Journal of General Virology (1992), 73, 559-566. Printed in Great Britain .... (L. dispar) cells were a gift from Dr M. Shapiro (Insect Pathology. Laboratory, U.S. ...
Journal of General Virology(1992), 73, 559-566. Printedin GreatBritain
559
The predicted amino acid sequence of the spheroidin protein from Amsacta moorei entomopoxvirus: lack of homology between major occlusion body proteins of different poxviruses Myriam Banville, 1 France Dumas, 2 Silvana Trifiro, 1 Basil Arif 3 and Christopher Richardson T M 1Virology Group and 2peptide Synthesis and Protein Sequencing Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, 3Forest Pest Management Institute, Forestry Canada, 1219 Queen Street East, Sault Ste Marie, Ontario P6A 5M7 and 4Department of Microbiology and Immunology, MeGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada
Entomopoxviruses replicate in the cytoplasm of insect cells and characteristically produce occlusion bodies which serve to protect the virion from the environment; the major component of these bodies is a protein called spheroidin. We have previously identified and sequenced the gene encoding the major occlusion body protein of eastern spruce budworm (Choristoneura biennis) entomopoxvirus (CbEPV) and found it to encode a 47K polypeptide which aggregates due to the formation of intermolecular disulphide bonds. In this publication we demonstrate that the insect poxvirus of Amsacta moorei produces spheroidin with a unit Mr of 114.8K. The gene for this protein was cloned and sequenced, and the predicted polypeptide was demonstrated to contain 38 cysteine residues, a leucine zipper for possible protein-protein interactions and 14 potential Asn-linked glycosylation sites. Other than possess-
ing a large number of sulphydryl groups, this protein showed no homology to its analogue found in cells infected with CbEPV. Antibodies directed against occlusion body proteins of the two viruses also failed to cross-react significantly on Western blots. In addition, nucleic acid probes prepared from the two different genes did not cross-hybridize on Southern blots of genomic D N A prepared from the viruses. Finally, the occlusion body proteins from the two insect viruses were compared with the A-type inclusion body protein of cowpox virus. Again, little homology between these proteins was evident, with the exception of a generally high cysteine content and a similarity between their late gene promoters. We conclude that the major occlusion body proteins of different poxviruses possess diverse primary structures, but all are capable of yielding large aggregates through the formation of disulphide bonds.
Introduction
insect gut or under reducing conditions in the presence of sodium carbonate (McCarthy et al., 1974). Little is known about their molecular biology, but the entomopoxvirus of Amsacta moorei (AmEPV) is the most extensively studied member of the insect poxvirus family (Granados & Roberts, 1970; McCarthy et al., 1974, 1975; Langridge & Greenberg, 1981; Langridge et al., 1983; Langridge 1983a, b, c). The D N A genome of AmEPV is 222 to 242 kb in length and is rich in A + T residues (78 to 82%). Recently, the genome of this virus was purified by field inversion electrophoresis and the DNA was mapped by restriction endonuclease analysis (Hall & Hink, 1990). This particular entomopoxvirus can be propagated in cell lines derived from saltmarsh (Estigmene acrea), cabbage looper (Trichoplusia ni) and gypsy moth (Lymantria dispar) caterpillars (McCarthy et al., 1974; Langridge & Greenberg, 1981 ; Goodwin et al., 1990). Occlusion bodies are produced during infections
Insect poxviruses, known as entomopoxviruses, have been isolated from at least 31 host species (Granados, 1981; Arif, 1984; Arif & Kurstak, 1991). These viruses resemble their mammalian counterparts both biochemically and morphologically (Pogo et al., 1971 ; McCarthy et al., 1975; Mathews, 1982), but they have a limited host range and will infect only the larvae from certain species of Lepidoptera, Coleoptera, Diptera and Orthoptera. Entomopoxviruses characteristically form occlusion bodies or spheroids which serve to protect the virions from the environment. Virus can be released from the protective bodies in the alkaline environment of the The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBLand GenBank databasesand assigned the accessionnumber M75889. 0001-0564 © 1992SGM
