Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India1
Department of Microbiology, University of Washington, Seattle, WA 98195, USA2
Author for correspondence: Ananta Ghosh. Fax +91 3222 778707. e-mail aghosh{at}hijli.iitkgp.ernet.in
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Abstract |
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Introduction |
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The genomes of CPVs, like those of other members of the Reoviridae, are usually composed of 10 double-stranded (ds) RNA segments (S1S10) (Payne & Mertens, 1983 ) although, in some cases, a small, eleventh segment (S11) has been reported (Arella et al., 1988
). Each dsRNA segment is composed of a plus-strand mRNA and its complementary minus strand in an end-to-end base-paired configuration except for a protruding 5' cap on the plus strand. On the basis of electrophoretic migration patterns of the dsRNA segments in agarose or acrylamide gels, they have been classified into 14 different types (Belloncik et al., 1996
; Fouillaud & Morel, 1994
; Payne & Rivers, 1976
; Payne & Mertens, 1983
).
Among the family Reoviridae, complete nucleotide sequences of dsRNA genomes have been reported for members of the genera Orthoreovirus, Rotavirus, Orbivirus and Phytoreovirus and putative members of Fijivirus and Cypovirus (Duncan, 1999 ; Estes & Cohen, 1989
; Roy et al., 1990
; Suzuki, 1995
; Nakashima et al., 1996
). From the cypoviruses, segment 10, encoding the polyhedrin gene, has been cloned and sequenced from Bombyx mori CPV (BmCPV), Euxoa scandens CPV, Orgyia pseudotsugata CPV, Heliothis armigera CPV and Choristoneura fumiferana CPV (Arella et al., 1988
; Echeverry et al., 1997
; Fossiez et al., 1989
; Galinski et al., 1994
; Mori et al., 1989
). However, no sequence similarities were found. In the case of BmCPV, segments 9, 8 and 5, encoding non-structural proteins NS5, p44 and p101 (Hagiwara et al., 1998a
, b
, 2001
), and segments 4, 6 and 7, encoding structural proteins VP3, VP4 and VP5 (Hagiwara & Matsumoto, 2000
; Ikeda et al., 2001
), have also been cloned and sequenced. Recently, nucleotide sequences of segments 1, 2 and 3 of BmCPV and complete sequences of Lymantria dispar CPV, Trichoplusia ni CPV and Choristoneura fumiferana CPV have also been deposited in GenBank.
The Indian non-mulberry Saturniidae silkworms Antheraea mylitta, Antheraea assamensis and Antheraea proylei are wild in nature and produce exotic varieties of silk called Tasar and Muga silk (Jolly et al., 1974 ). A. mylitta is a tropical variety whereas A. assamensis and A. proylei are semi-temperate and temperate varieties. Each year, CPV infection destroys a major population of these silkworms and reduces the yield of tasar silk (Jolly et al., 1974
).
We have previously characterized the structure of CPVs from these three silkworm species by electron microscopy and their genomes by electrophoresis and found that the genomes are similar to that of a type IV CPV that infects Actias selene and consist of 11 dsRNA molecules. The molecular sizes of the different RNA segments of Antheraea mylitta CPV (AmCPV), Antheraea assamensis CPV (AaCPV) and Antheraea proylei CPV (ApCPV) isolates are similar (3·9, 3·8, 3·6, 3·3, 2·15, 1·9, 1·8, 1·7, 1·45, 1·4 and 0·35 kb) but are quite different from those of BmCPV, which is a type 1 CPV (Qanungo et al., 2000 ).
In order to compare the AmCPV, AaCPV and ApCPV isolates further with each other and with other known members of the genus Cypovirus such as BmCPV, we describe the molecular cloning and sequencing of RNA segment 9 from these three isolates and the characterization of their encoded proteins by expression in E. coli and insect cells. We show that a new CPV infects all three Indian Saturniidae silkworms and its segment 9 encodes a novel protein. By immunoblot and immunofluorescence analysis, we also show that the product of segment 9 is expressed in infected cells as a non-structural protein and binds viral RNA. Thus, it appears that it may play a role in the regulation of genomic RNA function and packaging.
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Methods |
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Purification of polyhedral bodies, isolation of total genomic RNA and extraction of segment 9 RNA.
