Characterization of Perina Nuda Nucleopolyhedrovirus (Penunpv) Polyhedrin Gene
Total Page：16
File Type：pdf, Size：1020Kb
JOBNAME: JIP 67#3 96 PAGE: 1 SESS: 51 OUTPUT: Tue May 28 17:14:06 1996 /xypage/worksmart/tsp000/69984c/12 JOURNAL OF INVERTEBRATE PATHOLOGY 67, 259–266 (1996) ARTICLE NO. 0041 Characterization of Perina nuda Nucleopolyhedrovirus (PenuNPV) Polyhedrin Gene CHIH-MING CHOU,* CHANG-JEN HUANG,† CHU-FANG LO,* GUANG-HSUNG KOU,* AND CHUNG-HSIUNG WANG‡,1 *Department of Zoology, National Taiwan University, Taipei, Taiwan 107; †Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; and ‡Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan 107 Received March 21, 1995; accepted February 13, 1996 leoptera, Neuroptera, Thysanura and Trichoptera as The Perina nuda nucleopolyhedrovirus (PenuNPV) well as from the crustacean order Decapoda (shrimp) polyhedrin gene was located in EcoRI-G (6.3 kilobase (Murphy et al., 1995). In vivo and in vitro infective pairs; kbp) and PstI-G (4.3 kbp) fragments of its geno- studies of NPVs have shown that most NPVs are effec- mic DNA. A portion of 1333 nucleotides (nt) containing tively replicated only in a limited number of host spe- this gene was sequenced. An open reading frame of 735 cies. Currently, over 600 baculoviruses have been re- nt encoded a 245-amino-acid-long polyhedrin. A con- ported in the literature, but fewer than 20 have been served TAAG motif which is associated with transcrip- studied at the molecular level (Vlak and Rohrmann, tional start sites was identified 51 nt upstream of the 1985; Miller, 1988). translation initiation codon of PenuNPV polyhedrin The baculoviruses produce polyhedrin and p10 at gene. A putative polyadenylation signal, AATAAA, was found 116 nt downstream of the termination codon very high levels in the late phase of infection. Polyhed- (TAA). Comparison of the amino acid sequences of rin is the major crystalline protein matrix of the viral PenuNPV polyhedrin with those of other NPVs occlusion bodies which serve to protect the viruses from showed that PenuNPV poyhedrin was most close- the environment outside the host insects (Vlak and ly related to Orgyia pseudotsugata multiple NPV Rohrmann, 1985; Rohrmann, 1986a). The molecular (OpMNPV) polyhedrin. © 1996 Academic Press, Inc. mass of polyhedrin is approximately 29,000 Da (dal- KEY WORDS: Polyhedrin gene; Perina nuda nucleo- tons), and it may be produced in greater amounts than polyhedrovirus; baculovirus. any other proteins in virus-infected insect cells (Rohr- mann, 1986a). By using a strong promoter of the poly- hedrin gene to drive the expression of a foreign gene, INTRODUCTION the baculovirus expression system is now widely used Perina nuda nucleopolyhedrovirus (PenuNPV) is a for large-scale production of eukaryotic proteins member of the family Baculoviridae and belongs to the (Luckow and Summers, 1988). genus Nucleopolyhedrovirus (Murphy et al., 1995). Ficus transparent wing moth, P. nuda Fabricius Baculoviruses are large, enveloped, double-stranded (Lepidoptera: Lymantriidae), is a serious pest of DNA viruses with genomes ranging in size from 90 to banyan (Ficus spp.) and is a major defoliator of forest 160 kbp (kilobase pairs). Baculoviridae have been di- and shade trees in Southeastern Asia (Su et al., 1983; vided into two genus: Granulovirus (GV) and Nucleo- Lo et al., 1990; Wang and Tsai, 1995). An epizootic polyhedrovirus (NPV). Both NPVs and GVs can pack- disease of nuclear polyhedrosis of P. nuda occurs from age their virus particles (occluded virus) into occlusion spring to early summer in Taiwan and mainland bodies (polyhedra or granules) in the late phase of in- China. The main pathogen was found to be the bacu- fection. However, baculoviruses have been isolated lovirus, PenuNPV. PenuNPV is a pathogen with high only from arthropods, primarily from insects of the or- virulence and specificity to this moth. PenuNPV al- der Lepidoptera, but also Hymenoptera, Diptera, Co- ready inhibits outbreaks of P. nuda in nature and is therefore a good candidate for biological control of this moth (Su et al., 1983). PenuNPV has been isolated from The sequence data in this paper have been submitted to the P. nuda larvae in Taiwan (Lo et al., 1990; Wang and EMBL/GenBank Data Libraries under Accession No. U22824. 