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M. Banville and others
in all three of these cell types. The major occlusion body protein (called spheroidin) of AmEPV has been characterized from E. acrea and T. ni cells, and found to have an Mr of 110000 (Langridge, 1983a, b). Eastern spruce budworm (Choristoneura biennis) is infected by another entomopoxvirus (CbEPV) and its molecular composition has also been studied (Arif, 1976; Bilimoria & Arif, 1979). Genomic D N A of this virus has been purified and determined to possess a high A + T content, 73-4 to 74.6~o. Our laboratory recently cloned and sequenced the major occlusion body protein of CbEPV (Yuen et al., 1990). We found the protein to be rich in cysteine residues and to have a predicted Mr of 38"5K. The post-translationally modified protein migrates on SDS-polyacrylamide gels as a 47K monomer which aggregates to form dimers and trimers in the absence of reducing agent. Amino-terminal sequencing of the protein demonstrated that CbEPV spheroidin possesses a signal peptide which is cleaved during maturation. The apparent difference in Mrs between the spheroidin proteins of AmEPV and CbEPV prompted us to investigate the relationship between these two viruses more closely. In this report we have compared the major occlusion body proteins of CbEPV and AmEPV using SDS-PAGE and immunoblot techniques. In addition, D N A libraries were prepared from m R N A and genomic fragments, and the spheroidin gene of AmEPV was subsequently cloned and sequenced.
Methods Virus, insect celllines and larvae. Occlusion bodies for CbEPV and C. fumiferana entomopoxvirus (CfEPV) were supplied by the Forest Pest Management Institute, Forestry Canada (Sault Ste Marie, Ontario, Canada). Non-occluded and occluded AmEPV were a kind gift from Drs R. L. Hall and W. F. Hink (Department of Entomology, Ohio State University, Columbus, Ohio, U.S.A.). This virus originated from the laboratory of Dr R. R. Granados (Boyce Thompson Institute for Plant Research, Ithaca, New York, U.S.A.). Viruses were purified by plaque selection. The BTI-EAA (E. acrea) haemocyte cell line also came from the laboratory of Dr R. R. Granados and was subcloned in our laboratory to give a homologous population which supported the growth of AmEPV. These cells were propagated in Grate's insect medium (Gibco) supplemented with 10 ~ foetal calf serum (FCS). IPLB-LD-652 (L. dispar) cells were a gift from Dr M. Shapiro (Insect Pathology Laboratory, U.S. Department of Agriculture, Beltsville, Md., U.S.A.) and were cultivated in EX-CELL 400 medium (JRH Biosciences). The FPMI-CF 16 cell line from C.fumiferana supported the growth of both CbEPV and CfEPV and was obtained from Dr Sardar Sohi (Forest Pest Management Institute, Sault Ste Marie, Ontario, Canada). C. fumiferana and E. acrea larvae were also supplied by the Forest Pest Management Institute.
Purification of occlusion bodies from larvae. CbEPV occlusion bodies were harvested from infected C. fumiferana larvae and AmEPV spheroids were obtained from infected E. acrea caterpillars. Larvae were infected by adding small quantities of the occlusion bodies to an
artificial diet supplied by Bio-Serv. After death, the caterpillars were frozen at - 20 °C and lyophilized for 24 h. The dried insects (5 g) were subsequently ground in a mortar and pestle, and stirred in 100 ml water containing 0.5 ~ SDS for 2 h. The suspension was subsequently filtered three times through multiple layers of cheesecloth. Filtrates were centrifuged at 1000 g for 15 rain at room temperature, and the resulting pellet was resuspended in 100 ml distilled water and recentrifuged. The washing process was performed three times. Occlusion bodies were suspended in 20 ml of water, large particulate matter was allowed to settle for 5 min and the supernatant was placed on discontinuous sucrose gradients consisting of 10 ml 6 5 ~ (w/w) sucrose, 12 ml 5 0 ~ (w/w) sucrose and 3 ml 3 5 ~ (w/w) sucrose in water. A sample (10 ml) was layered onto each gradient and the tubes were centrifuged at 25 000 r.p.m, using a Beckman SW28 swinging bucket rotor. Purified occlusion bodies at the 65~/50% sucrose interface were aspirated, diluted with water and collected by centrifugation at 1000 g.
