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Molecular Cloning and Characterization of Cytoplasmic Polyhedrosis Virus Polyhedrin and a Viable Deletion Mutant Gene

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Molecular Cloning and Characterization of Cytoplasmic Polyhedrosis Virus Polyhedrin and a Viable Deletion Mutant Gene JOURNAL OF VIROLOGY, Jan. 1988, p. 211-217 Vol. 62, No. 1 0022-538X/88/010211-07$02.00/0 Copyright © 1988, American Society for Microbiology Molecular Cloning and Characterization of Cytoplasmic Polyhedrosis Virus Polyhedrin and a Viable Deletion Mutant Gene MAX ARELLA,1 CLAUDE LAVALLEIE,12 SERGE BELLONCIK,l AND YASUHIRO FURUICHI2* Department of Virology, Institut Armand-Frappier, University of Quebec, Montreal, Quebec, Canada,' and Nippon Roche Research Center, Department of Molecular Genetics, Kamakura, Japan2 Received 12 May 1987/Accepted 21 September 1987 The double-stranded RNA genome of Bombyx mori cytoplasmic polyhedrosis virus (CPV) was converted to double-stranded DNA and cloned into plasmid pBR322. The complete nucleotide sequence of cloned genome segment 10, which encodes virus polyhedrin polypeptide, was determined. The CPV polyhedrin gene consists of 942 based pairs and possesses a long open reading frame that codes for a polypeptide of 248 amino acids (molecular weight, 28,500), consistent with an apparent molecular weight of 28,000 previously determined for purffied polyhedrin. No sequence homology was found between CPV polyhedrin and polyhedrins from several nuclear polyhedrosis viruses. In addition to the polyhedrin gene, we completed the sequence analysis of a small deletion mutant gene derived from the polyhedrin gene. This mutant gene consists of two subset domains of the polyhedrin gene, i.e., the 5'-terminal 121 base pairs and the 3'-terminal 200 base pairs. An in vitro transcription demonstrated that the small mutant gene is transcribed by virion-associated RNA polymerases. These data confirm the importance of CPV terminal sequences in virus genome replication. Cytoplasmic polyhedrosis viruses (CPVs), one genus of reported previously (12). During the process, unexpectedly, the family Reoviridae, infect midgut cells of a wide range of we found in a CPV dsRNA mixture an extra subgenomic insects (22). Cytoplasmic polyhedrosis disease caused by segment of about 300 base pairs referred to here as the SP CPV infection results in the accumulation of occlusion gene (small polyhedrin gene). Since the SP gene appeared to bodies or polyhedra in the cytoplasm which are formed by be related in sequence to the polyhedrin gene, we also the crystallization of the viral polyhedrin polypeptide. In the cloned the cDNA into Escherichia coli plasmid pBR322 and late infection stage, newly formed viruses are occluded in investigated it together with the polyhedrin gene. A direct the polyhedron complex, perhaps to stabilize infectious sequencing and a comparative study clearly indicated that virus particles. Greater biological meaning of the virus the SP gene is a deletion mutant derived from the polyhedrin occlusion bodies may lie in the accurate delivery of virus to gene. This report describes the complete sequence informa- the target intestinal cells, where the occlusion bodies are tion of CPV polyhedrin as well as its deletion mutant gene. solubilized by intestinal alkaline pH and the infectious virus particles are released. MATERIALS AND METHODS The genome of CPV, like that of other members of the Preparation of CPVs and genomic dsRNAs. The type 1 Reoviridae, consists of 10 double-stranded RNA (dsRNA) CPV of B. mori was propagated by infecting fifth-instar segments (4). Each genome dsRNA segment is composed of larvae with purified CPV. The polyhedra that contain virus an mRNA (plus strand) and its complement (minus strand) in particles were isolated from infected midguts, and the virus an end-to-end base-paired configuration, except for the was purified from polyhedra as described previously (6, 27). protruding 5' cap in the plus strand (6, 7). The segments are Other CPVs were prepared similarly from infected Euxoa transcribed by virus-associated RNA polymerase to form scandens (type 5 virus) and from Inachis io and Spodoptera capped mRNAs which also function as templates for a exigua (type 2 and type 11 viruses, respectively) (22) and replicase in virus-infected cells. Thus, each genome segment were kindly provided by C. C. Payne at the Glasshouse should contain recognition sites for genome transcription, Crops Research Institute, Littlehampton, United Kingdom. mRNA translation, duplex segment replication, and correct Molecular cloning of CPV polyhedrin and SP dsRNA genes. assembly into virus particles. Consistently, a terminal se- The cDNA for the polyhedrin gene was prepared from quence of denatured CPV dsRNA and cloned into E. coli plasmid m7GpppAmGUAAA-----GUUAGCC pBR322 (at the PstI site) as described previously for human U CAUUU-----CAAUCGG reovirus (2), human rotavirus (12), and wound tumor virus (1). E. coli clones that contained the recombinant plasmids was identified for segments of the