative-sense -5’
6 CGCAGTTGCTGATCTTAGCGTG 3’, were synthesised to incorporate
7 nucleotide substitutions encoding the amino acid alterations (shown in bold).
8 Template DNA was prepared from E.coli DH5a containing pCAU269/7
9 cultured at 37 °C in Luria Bertani broth (LB) containing 50 m g ampicillin /ml.
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10 Plasmid was purified using a Qiagen Midi kit according to the manufacturer’s ow
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11 instructions. The PCR was carried out in two stages. The first stage consisted ed
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12 of a set of 2 PCR reactions: one from the upstream flanking primer CAV.3 -5’ http
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13 CTATCGAATTCCGAGTGGTTACTAT 3’ w
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14 to the negative-sense mutagenesis primer, and one from the downstream rg
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15 flanking primer CAV.4 -5’ TGCTCACGTATGTCAGGTTC 3’ to the positive- es
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16 sense mutagenesis primer. In the second stage of mutagenesis, PCR pril 1
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17 products from the first pair of reactions acted as template for a PCR reaction 0
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18 using only the flanking primers. The PCR product generated was bounded by
19 the flanking sequences and incorporated in both strands the mutations
20 introduced into the template in the first stage of PCR.
21 In the first stage, a 100 m l reaction mixture was prepared containing 300 m M
22 each of dATP, dCTP, dGTP and dTTP, 2 mM MgSO , 0.2 m M of each primer,
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23 10 m l of 10 · Platinum Pfu Taq DNA polymerase buffer, 2 U of Platinum Pfu
24 Taq DNA polymerase (Promega), and 1 m l of template DNA. The reaction was
25 incubated at 95 °C for 2 min, followed by 30 cycles of 95 °C for 40 s, 60 °C for
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1 60 s and 68 °C for 40 s, with a final incubation at 68 °C for 5 min. First stage
2 template was removed by digestion with DpnI (Boehringer Mannheim,
3 Mannheim, Germany). The PCR products were analysed by agarose (1%) gel
4 electrophoresis and the bands corresponding to the stage one products were
5 excised and purified using a Qiaex II gel extraction kit according to the
6 manufacturer’s instructions.
7 For the second stage PCR, a 100 m l reaction mixture was prepared containing
8 300 m M each of dATP, dCTP, dGTP and dTTP, 2 mM MgSO , 0.2 m M of each
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9 primer, 10 m l of 10 · Platinum Pfu Taq DNA polymerase buffer, 2 U of
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10 Platinum Pfu Taq DNA polymerase, and 1 m l of each template DNA. The PCR ow
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11 reaction was incubated at 95 °C for 2 min, followed by 1 cycle of 95 °C for 40 d
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12 s, 57 °C for 90 s and 68 °C for 40 s, then 15 cycles of 95 °C for 40 s, 57 °C for ttp
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13 60 s and 68 °C for 40 s, with a final incubation at 68 °C for 5 min. w
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14 The PCR products were digested with StuI (New England Biolabs, Beverly, brg/
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15 MA., USA) and BsmI (New England Biolabs) and analysed by agarose (1%) s
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16 gel electrophoresis. A band of the expected size of 357 bp was excised and ril 1
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17 purified using a Qiaex II gel extraction kit according to the manufacturer’s 01
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18 instructions. The VP2-pGEX-4T-2 clone was similarly digested with StuI and
19 BsmI to remove the region of 357 bp to be replaced with the mutant sequence
20 and a band of the expected size of 5260 bp was purified from a 1% agarose
21 gel. The digested PCR product was then ligated to the digested VP2-pGEX-
22 4T-2 backbone. E.coli DH5a was transformed by electroporation with the
23 ligated plasmid and cultured at 37 °C on LA containing ampicillin at 50 m g/ml.
24 Plasmid was purified from selected clones using a Qiagen Midi kit according
25 to the manufacturer’s instructions. Clones were screened for the presence of
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1 insert by PCR using the forward primer CAV.2 and a reverse primer
2 overlapping the VP2 pGEX multicloning site -5’
3 CTCTAGAGGATCCTCACACTATACGTACCGGGGC 3’. The cloned DNA
4 was sequenced using a Taq Dye Deoxy Terminator Cycle Sequencing kit
5 using the above primers.
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7 Protein expression and purification
8 CAV VP2 was produced as a C-terminal fusion with glutathione S-transferase.
9 Briefly, 1 L cultures of E. coli DH5a possessing the CAV VP2 pGEX-4T-2
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10 construct were cultured in LB containing ampicillin at 50 m g/ml. Expression nlo
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11 was induced by adding isopropyl-b -D-thiogalactopyranoside (IPTG) to a final fro
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12 concentration of 1 mM when the culture reached an optical density of 0.6 at ://w
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13 600 nm, and the culture incubated an additional hour prior to harvest. Bacteria .jbc
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14 were recovered by centrifugation at 6000 g for 30 min and the pellets washed y
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15 twice in phosphate buffered saline (PBS). The cells were resuspended in 25 n
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16 ml of PBS containing 0.3 M EDTA, 200 mg lysozyme, and 100 m g of phenyl ril 14
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17 methyl sulfonyl fluoride (Sigma Aldrich, St. Louis, MO) / ml and lysed by 10 s
18 bursts of sonication at low frequency. The lysate was solubilized in 0.1%
19 Triton X-100, incubated a further 10 min at 4(cid:176) C, and the cellular debris
20 removed by centrifugation at 10 000 g for 30 min. The fusion protein was
21 affinity purified using glutathione sepharose resin (Promega) following the
22 manufacturer’s protocol. The eluate was extensively dialyzed against a buffer
23 containing 137 mM NaCl, 2.7 mM KCl and 25 mM Tris HCl pH 7.4 (TBS).
24 CAV GST-VP2 mutagenised to contain serine at position 95 and negative
25 control glutathione-S-transferase were purified from E. coli DH5a transformed
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