Purification of occlusion bodies from an E. acrea cell line. BTI-EAA cells were infected with non-occluded virus at a multiplicity of 1 to 5 p.f.u./cell. The infected cells were cultivated for 96 h in Grace's medium which contained 1% (v/v) rather than the usual 10~ (v/v) FCS. Occlusion bodies were apparent by this time and 5 x 108 cells were harvested and collected by low speed centrifugation at 1000g. The cell pellet was lysed in 50 ml of water containing 0.5~ (w/v) SDS with a Dounce homogenizer containing a loose pestle. Occlusion bodies were collected by centrifugation at 1000g for 15 rain, resuspended in 10 ml of water and purified on the sucrose gradient described above. Southern blot analysis of entomopoxvirus DNA with probes constructed from the major occlusion body protein genes of CbEPV and AmEPV. Genomic DNA was digested with restriction endonucleases (EcoRI, HindlIl or XmnI, or a combination of the three). DNA fragments were resolved on 0.7% agarose gels in the presence of ethidium bromide and transferred to nitrocellulose (Southern, 1975). Radioactive probes were constructed from the entire regions encoding the major occlusion body proteins using the Multiprime kit (Amersham). Filters were hybridized at 37 °C as previously described (Vialard et al., 1990). After 24 h, the blots were washed four times with buffer composed of 2 × SSC and 0.1% SDS at 28 to 30°C, and exposed to film for 4 to 6 h.
Purification of AmEPV DNA and construction of genomic libraries. Genomic DNA was purified by suspending approximately 10 t° occlusion bodies and dissolving them in 2 mt of a buffer containing 0-8 M-sodium carbonate and 0-02 M-sodium thioglycotate for 1 h. The pH was lowered to pH 8-5 by adding dilute HCI, and D N A was released from virions by adjusting the solution to 1% SDS, 0.05 M-EDTA and 2 mg/ml proteinase K. The mixture was incubated at 65 °C for 2 h, then diluted to 5 ml with water, and extracted with phenol and chloroform. Genomic DNA was precipitated with ethanol and sodium acetate, digested with EcoRI, HindlII or XmnI, or a combination of these enzymes, and ligated to the pUC19 cloning vector digested similarly.
Purification of mRNA from infected E. acrea and L. dispar cells. Messenger RNA from BTI-EAA and IPLB-LD-652 cells infected with AmEPV was preparated using an mRNA Purification Kit from Pharmacia LKB. Total RNA was extracted from 5 x 109 cells using guanidinium thiocyanate and RNA was sedimented by ultracentrifugation through caesium trifluoroacetate. Poly(A)+ R N A was selected from total R N A using oligo(dT)-cellulose spin column chromatography. Synthesis of dsRNA from infected cell mRNA and construction of cDNA libraries. A cDNA library was constructed from infected cell mRNA using the eDNA Synthesis Kit from Pharmacia LKB. Double-stranded DNA was synthesized from poly(A) + RNA using murine leukaemia virus reverse transcriptase and the Klenow fragment of DNA polymerase I. EcoRI/NotI linkers were added to the dsRNA and the
Entomopoxvirus occlusion body protein
561
product was ligated to the pUC19 vector which had been digested with EcoRI. Competent Escherichia coli DH5ct was transformed with the ligated DNA.