Polyhedra from infected larvae were purified by sucrose-density-gradient centrifugation according to a modification (Qanungo et al., 2000 ) of the method of Hayashi & Bird (1970)
. The purity of the polyhedral preparations as checked by light and scanning electron microscopy was more than 95%. Genomic RNA was extracted from purified polyhedra by a standard guanidium isothiocyanate method (Ausubel et al., 1995
). RNA was then fractionated in 10% polyacrylamide gels and the segment 9 RNA was excised from ethidium bromide-stained gels and eluted by the crush and soak method (Sambrook et al., 1989
).
Molecular cloning and sequencing of segments 9 of the AmCPV, AaCPV and ApCPV genomes.
cDNA of the segment 9 genomic RNA of AmCPV was prepared as described previously for the cloning of the polyhedrin gene of BmCPV (Arella et al., 1988 ). In brief, approximately 200 ng segment 9 RNA (1·5 kb) was denatured by heating at 95 °C in the presence of 95% DMSO and then polyadenylated with ATP and poly(A) polymerase (Life Technologies).
First-strand cDNAs of both RNA strands were synthesized using the primer 5' AAGCAGTGGTAACAACGCAGAGTACT30VN 3' (N=A, C, G or T; V=A, G or C) (Clontech) and Superscript II AMV reverse transcriptase (Gibco) according to the manufacturer's protocol. RNA was then removed with RNase H (Roche) at 37 °C for 20 min. The resulting cDNAs of the two strands were annealed at 65 °C for 60 min, ends were repaired by Taq DNA polymerase (Bioline) and the cDNAs were cloned into pCR2.1 TOPO (Invitrogen) to create recombinant plasmid pCR2.1 TOPO/AmCPV-9. After transforming E. coli TOP 10 cells (Invitrogen), plasmids were isolated and characterized by EcoRI digestion. A recombinant plasmid containing the full-length insert (1·5 kb) was then sequenced by using an ABI 377 automated DNA sequencer with M13 forward and reverse primers as well as internal primers designed from deduced sequences. Homology searches were done using BLAST (Altschul et al., 1997
) and the secondary structures of segment 9-encoded proteins were predicted using PHD (Rost & Sander, 1994
).
For cloning segment 9 RNA from ApCPV and AaCPV, primers AG8 (5' GAATCGACGTAGTCCTTGAAC 3'; forward primer) and AG9 (5' TCATACGACGCAAGTGCTCAT 3'; reverse primer) were synthesized on the basis of terminal RNA sequences from AmCPV segment 9. Purified segment 9 dsRNA (200 ng) from AaCPV and ApCPV was denatured at 100 °C for 5 min, chilled rapidly on ice and then converted to cDNA by incubating at 50 °C for 60 min in a reaction mixture containing 20 pmol forward and reverse primers, 20 U RNase inhibitor, 40 U Thermoscript reverse transcriptase (Gibco), 10 mM DTT and 2 mM of each dNTP. The reaction was stopped by heating at 95 °C for 10 min and, after cooling, RNA was removed with RNase H at 37 °C for 20 min. The resulting cDNAs of the two strands were then annealed, ends were repaired with Taq DNA polymerase and cDNAs were cloned into pCR2.1 TOPO as described above.
Northern hybridization.
In order to verify cloning of the segment 9 cDNA from the corresponding RNA of AmCPV, all of the genomic dsRNA segments were separated in a 10% polyacrylamide gel and observed by staining with ethidium bromide. The RNA segments in the gel were then denatured by brief treatment with 0·1 M NaOH, neutralized in TAE buffer and electroblotted onto nitrocellulose membrane. The membrane was then hybridized with 32P-labelled cloned segment 9 cDNA from AmCPV, washed and autoradiographed (Bittner et al., 1980 ; Feinberg & Vogelstein, 1983
; Qanungo et al., 2000
).
Expression of AmCPV segment 9 in E. coli.