prototype CPV that infects which harbor polyhedrin cDNA were detected by colony Bombyx mori, suggesting that the conserved terminal se- hybridization (8) with [32P]pCp-end-labeled segment 10 quence is important for viral replication (15). dsRNA which was purified by polyacrylamide gel electro- The polyhedrin protein is coded for by the smallest phoresis. genome segment, segment 10, and its expression appears to For SP gene cloning, SP dsRNA was first purified from the occur most extensively in the late infection stage (21). In an mixture of CPV dsRNA by polyacrylamide gel electropho- effort to understand the regulation of polyhedrin gene resis. After cDNA synthesis, it was cloned into pBR322 in expression and to characterize the genome structure, we the same way as the polyhedrin gene and the bacteria which cloned the segment 10 cDNA by the method which we have harbored a recombinant SP cDNA-pBR322 were identified by colony hybridization with [32P]pCp-end-labeled segment * Corresponding author. 10 and SP dsRNAs. 211 212 ARELLA ET AL. J. VIROL. A B RESULTS Presence in B. mori CPV of small polyhedrin-related gen- G t.- n tri to- ome segment. A mixture of genome dsRNA segments of B. '_;,'1:'.'.a% mori CPV (BmCPV) extracted from purified virus were resolved into nine discrete bands by polyacrylamide gel ' 'M electrophoresis (Fig. 1A). Recently, we found an extra, small genomic dsRNA segment in a CPV genome RNA ltx preparation (Fig. 1B). This small segment had been over- looked before, since it ran off the gel under the normal 2 .z: WmA'InIt.aNk, electrophoresis conditions, which were intended to resolve SP dsRNA high-molecular-weight segments. A preliminary 3'-terminal analysis of the [32P]pCp-end-labeled small RNA indicated that it contained 3'-ACU-OH and 3'-GCC-OH, the common '5. 3'-terminal sequences for CPV genome dsRNA segments : (data not shown). When the [32P]pCp-3'-end-labeled small RNA was isolated from gels and used for hybridization to the other individual genome dsRNA segments, which were denatured and fixed on a nitrocellulose filter, it hybridized to genome segment 10 and segments 1 to 3 (Fig. 2A). The labeled segment 10 also hybridized specifically with this small RNA (Fig. 2B). Molecular cloning of polyhedrin and its related SP genome RNAs. A mixture of duplex cDNA of CPV genome dsRNAs 7- was prepared as described before (6) except that the 3' tailing of the template dsRNAs was done by polyadenylation 9- with E. coli poly(A) polymerase as described by Cashdollar et al. (2). The cDNAs were then poly(dC) tailed with E. coli terminal deoxytransferase, annealed to poly(dG)-tailed pBR322 plasmid DNA, and cloned into E. coli RR1 cells. Clones that contained the recombinant plasmids harboring the polyhedrin cDNA gene were isolated after colony hy- bridization with 3'-[132P]pCp-labeled polyhedrin gene FIG. 1. Detection of small dsRNA molecules in CPV genome (genome segment 10) as a probe. The CPV polyhedrin cDNA RNA. CPV genome RNA was prepared from purified virus, and the was excised with PstI as a single band which migrated 3' termini were radiolabeled with [32P]pCp with RNA ligase as slightly slower than genome segment 10 (Fig. 3). described before (28). The labeled CPV genome segments were For cloning of SP dsRNA, the mixture of CPV genome separated by 5% polyacrylamide gel electrophoresis in 50 mM Tris-phosphate buffer (pH 8) containing 5 mM EDTA at approxi- RNA was first resolved by polyacrylamide gel electrophore- mately 5 to 7 V/cm. Electrophoresis was run for 4 h (lane B) and 18 sis and the SP dsRNA was extracted from the gel to avoid h (lane A). contamination of the polyhedrin gene. A small amount (approximately 100 ng) of purified SP dsRNA was 3' poly- adenylated, and the cDNA was prepared as the polyhedrin gene was. Clones containing the SP cDNA gene were Determination of nucleotide sequence. Cloned polyhedrin detected with [32P]pCp-labeled polyhedrin and SP RNAs. and SP cDNA genes were excised from the recombinant Several cDNA clones that contained the full length were pBR322 DNA by digestion with endonuclease PstI and their obtained for SP RNA genes as determined by agarose gel nucleotide sequences were analyzed by the Maxam-Gilbert (18) and M13 chain termination procedures (26). Sequence analysis was done for both plus and minus strands. GENOME SEGmE\TS CPV in vitro transcription. CPV mRNAs were synthesized in the in vitro transcription reaction as described before (5, 2 28). Purified CPV (-10 pg) was incubated at 30°C in a 1 3 4 5 6 7 a 9 C transcription reaction mixture (100 Rl) consisting of 10 mM Tris hydrochloride (pH 8.0), 10 mM MgCl2, 1 mM each ATP, A *. * 0, - CTP, and GTP, 0.4 mM UTP, 10 pLCi of [a-32P]UTP (specific activity, 3,000 Ci/mmol; Amersham Corp., Arlington Heights, Ill.), 1 mM S-adenosylmethionine, and 50 units of B . RNasin (catalog no. 121800; Promega Biotec, Madison, Wis.) per ml. After 1 h, the reaction mixture was diluted FIG. 2. Characterization of small dsRNA by dot-blot hybridiza- fivefold with 50 mM Tris hydrochloride buffer (pH 8.0) and tion.
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