Electrophoretic purification and protein sequencing o f A m E P V protein. AmEPV occlusion bodies (106) were suspended in denaturation buffer consisting of 20 mM-DTT, 6 M-guanidine hydrochloride, 1 mg/ml EDTA and 100 mM-Tris-HCl pH 8-5, and were incubated at 37 °C for 2 h. Reduced disulphide groups were alkylated by adding 80 mMiodoacetamide for a period of 0.5 h. Excess alkylating agent was subsequently allowed to react with 200 mM-DDT. The sample was dialysed three times against 100 mM-Tris-HC1 pH 8.5 at 4 °C. Reduced and alkylated proteins were subjected to electrophoresis in the presence of SDS on a minigel consisting of 7 ~ potyacrylamide. Proteins were transferred to polyvinylidene difluoride membranes (Matsudaira, 1987). The protein band which migrated with an Mr of 110K was cut out for sequencing. An acetyl blocking group on the amino terminus of AmEPV spheroidin was removed using anhydrous trifluoroacetic acid (Wellner et al., 1990). The deblocked protein was sequenced by automated Edman degradation on an Applied Biosystems Model 470A gas phase sequencer (Hewick et al., 1981). Internal sequences were determined from peptides following lys-c endoprotease digestion. Prior to sequencing, peptide fragments were resolved on a Brownlee HPLC microbore column (RP-300, 2.1 mm × 30 mm) using an Applied Biosystems 130A Separation System. Identification o f DNA clones corresponding to the A m E P V spheroidin gene. Two degenerate oligonucleotide probes were constructed from the peptide sequence data. AA(C/T)GTICCNCTIGCNACIAA, corresponding to NVPLATK, and T A ( C / T ) A C I A A ( C / T ) T T ( C / T ) A C N A A , corresponding to YTNFTK, where N is any one of the four nucleotides and I represents the non-hydrogen-bonding nucleotide base inosine. Plasmids containing cDNA inserts derived from mRNA were digested with EcoRI, and DNA was resolved on a 0.7~ agarose gel by electrophoresis and transferred to nitrocellulose by Southern blotting. These blots were subsequently probed with the above oligonucleotides which had been labelled at their 5" ends with T4 polynucleotide kinase and [y-32p]ATP. Hybridization was overnight at 37 °C in buffer containing 6 × SSC and 100 gg/ml yeast tRNA and washing was performed with 2 × SSC at room temperature. Genomic libraries were subsequently probed with positive clones from the mRNA library. DNA sequencing of cDNA and cloned genomic fragments was performed on double-stranded plasmid DNA using synthetic oligonucleotide primers, [~-32p]dATP (Amersham, 3000 Ci/mmol) and the T7 DNA polymerase sequencing kit from Pharmacia. Both DNA strands were sequenced to ensure accuracy.
Results Cultivation of AmEPV in cultured insect cell lines and isolation of occlusion bodies At the start of our studies with AmEPV we had difficulty propagating the virus in BTI-EAA cells. Occlusion bodies appeared in only a proportion of the cells even after 1 week of incubation. Therefore, the cell line was cloned using a fluorescence-activated cell sorter to produce homogeneous populations which supported the replication of the entomopoxvirus and permitted the formation of occlusion bodies. Three selected cell lines totally supported the replication of the virus and a typical illustration of an AmEPV infection in culture is shown in
Fig. 1. Occlusion bodies of AmEPV produced in an E. acrea cell line (BTI-EAA). (a) Cells were infected at a multiplicity of 1 p.f.u./cell and the infection was allowed to proceed for 60 h, after which a phase contrast photomicrograph was taken. Bar marker represents 40 Ixm. (b) Occlusion bodies from infected cells were harvested after 96 h of infection, purified as described in Methods and suspended in distilled water, after which a photomicrograph was taken. Bar marker represents 20 ~tm for (b) and 40 ~tm for (a).
Fig. l(a). Eventually all the cells became round and each contained 20 to 30 cytoplasmic occlusion bodies. We also observed that reducing the level of FCS from 10~ to 1 ~o during infection dramatically accelerated the process of occlusion body formation. This finding may be related to the observation by other investigators that epidermal growth factor inhibits vaccinia poxvirus infection through competition for cellular receptors (Eppstein et al., 1985). A gypsy moth cell line (IPLB-LD-652) also efficiently supported infections (Goodwin et al., 1990). Both cell lines yielded similar titres of virus (107 to 108 p.f.u./ml) but the occlusion bodies in IPLB-LD-652 cells were one-quarter of the diameter of those produced in BTI-EAA cells. Growth of AmEPV in the gypsy moth cells was slightly faster (by 24 h), possibly due to the fact that virus was cultured in serum-free medium. Occlusion bodies of AmEPV (shown in Fig. 1b) were isolated from infected BTI-EAA cells as described in Methods and used in the subsequent experiments.