1 Tsai, 1995), and the genome size was estimated to be To whom correspondence should be addressed at Department of 121 kbp (Wang, personal communication). Recently, a Plant Pathology and Entomology, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei 107, Taiwan. Fax: 2-363-8179; E-mail: highly susceptible cell line (NTU PN-HH) derived from [email protected]. P. nuda pupal ovaries was established (Wang et al., 259 0022-2011/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved. JOBNAME: JIP 67#3 96 PAGE: 2 SESS: 52 OUTPUT: Tue May 28 17:14:06 1996 /xypage/worksmart/tsp000/69984c/12 260 CHOU ET AL. 1996, in press). The characteristics of PenuNPV, in- merase (Promega), 100 ng of viral DNA, 0.5 g of each cluding its host ranges in vitro and in vivo, the protein primer, 200 M of four dNTP, and 1× reaction buffer profiles of its polyhedra and virions, the restriction en- (10 mM Tris–HCl, pH 9, 50 mM KCl, 1.5 mM MgCl2, zyme profiles of its DNA, and the location of the poly- 0.1% Triton X-100). Amplifications were performed hedrin gene in the genome have been studied and it is over 30 cycles of denaturing at 94°C for 1 min, followed suggested to be a distinct NPV (Wang, personal com- by annealing at 50°C for 1 min, and elongating at 72°C munication). for 3 min. There was a final extension step of 5 min at In this paper we describe the isolation, cloning, 72°C in a MJR PTC-100 Thermocycler (Watertown, and characterization of the polyhedrin gene from MA). PCR products were analyzed in 1.5% agarose gels PenuNPV. In addition, the nucleotide sequence and in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH transcriptional analysis of this gene are reported. Our 8.0) containing 0.5 g/ml ethidium bromide and visu- data indicate that PenuNPV polyhedrin is very similar, alized with short-wave ultraviolet light. In addition, at the amino acid level, to that of OpMNPV. PCR products of approximately 680 bp in length were subcloned into the SmaI site of plasmid pUC18 MATERIALS AND METHODS (Yanisch-Perron et al., 1985) and sequenced by the dideoxy chain termination method (Sanger et al., 1977) Viruses, Insects, and Cells using a Sequenase kit (United States Biochemical An isolate of P. nuda NPV (PenuNPV) was obtained Corp. Cleveland, OH) with either universal primers or from infected larvae with nuclear polyhedrosis symp- specific internal primers. Oligonucleotides were syn- tom. Infected larvae P. nuda specimens were collected thesized commercially on an Applied Biosystems DNA from the campus of National Taiwan University and synthesizer. other sites in Northern Taiwan (Lo et al., 1990; Wang Southern Blot, Cloning, and Sequence Analysis and Tsai, 1995). The P. nuda cell line, NTU PN-HH, was established from P. nuda pupal ovary (Wang et al., The genomic DNA of PenuNPV was digested with 1996, in press). The virus was multiplied in NTU PN- several restriction enzymes and then electrophoretical- HH cells and fourth instar larvae of P. nuda were fed ly separated in 0.8% agarose gels and transferred to with polyhedra or inoculated with viral suspension for Hybon N nylon membrane (Amersham) by using a mass production of PenuNPV polyhedra (Lo et al., vacuum transfer unit (Hoefer TE-80) for 60 min. The 1990). Autographa californica nuclear polyhedrosis vi- blot was hybridized at 37°C for 16 hr to DIG-labeled rus (AcMNPV) strain E2 (Smith and Summers, 1978) probes (Sambrook et al., 1989). The AcMNPV polyhe- was kindly supplied by Dr. M. J. Fraser at Department drin gene fragment was used as control. After hybrid- of Biological Sciences (University of Notre Dame, ization and blocking membranes were incubated with U.S.A.) and propagated in IPLB SF-21 AE cells. NTU alkaline phosphatase-conjugated anti-DIG antibody PN-HH and IPLB SF-21 AE cells were maintained as (1:10,000 dilution in buffer 2) for 1 hr and followed by monolayers in plastic tissue culture flasks (Nunc and/ two 15-min washes in washing buffer (100 mM maleic or Cloning Co.) and incubated at 28°C in TNM-FH me- acid, 150 mM NaCl, pH 7.5, 0.3% Tween 20). The mem- dium (Hink, 1970) supplemented with 100 IU/ml peni- brane was then equilibrated with buffer 3 (100 mM cillin, 100 g/ml streptomycin, 1.25 g/ml fungizone, Tris–HCl, 100 mM NaCl, 50 mM MgCl2; pH 9.5) and and 10% fetal calf serum. The polyhedra were isolated incubated with Lumigen-PPD solution (1:200 dilution and purified as described by Lee and Miller (1978). in buffer 3) for 5 min. Membranes were exposed to Kodak XAR-5 film at 37°C for 15–60 min and then the PCR Amplification of Viral DNA Polyhedrin Gene film was developed. Cloning of positive restriction en- zyme fragments of PenuNPV DNA was achieved by In order to characterize PenuNPV polyhedrin gene, isolated from agarose gel and subcloning into the plas- the amino acid sequences of the polyhedrin gene from mid pUC18 (Yanisch-Perron et al., 1985). DNA se- several baculoviruses (Rohrmann, 1986a; Vlak and quence analysis was performed using the Sequenase Rohrmann, 1985; Rohrmann et al., 1981) were com- kit as described above. pared and appropriate highly conserved regions were chosen to design primers (primers 35 and 36) for the RNA Isolation polymerase chain reaction (PCR, Mullis and Faloona, 1987). The primer sequences were as follows: primer For Northern blot hybridization and primer exten- T T A T 35, 5Ј -AC /C TA /C GTG TAC GAC AAC AA /G TA /C sion analysis, total RNA was isolated from PenuNPV- T T T TAC AAA-3Ј; primer 36, 5Ј -GG /C GCG TC /G GG /C infected NTU PN-HH cells at 0, 12, 24, 48, 72, 96, 120, T T A T A GCA AA /C TC /C TT /T AC /C TT /G AA-3Ј.