Laboratory of Veterinary Microbiology, Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065 Japan1
Tsukuba Central Laboratories, Kyoritsu Shoji Corporation, 2-9-22 Takamihara, Kukizaki-Machi, Inashiki-Gun, Ibaraki-Ken, 300-1252 Japan2
Laboratory of Clinical Microbiology, Kyoritsu Shoji Corporation, 1-12-4 Kudan-Kita, Chiyoda-ku, Tokyo, 102-0073 Japan3
Author for correspondence: Yukinobu Tohya. Fax +81 99 285 8725. e-mail ytohya{at}vet.agri.kagoshima-u.ac.jp
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Abstract |
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CaCV No. 48 strain was propagated in Miyazaki University canine mammary gland mixed tumour (MCM-B2) cells (Priosoeryanto et al., 1995 ). In order to inhibit proteolytic processing, CaCV-infected cells were treated at an elevated temperature of 45 °C as previously described (Carter, 1989
; Shin et al., 1993
).
A serum monospecific to the capsid protein was obtained from BALB/c mice that had been immunized with approximately 10 µg/mouse CaCV capsid protein. The capsid protein used for the immunization was prepared as follows. CsCl-purified virus was separated by 10% SDSPAGE and stained with Coomassie brilliant blue R250 as described previously (San Gabriel et al., 1997 ). The band of the capsid protein was sliced and eluted in an electro-eluter (Model 422; Bio-Rad). The eluted protein was precipitated with acetone and resuspended in PBS for future use.
The CaCV ORF2 expression plasmid was constructed as follows. CaCV RNA was extracted from the purified CaCV suspension as previously described (Roerink et al., 1999 ). Reverse transcription was carried out using oligo(dT) primer. A cDNA fragment was produced by PCR using synthetic oligonucleotides CaCV1 (5' GAACTCGAGATGGCTCGC TATCTTGAACT 3') and CaCV2 (5' GCTCTAGATCAT AGTGTTGTAGCGCTAC 3') as primers. Primer CaCV1 corresponded to nt 120 of the CaCV ORF2 with a XhoI restriction enzyme site upstream from the first ATG initiation codon (underlined), and primer CaCV2 was complementary to nt 20572076 of the CaCV ORF2 and contained an XbaI restriction enzyme site. The PCR-amplified fragment was digested by XhoI and XbaI, and cloned between the XhoI and XbaI sites of the pME18S expression vector (Shin et al., 1993
). The constructed plasmid was designated pDCV-II, and contained the first ATG of ORF2 directly under the control of the SR
promoter (Takebe et al., 1988
). pMCV-3C, which expresses the putative 3C region of FCV strain F4 (Oshikamo et al., 1994
), was constructed as follows. A cDNA fragment including the putative 3C region was generated by PCR with synthetic oligonucleotides XHO-ATG5'3C (5' AATCTCGAGATGT CTGGGCCTGGCACTAA 3') and PST-TCA-3'3C (5' GGCTGCAGTCATTCCAAGAAGATGTTCAT 3') as primers and a plasmid containing the ORF1 of FCV F4 as template. Primer XHO-ATG5'3C corresponded to nt 32143230 of the FCV ORF1 with a XhoI restriction enzyme site and an initiation codon, and primer PST-TCA-3'3C was complementary to nt 40184035 of the FCV ORF1 and contained a PstI restriction enzyme site and a termination codon. The PCR-amplified fragment was digested by XhoI and PstI, and cloned between the XhoI and PstI sites of pME18S. The FCV ORF2 expression vector pMCV-II was constructed as described previously (Shin et al., 1993
). Three micrograms of pDCV-II or pMCV-II and 1 µg pMCV-3C or pME18S were co-transfected into COS-7 cells according to previously described methods with minor modifications (Seed & Aruffo, 1987
; Shin et al., 1993
).
Detection and identification of the CaCV capsid precursor were performed by immunoblot analysis using the serum monospecific to the capsid protein (Fig. 1a, lanes 13, and 5). The serum showed specific reactivity with 57 kDa capsid protein in CaCV-infected cells (Fig. 1a
, lane 2). In CaCV- infected cells, which were treated at an elevated temperature of 45 °C, 75 kDa and 57 kDa proteins were specifically detected (Fig. 1a
, lane 3). This phenomenon was also seen in similar experiments using SMSV- and FCV-infected cells (Fretz & Schaffer, 1978
; Carter, 1989
; Shin et al., 1993
). The 75 kDa protein was identified as the precursor of the 57 kDa CaCV capsid protein in this study. When the ORF2 of CaCV was expressed transiently in COS-7 cells by transfection with pDCV-II, the 75 kDa protein was detected in the immunoblot analysis (Fig. 1a
, lane 5). The additional bands above the 75 kDa protein may be polypeptides translated by incorrect initiation from an ATG codon in-frame and upstream from the first ATG of CaCV ORF2 or by read-through of the termination. In addition, low density minor bands were considered to be degraded products of the capsid precursor as described previously (Shin et al., 1993
). The molecular mass of the 75 kDa protein detected in the pDCV-II-transfected cells was similar to that expected if the CaCV ORF2 was translated from the first ATG in ORF2 to its termination codon (i.e. 76180 Da). The mobility of the 75 kDa protein in the transfected cells was identical to that of the capsid precursor (Fig. 1a
, lanes 3 and 5). The expressed protein therefore seems to have the same basic characteristics as the CaCV capsid precursor both in antigenicity and in electromobility. This result also suggests that no post-translational processing by autocatalytic or host cell-mediated cleavage has occurred in this system.
