Sequence analysis of the BamHI F fragment of the genome of Bombyx mona nuclear polyhedrosis virus. (BmNPV) revealed an open reading frame whose ...
JOURNAL OF VIROLOGY, Oct. 1994, p. 6619-6625
Vol. 68, No. 10
0022-538X/94/$04.00+0 Copyright X 1994, American Society for Microbiology
A Cysteine Protease Encoded by the Baculovirus Bombyx mon Nuclear Polyhedrosis Virus TARO OHKAWA, KEI MAJIMA,t AND SUSUMU MAEDA*
Department of Entomology, University of California, Davis, California 95616 Received 8 June 1994/Accepted 18 July 1994
Sequence analysis of the BamHI F fragment of the genome of Bombyx mona nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a ,1-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those ofwild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus.
Baculoviruses are characterized by double-stranded circular packaged within rod-shaped enveloped capsids. The family Baculoviridae is composed of three subgroups, nuclear polyhedrosis viruses (NPVs), granulosis viruses, and nonoccluded baculoviruses. NPVs have been used as vectors for the high-level expression of foreign genes (for a review, see reference 22). NPVs and granulosis viruses have also been studied as alternatives to chemical insecticides (for a review, see reference 6). Autographa califomica NPV (AcNPV) is the most wellcharacterized baculovirus, the next most well-characterized being Orgyia pseudotsugata NPV and Bombyx mori NPV (Bm NPV) (see reference 19). More than 60% of the AcNPV genome has been sequenced (13), resulting in the discovery of a number of interesting genes, including UDP-glucosyltransferase (21), apoptosis-blocking protein (2), and ubiquitin (7) (see reference 22). Nucleotide sequence analysis of the Bm NPV genome in our laboratory (5) indicates that AcNPV and BmNPV share about 95% homology in 90% of the sequenced regions (see references 10 and 19). BmNPV and AcNPV are especially useful models for the study of virus host range determinants, since BmNPV and AcNPV have nonoverlapping hosts but very high nucleotide sequence homology. Proteases from a number of viruses related to the papain, trypsin, and aspartate proteinase superfamilies have been identified (for a review, see reference 31). But with the exception of the adenovirus L3 23-kDa proteinase and human immunodeficiency virus type 1 protease, which have been
shown to cleave host intermediate filament proteins (1, 27), viral proteases have been implicated only in the cleavage of polyprotein precursors. Previously described viral proteases show a remarkable specificity for sites where precursor cleavage will generate mature proteins and have not been found to play a role in protein turnover or degradation. Although these viral proteases have catalytic-site residues identical to those of cellular proteases, the overall sequence homologies to cellular proteases are low. Viruses which encode trypsin-like proteases include alphaviruses, flaviviruses, picornaviruses, and a number of plant viruses (for reviews, see references 8 and 14). Viruses with papain-like proteases include alphaviruses, coronaviruses, and tobacco etch virus (for a review, see reference 14). Aspartate proteases have been found in retroviruses (for reviews, see reference 23 and 28). The viral papain-like cysteine protease genes in general show limited sequence similarity only for active-site residues. Furthermore, the spacing between the conserved catalytic residues is different from that found in papain superfamily cysteine proteases. In the AcNPV genome, a partial papainlike sequence has been identified (25) upstream of the AcNPV gp64 gene (34). The deduced amino acid sequence of the incomplete open reading frame showed 32% identity to that of papain and had conserved catalytic residues and cysteine residues putatively involved in the formation of disulfide bridges. However, an increase in cysteine protease activity was not found in AcNPV-infected SF9 cells. Stimulation of acidic cysteine protease activity in BmNPVinfected isolated pupal abdomens of the silkworm, B. mon, has been reported previously (12). It was also reported that cysteine protease activity is stimulated by ecdysone injection into isolated pupal abdomens. The virus-stimulated protease activity was hypothesized to be of host origin, although the chromatographic patterns of activity differed from those of the ecdysone-stimulated activity.
genomes
Corresponding author. Mailing address: Department of EntomolUniversity of California, Davis, CA 95616. Phone: (916) 752-0483. Fax: (916) 752-1537. Electronic mail address: SMAEDA@UCDAVIS. *
ogy,
CEDU. t Present address: Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan. 6619
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OHKAWA ET AL.
We report here a functionally active BmNPV cysteine protease gene whose deduced amino acid sequence has significant homology to those of the papain family of cysteine proteases, including residues highly conserved among this superfamily. Viral growth and polyhedron production of Bm CysPD was normal in BmN cells. However, deletion of the cysteine protease gene resulted in dramatic differences in symptoms (i.e., degradation of host tissues) induced by Bm CysPD infection of larvae, indicating the importance of the cysteine protease in horizontal transmission of the virus.