562
M. Banville and others (a) 1
(b) 2
M
1
97K 66K
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2
M
97K 661(
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Fig. 2. Occlusion body proteins of CbEPV (a) and AmEPV (b) separated by SDS-PAGE. Occlusionbodies (10 6) were suspendedin 200 ~tl of sample buffercontaining5% 2-mercaptoethanol,and 10 til (lanes 1) and 20 ~tl (lanes 2) of each preparation was loadedonto the 10%gel. The gel was stainedwith Coomassieblue. The Mrs of protein standards (lanes M) are shown to the right of each gel.
Comparison of AmEPV and CbEPV occlusion body proteins by SDS-PAGE Approximately 106 occlusion bodies from AmEPV and CbEPV infections were solubilized in sample buffer containing 5% 2-mercaptoethanol and subjected to electrophoresis on SDS-polyacrylamide gels as shown in Fig. 2. The migration of protein bands for the two types of occlusion bodies was different. The major occlusion body protein of CbEPV consists of a 47K monomer which tends to aggregate in the absence of reducing agent (Yuen et al., 1990). The process of monomer formation was reversible and dimers could be reformed by removing 2-mercaptoethanol by dialysis. Peptide antibodies directed against the carboxy terminus of monomeric spheroidin also reacted with the larger aggregates (unpublished data) and the amino termini of both the monomer and dimers were shown to be identical
by gas-phase sequencing techniques. In contrast, it was demonstrated that the occlusion bodies of AmEPV were composed primarily of a 114K polypeptide with minor protein bands being present at 38K and 16K, as shown in Fig. 2. To determine the primary sequence of AmEPV spheroidin and compare it to that of the CbEPV analogue, AmEPV spheroidin was gel-purified and sequenced by Edman degradation as described in Methods. Its amino terminus was found to be SNVPLA?KTI ?K; six internal peptides [ELLFP ?NVNEAQV?KYV, ISEYTNFTKS, LVDSSVQSQDVLQGLLNT(E/C)NTID, ASRLGNLGLVNRIN(E/C)SN, GYRGVYENNK and IFD(E/C)NPNNN] were also sequenced following lys-c endoprotease digestion. None of these amino acid sequences was found in the protein sequence for the CbEPV occlusion body protein published previously (Yuen et al., 1990).
Antibodies directed against AmEPV and CbEPV occlusion body proteins do not cross-react Antisera against all the solubilized occlusion body proteins from AmEPV and the gel-purified 47K occlusion body protein of CbEPV were produced in rabbits. A closely related entomopoxvirus (CfEPV) of another species of spruce budworm (C.fumiferana) was also being studied in our laboratories. The three different types of occlusion bodies were solubilized in the presence of reducing agent and the proteins were resolved on SDSpolyacrylamide gels. Immunoblots were probed with the anti-AmEPV and anti-CbEPV antibodies, and binding was detected with 125I-labelled Protein A (data not shown). Anti-AmEPV antibodies reacted only with the AmEPV occlusion body proteins and failed to react with the 47K spheroidin protein of CbEPV and CfEPV. Antibodies directed against the 47K protein of CbEPV failed to react with AmEPV occlusion body proteins, and reacted primarily with both the major occlusion body proteins from CbEPV and CfEPV. Although the occlusion body proteins of CbEPV and CfEPV were similar antigenically, the spruce budworm entomopoxvirus occlusion body protein was clearly unrelated to the proteins of AmEPV.