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In FCV, the capsid precursor is cleaved by a virus-encoded proteinase between amino acid positions 124 and 125 (Glu124 and Ala125) (Sosnovtsev et al., 1998 ), and the 14 kDa N- terminal polypeptide was identified in infected cells (Tohya et al., 1999
). In CaCV, a corresponding cleavage site (Glu157/Ser158) was suggested by homology analysis with FCV and SMSV (Roerink et al., 1999
). In order to determine whether the FCV proteinase cleaved at the corresponding site in the CaCV capsid precursor, we prepared a serum against an N-terminal polypeptide (NTP) corresponding to amino acids Met1 to Glu157 of the capsid precursor using a glutathione S-transferase (GST)/NTP fusion protein. The GST/NTP fusion protein expression plasmid was constructed as follows. The 5'-end 471 nt region of ORF2 encoding the NTP was PCR-amplified using plasmid pDCV-II as the template and synthetic oligonucleotides CaCV ORF2-A (CTCAGGATCCAGATG GCTCGCTATCTTGAA) and CaCV ORF2-AR (ATACT CGAGTTCCGCGCGGAATTGGAATTC) as primers. Primer CaCV ORF2-A corresponded to nt 118 of the CaCV ORF2 with a BamHI restriction enzyme site and additional nucleotides (AG) upstream of the ATG initiation codon to adjust the frame, and CaCV ORF2-AR was complementary to nt 451471 of the ORF2 and contained a XhoI restriction enzyme site. The PCR-amplified fragment was cloned between the BamHI and XhoI sites of the vector pGEX-5X-1 (Pharmacia Biotech). Expression and purification of the fusion protein and immunization of mice were performed as described previously (Tohya et al., 1999
).
The results of immunoblot analysis using the anti- GST/NTP serum are shown in Fig. 1(b). The serum showed specific reactivity with a 22 kDa polypeptide in the CaCV- infected cells (Fig. 1b
, lane 2). The anti-GST serum prepared as a control showed no specific reactivity with the proteins in the cells (data not shown). In addition to the 22 kDa polypeptide, the 75 kDa capsid precursor was detected in CaCV-infected cells which were treated at the elevated temperature (Fig. 1b
, lane 3), indicating that the cleaved N-terminal part of the capsid precursor has a molecular mass of 22 kDa, although the molecular mass of the 22 kDa polypeptide was a little larger than the mass of 18256 Da deduced from sequence analysis. The 22 kDa polypeptide, in addition to the 75 kDa capsid precursor, was also detected only in the pDCV-II and pMCV-3C co-transfected COS-7 cells, and not in the pDCV-II and pME18S co-transfected cells (Fig. 1b
, lanes 4 and 5). The result suggested that the proteinase of FCV cleaves at the authentic site in the capsid precursor of CaCV and that CaCV may have a similar proteinase possibly encoded in its ORF1.
A feature of the FCV proteinase is its high substrate specificity that is determined by the structure of the cleavage site sequence (Sosnovtsev et al., 1998 ). In order to confirm the suggested cleavage site (Glu157/Ser158), we investigated whether processing of the CaCV capsid precursor could be blocked by the co-expression system. The XhoI/EcoRV fragment (924 bp) containing the 5'-end of ORF2 was excised from pDCV-II and ligated into pBluescript II KS(+) (Stratagene). The Glu 157 mutation (GAA to AAA for Lys) and the Ser158 mutation (TCC to CCC for Pro) were introduced, respectively, into the fragment using mutagenic oligonu- cleotides designed to create substitutional mutations and the TaKaRa LA PCR In Vitro Mutagenesis kit (TAKARA) according to the manufacturers instructions. After both mutations in the fragments were confirmed by sequence analysis, the mutated fragments were used to replace the parent fragment in pDCV-II. The mutated expression plasmids were designated pDCV-II mut EK and pDCV-II mut SP for the Glu157 and Ser158 mutations, respectively. In the COS-7 cells co-transfected with the mutated plasmids and the pMCV-3C, the mature 57 kDa capsid protein was not detected by immunoblot analysis using the serum monospecific to the capsid protein (Fig. 2
, lanes 2 and 3). The result shows that processing by the FCV proteinase was blocked by changing the amino acids at the site and strongly suggests that the Glu157/Ser 158 site is the cleavage site in the CaCV capsid precursor by the virus-encoding proteinase.
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Acknowledgments |
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References |
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Received 26 April 1999;
accepted 16 September 1999.