The entire 345 amino acid protein-coding region of AmCPV segment 9 cDNA, from nucleotide 31 to 1068, was amplified by PCR from plasmid pCR2.1 TOPO/AmCPV-9 by using Advantage Taq DNA polymerase (Clontech) and two synthetic primers, AGQ1 (5' GTAGTCCTGGATCCAGACTAGACATG 3'; forward primer) and AGQ2 (5' CGGCATGTTAAGCTTGAATTACTTTC 3'; reverse primer), complementary to bases 833 and bases 10611086, respectively, and containing BamHI (in the forward primer) and HindIII (in the reverse primer) restriction enzyme sites (underlined). The amplified PCR product (1·1 kb) was digested with BamHI and HindIII, separated on a 1% agarose gel and purified from the gel by using a Qiaquick gel extraction kit (Qiagen). The purified DNA was ligated to BamHI/HindIII-digested pQE-30 vector (Qiagen) in-frame with a sequence encoding six histidine residues at the N terminus. The resulting recombinant plasmid, pQE-30/NSP38, was then transformed into E. coli M15 and colonies were screened following BamHI and HindIII digestion.
For protein expression, recombinant bacteria were grown in 5 ml LB medium containing ampicillin (60 µg/ml) for 4 h at 37 °C and then induced with 1·5 mM IPTG for an additional 5 h at the same temperature. Bacteria was harvested by centrifugation, lysed by boiling with sample loading buffer (60 mM TrisHCl, pH 6·8; 10% glycerol; 2% SDS; 5% -mercaptoethanol and 1 µg/ml bromophenol blue) for 3 min and then loaded onto a 3·5% stacking gel cast above a 10% resolving SDSpolyacrylamide gel (Laemmli, 1970
). After electrophoresis, the protein bands in the gel were stained with Coomassie brilliant blue (Ausubel et al., 1995
). The molecular mass of the recombinant NSP38 was determined by comparison to standard protein molecular mass markers and by using Quantity One software in Gel-Doc 2000 (Bio-Rad).
Purification of His-tagged protein.
Recombinant bacteria containing pQE-30/NSP38 were grown in 1 l LB medium and induced with IPTG as described above. The insoluble 6xHis-tagged NSP38 fusion protein was first prepared to 60% purity from inclusion bodies (Caligan et al., 1995 ). After solubilizing the inclusion bodies in 6 M guanidine hydrochloride, further purification of protein was carried out by using a NiNTA agarose kit (Qiagen) according to the manufacturer's protocol and then by FPLC using a Superdex 75 column (Pharmacia) in the presence of 8 M urea. The total amount of purified NSP38 was determined by the method of Bradford (1976)
using BSA as the standard and purity was checked by SDS10% PAGE (Laemmli, 1970
).
Rabbit immunization and production of polyclonal antibodies.
One rabbit was immunized with bacterially expressed, purified, recombinant His-tagged NSP38 protein by standard methods (Harlow & Lane, 1988 ). In brief, purified protein (625 µg) was mixed with Freund's complete adjuvant and injected intramuscularly at multiple sites. Three booster doses with Freund's incomplete adjuvant and the same amount of protein were administered via the same route at 4-week intervals. Twelve days after the final booster, blood was collected, serum was prepared and the antibody titre was determined by ELISA (Harlow & Lane, 1988
). Specific antibody was purified by antigen (NSP38Sepharose) affinity chromatography (Harlow & Lane, 1988
; Sambrook et al., 1989
).
Transient expression of NSP38 in insect cells.
The NSP38 ORF was amplified from pCR2.1 TOPO/AmCPV-9 by PCR by using the primers AG10S (5' AATCGAGGTACCCCTTGAACAAGC 3'; forward primer) and AG12S (5' TGTTGATTCCGCGGCATGTTGATG 3'; reverse primer) and PWO high-fidelity Taq polymerase (Roche). KpnI and SacII sites were introduced in the forward and reverse primers, respectively (underlined). The PCR-amplified product (1·1 kb) was digested with KpnI and SacII, gel-purified (Qiagen) and cloned into pITZ/V-5His vector (Invitrogen) to make the recombinant plasmid pITZ/V-5His-NSP38.
Sf9 cells (2x106) were transfected with purified pITZ/V-5His-NSP38 plasmid (1 µg) by Bacfectin (Clontech) and cells were checked 48 h after transfection for the expression of green fluorescent protein (GFP) in the transfected cells by fluorescence microscopy. Three days post-transfection, the cells were harvested and a cytoplasmic extract was prepared by the method of Behrens et al. (1996) . In brief, cells were resuspended (7·5x106 cells/ml) in buffer A (10 mM TrisHCl, pH 8·0, 1·5 mM MgCl2, 10 mM NaCl, 1 mM DTT, 1 mM PMSF) and allowed to swell on ice for 30 min. After vortexing vigorously, glycerol (10%, v/v), NP-40 (1%, v/v) and CHAPS (0·5%, v/v) were added and the mixture was incubated on ice for an additional 1 h with occasional shaking. Cell debris was removed by centrifugation at 8000 g for 10 min and the supernatant was assayed for NSP38 expression by immunoblotting and poly(rI).(rC)agarose binding (Hagiwara et al., 1998a
).