MATERIALS AND METHODS Cell lines, viruses, and silkworms. The BmN (BmN-4) cell line (16) was maintained with TC-100 supplemented with 10% fetal bovine serum as described previously (16). The BmNPV T3 isolate (17) and the BmNPV mutant BmCysPD, lacking the putative cysteine protease gene (see below), were propagated on BmN cells. Larvae of the silkworm B. mori were reared on an artificial diet at 27°C (17). Viral infection and plaque assay in BmN cells. BmN cells were infected with BmNPV T3 or BmCysPD at a multiplicity of infection of 10 as described previously (16). In all experiments, time zero was defined as the point at which fresh medium was added following the 1-h viral absorption period. Viral replication was examined by infection of 3.8 x 106 BmN cells per 60-mm-diameter culture dish with BmNPV T3 or BmCysPD. At 1, 5, 12, 24, 36, 48, and 72 h postinfection (p.i.), 100 ,ul of culture supernatant was removed, centrifuged briefly to remove cell debris, and stored at -80°C prior to plaque assay. Viral growth curves were determined by plaque assay with serially 10-fold-diluted viral suspensions by modifications of the method described previously (16). The production of polyhedra in infected BmN cells was observed under light microscopy at 48 h p.i. Sequencing of the BmNPV BamHI F fragment. To determine the nucleotide sequence of the putative cysteine protease, either the entire BamHI F fragment of BmNPV (18) or a part of it was inserted adjacent to a nonessential lambda DNA fragment which had already been inserted into the multiple cloning region of a pUC-derived plasmid (pTZ18R; Pharmacia LKB, Piscataway, N.J.) (19). Following cleavage at the BamHI F (or appropriate viral) fragment-lambda DNA junction, nested deletions of the BamHI F fragment were made by treatment with Bal 31 for up to 5 min at 31°C. The remaining lambda DNA was then removed (by cleavage with SmaI), and the remaining plasmid containing the BamHI F (or appropriate viral) fragment with nested deletion was selfligated and transformed into competent Escherichia coli cells. Plasmids of appropriate sizes for sequencing were propagated, isolated, and sequenced with a reverse pUC primer by the dideoxy chain termination method as described previously (19). Nucleotide sequencing analysis and homology searches were carried out with the National Biomedical Research Foundation (NBRF)-PIR database and the DNASIS/PROSIS programs (Hitachi America, San Bruno, Calif.) as described
previously (19). Isolation of a BmNPV mutant (BmCysPD) with a deletion in the cysteine protease gene. The pTZ18R plasmid containing the BamHI F fragment of BmNPV was cleaved with restriction endonucleases ApaI and HpaI (within the coding region of the putative cysteine protease; see Fig. 2; restriction endonuclease sites are double underlined), treated with the Klenow fragment of DNA polymerase I, and ligated with a gene cassette containing a 3-galactosidase gene driven by a Drosophila melanogaster heat shock promoter (hsp7O; see references 19
and 33). The cysteine protease gene of BmNPV was deleted by homologous recombination of wild-type BmNPV DNA with this plasmid. Recombinant viruses were isolated by plaque assay with an overlay containing 0.2 mg of 5-bromo-4-chloro3-indolyl-3-D-galactoside (X-Gal) per ml as described previously (10). In order to confirm the insertion of the ,-galactosidase gene cassette at the cysteine protease gene locus, BmCysPD DNA was analyzed by restriction endonuclease and Southern blot analyses as described previously (19). Briefly, genomic DNA of BmNPV T3 or BmCysPD extracted from viral particles was digested with one or two of several restriction endonucleases, electrophoresed on 0.7% agarose gels in Tris buffer, transferred to nylon membranes, and subjected to Southern blot hybridization with a probe specific for the BmNPV-CP gene which was generated by cleavage of the BmNPV BamHI F fragment with ApaI and HpaI (see double-underlined sites in Fig. 2) and subsequent purification and radioactive labeling of the 0.4-kb resulting fragment. Blots were washed twice with 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)0.1% sodium dodecyl sulfate (SDS) for 20 min and once with 0.1 x SSC-0.1% SDS, dried, and exposed to X-ray film. Cysteine protease assay. The cysteine protease assay was performed by a modification of the method described by Kobayashi et al. (12). BmN cells were infected with BmNPV T3 or BmCysPD at a multiplicity of infection of 10 as described previously (16). At 40 h p.i., cells were scraped from culture dishes with a rubber policeman and centrifuged (2,000 X g, 10 min), and the medium was removed. Cell pellets were washed twice with Tris-buffered saline. Approximately 3 x 107 cells of each type were homogenized in 10 mM sodium phosphate buffer (pH 6.8)-i mM dithiothreitol by 50 strokes of a Dounce homogenizer with an A pestle. The homogenates were centrifuged (10,000 x g, 20 min) at 4°C, and the supernatant was assayed by adding 250 RI to 400 ,ul of Azocoll (6 mg/ml in 100 mM succinic acid-NaOH, pH 4.1) (Sigma, St. Louis, Mo.). A mock-enzyme control was done in which 250 ,ul of 10 mM sodium phosphate buffer (pH 6.8)-i mM dithiothreitol without cell extract was added to 400 RI of Azocoll solution and assayed. After shaking (200 rpm) for 5 h at 37°C, 600 RI of 10% SDS was added; the samples were vortexed and then centrifuged (13,000 x g, 2 min). The A520 of the supernatant was measured by using the control as a blank. All samples were assayed in the presence and absence of E-64 (2.8 x 10' M)
(Sigma).
Effects of BmCysPD on B. mori larvae. Fourth- or fifth-instar B. mon larvae were starved for several hours, anesthetized by immersion in ice-water, and injected subcutaneously with 10 RI of a viral suspension (-2 x 105 PFU) containing 6 mg of kanamycin per ml and BmCysPD or BmNPV T3. Infected larvae were placed in paper cups and reared on an artificial diet. The symptoms of infected larvae were observed during infection and after death. Larvae infected with BmCysPD or BmNPV T3 were dissected, and degradation of tissues and polyhedron production in fat body and hemolymph were
examined by light microscopy. Nucleotide sequence accession number. The nucleotide sequences reported in this article can be obtained from GenBank under the accession number U12688. RESULTS A putative BmNPV cysteine protease. A part of a papain-like sequence has been identified in the AcNPV genome at around 80 map units, immediately downstream of the AcNPV gp64 gene (34). The corresponding region in the BmNPV genome
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