Construction of recombinant baculovirus and expression of NSP38 in Sf9 cells.
The protein-encoding region of NSP38 was excised from pQE-30/NSP38 by BamHI and HindIII digestion and cloned into the baculovirus transfer vector pBluebac-His2A (Invitrogen) at the 3' end of baculovirus polyhedrin promoter. The resulting recombinant baculovirus transfer vector and BsuI-digested, linearized Autographa californica nuclear polyhedrosis virus DNA (Invitrogen) were co-transfected into Sf9 cells by using Insectin Plus according to the manufacturer's protocol (Invitrogen). Four days post-transfection, the culture medium was collected and recombinant baculovirus showing cytopathic effects but not the production of polyhedral occlusion bodies was isolated by plaque purification (OReilly et al., 1992 ). For the expression of NSP38, Sf9 cells (2x107 cells in a 1 l spinner flask) were inoculated with the recombinant baculovirus at an m.o.i. of 5. The cells were harvested 6 days post-infection by centrifugation and a cytosolic extract was prepared by the method of Behrens et al. (1996)
. Baculovirus-expressed, His-tagged NSP38 was then purified by NiNTA affinity chromatography (Invitrogen).
SDSPAGE and immunoblotting.
In order to localize NSP38 expression, guts were dissected from virus (AmCPV)-infected and uninfected fifth instar larvae and homogenized with PBS and supernatants were collected after centrifugation at 10000 g for 10 min. Recombinant baculovirus-infected Sf9 cells, Sf9 cells transiently transfected with pITZ/V-5His-NSP38 plasmid and uninfected Sf9 cells (2x106) were homogenized and lysates prepared in the same way. Each protein sample was boiled in SDSPAGE sample buffer and separated by SDS10% PAGE under reducing conditions. After electrophoresis, proteins were transferred to Duralose membrane (Stratagene) using a transblot cell (Pharmacia) according to Towbin et al. (1979) . The membrane was blocked for 1 h at room temperature with 0·02% casein in TTBS (100 mM TrisHCl, pH 7·5, 0·9% NaCl, 0·1% Tween 20), washed with TTBS and then incubated with 10000-fold diluted, affinity-purified anti-NSP38 polyclonal antibody for 1 h at room temperature. After washing with TTBS as above, the membrane was incubated with Protein A-conjugated horseradish peroxidase at a dilution of 1:200 for 1 h and then washed and colour development was done by using the HPO colour development kit (Bio-Rad).
Binding assay of NSP38 using poly(rI).poly(rC)agarose.
Poly(rI).poly(rC)agarose (Pharmacia) (100 µl) was washed three times with wash buffer (20 mM HEPESNaOH, pH 7·5, 150 mM KCl, 10% glycerol, 5 mM magnesium acetate, 1 mM DTT, 1 mM benzamidine hydrochloride and 0·5% NP-40) and then incubated with crude cytosolic extracts of Sf9 cells or baculovirus-expressed, purified, soluble, His-tagged NSP38 for 60 min at 4 °C with occasional gentle mixing. Poly(rI).poly(rC)agarose resin was then pelleted by centrifugation at 1000 g for 30 s and washed three times with wash buffer. Bound protein was recovered from the resin by boiling in sample loading buffer and then analysed on SDS10% PAGE. For competition assays, purified NSP38 was preincubated with 5 or 50 µg total AmCPV dsRNA as well as heat-denatured AmCPV ssRNA for 60 min before being mixed with poly(rI).poly(rC)agarose (Hagiwara et al., 1998a ).
Immunofluorescence assay.
Cells were either grown on a slide or a tissue smear was prepared, washed twice with PBS, fixed and permeabilized with ice-cold acetone for 2 min at -20 °C. Slides were dried in air, rehydrated in PBS and blocked with 0·2% casein in PBS for 60 min at room temperature. Slides were then incubated with affinity-purified anti-NSP38 polyclonal antibody (1:1000) in blocking solution in a moist chamber at room temperature for 60 min. After washing three times in PBS, the slides were incubated with FITC-conjugated goat anti-rabbit IgG (1:100) (Sigma), washed, mounted with glycerol and observed by fluorescence microscopy (Harlow & Lane, 1988 ).
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Results |
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Discussion |
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The AmCPV and AaCPV segment 9 cDNAs showed 100% nucleotide sequence identity, whereas the ApCPV sequence showed two nucleotide changes. One of these changed an asparagine to serine and the other was found in the 3' untranslated region. These data suggest that all three CPV isolates are the same and infect different Antheraea species in different geographical regions. However, whether other genome segments are the same is not yet known.
When the nucleotide and deduced amino acid sequences of AmCPV segment 9 were compared with sequences in the GenBank and EMBL/SwissProt databases using BLAST or FASTA programs (Altschul et al., 1997 ), no significant similarity was found to any gene or protein sequence. This indicates that segment 9 encodes a novel protein conserved among a new type of CPV that infects these three Saturniidae silkworms. Amino acid sequence comparisons showed three regions of similarity in NSP38 to the RNA-binding motifs of IBDV and BTV RNA-dependent RNA polymerases (Poch et al., 1989
; Roy et al., 1990
; Yamaguchi et al., 1997
). However, NSP38 had no GDD sequence motif for the NTP-binding site found in the polymerases of single-stranded and double-stranded RNA viruses (Bruenn, 1991
; Dolja & Carrington, 1992
), although a YDD sequence found at amino acids 324326, an LDD sequence at amino acids 2931 and an FDD sequence at amino acids 167169 of NSP38 may be important in binding to CPV RNA.
Immunoblotting and immunofluorescence analysis using anti-NSP38 antibody failed to detect NSP38 in virions or uninfected cells, but the protein was present in virus-infected cells. This suggests that NSP38 is not a structural protein, but may play a role in the regulation of AmCPV genome replication or function.
NSP38 was expressed in E. coli as insoluble inclusion bodies but in soluble form in transiently transfected insect cells or baculovirus-infected insect cells. Transiently produced or baculovirus-expressed and purified NSP38 bound poly(rI).poly(rC)agarose and this binding was abolished competitively by AmCPV dsRNA and ssRNA, indicating that NSP38 produced in insect cells possessed the ability to bind viral dsRNA and ssRNA. In BmCPV, the segment 9-encoded non-structural protein NS5 is expressed in virus-infected cells and binds viral dsRNA (Hagiwara et al., 1998a ). In mammalian orthoreovirus, the non-structural protein
NS binds preferentially to ssRNA, like a single-stranded DNA-binding protein, better than dsRNA and regulates the replication of the reovirus RNA genome (Gillian et al., 2000
). Specific binding of rotavirus non-structural proteins NSP1 and NSP3 to viral RNA has also been reported (Hua et al., 1994
; Poncet et al., 1993
), but their functions in assortment and replication of RNA are not understood. Rotavirus non-structural protein NSP2 has been reported to be expressed at high levels in infected cells, accumulates in the viroplasm in multimeric forms and binds strongly to viral ssRNA (Petrie et al., 1984
; Taraporewala et al., 1999
). It also possesses nucleoside triphosphatase activity, which may provide the energy necessary for the protein to function as a molecular motor that directs the packaging of viral mRNA (Petrie et al., 1984
; Taraporewala et al., 1999
). Another rotavirus protein, NSP5 (a glycosylated phosphoprotein), has been found to be autophosphorylated and to form a complex with NSP2 and viral polymerase that then participates in virus replication and assembly (Blackhall et al., 1997
; Fabbretti et al., 1999
). It is also rich in serine and threonine residues. Since NSP38, encoded by AmCPV segment 9, also contains a large proportion of serine (8·7%) and threonine (6·1%) residues and binds viral RNA, we hypothesize that NSP38 may undergo autophosphorylation during its expression in virus-infected cells and that phosphorylated NSP38 binds viral RNA to play a role in the regulation of viral mRNA function or replication and packaging of the viral RNA genome.
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Acknowledgments |
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Footnotes |
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Received 14 November 2001;
accepted 1 February 2002.
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