Southern blot analysis of entomopoxvirus genomic DNA with probes constructed from major occlusion body genes of CbEPV and AmEPV Genomic DNA was purified from occlusion bodies of the two different entomopoxviruses and digested with restriction endonucleases (EcoRI, HindIII or XmnI, or a combination of the three). DNA fragments were subsequently resolved on agarose gels. Radioactive probes were constructed from either the major occlusion
Entomopoxvirus occlusion body protein
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420 AC~CGATACA~AAAAGAAATGTGTq~G~TTA~G~CCCGTGT~TAJ~ACG~GCCC~GTATGGA~TATGT~GTCGA~A~GCTAGAT~TGTATCACAT~TGACGT~ H G D T L K E M C F E L L F P C N V N E A Q V W K Y V S R L L L D N V S H N D V
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540 ~TATAAA~AG~TTTTAGA~A~CTT~TGGAAAACATTTA3%~A~AAAAGAAATCGATC~CCG~ATTTATTTATTTTGTcGA~ATTTGGGAAA~ATGGA~A~A K Y K L A N F R L T L N G K H L K L K E I D Q P L F I Y F V D D L G N Y G L I T
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780 T~ATGT~AcAGATGC~CT~TATTACT~F7AGAT1~F~TAAAT~G~TATcGCAGTATCA~CTTG~TATATA~ACG~G~T~T~TG~CA~GAT~A~ F D V T T D A T ~ T L D F N K S V N I A V S F L D I Y Y E V N N N E Q ~
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900 ~GATTTACTT~GAGATACGGTG~TTTG~GT~AT~CGCAGATACTGGA~`~TTTA~CTAA~T~GTA~AAAAA~ATGATA~GTGA~C~GTAGAAAGG~GCCAG K D L L K R ¥ G E F E V Y N A D T G L I Y A K N L S I K N ¥ D T V I Q V E R L P
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1020 ~TTTGAAAG~AGAGcATATAcT~GGATGAAAATGGTcGC~T~ATGTTTGATGAAAAT~CAT~AGTAcAG~GTAGACCCcGAGTATGT~CTAGT~T~CTTTA~GG V N L K V R A Y T K D E N G R N L C L M K I T S S T E V D P E Y V T S N N A L L
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1140 GTACGcTCAGAGTATATAAAAAGTTTGATAAATCTCAT~AAAAAT~T~TGCAT~CAGAGG~GTGGT~TGTATTTCCA~GATCA~ATATCTGG~GT~TGT~AAAG G T L R V Y K K F D K S H L K I V M H N R G S G N V F P L R S L Y L E L S N V K
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1260 GATATcCAG~AAAGcAT~GATACTTCGAGATTAGATG~GGTATTTACAAA~AAATAAAAT~ATGTAGAT~CGAcGAAAATA~TTATATTGG~GAAA~G~GCAG~TATA G Y P V K A S D T S R L D V G I Y K L N K I Y V D N D E N K I I L E E I E A E Y
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1380 GATGC~GAC~GTA~CCACG~CGTGTAAAACTT~TAAACACC~TGTAAATATACTCCCAAATGTCCATTcC~TTTG~GTAAACAGCcCAGATACTACGATTCACTTATATG
1440
R C G R Q V F H E R V K L N K H Q C K Y T P K C p F Q F V V N S P D T T I H L ¥ 1500 GTATTT~TGTTTGT~AAAACCTAAAGTACCCAAAAATTT~GACTTTGGGGA~GA~AGA~GCGATACTT~AGATTTA~AAACATATGGcTGATGGAT~GATGATTTAG G I S N V C L K P K V P K N L R L W G W I L D C D T S R F I K H M A D G S D D L
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1620 ATCTT~ACG~AGGCTT~TAGAAATGATATATGTTTAAAAC~GCCATAAAAC~CA~ATA~TGT~ATA~AGAGTACGCAAATACATATCCAAA~GCACA~ATCA~GG D L D V R L N R N D I C L K Q A I K Q H Y T N V I I L E Y A N T Y P N C T L S L
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1740 GT~T~TAGATTT~T~TGTATTTGATATG~TGAT~CAAAACTATAT~GAGTATA~cT~ACA~GTAGAc~GACC~T~CATGTCATGTATA~AGG~TAAACA
1800
G N N R F N N V F D M N D N K T I S E Y T N F T K S R Q D L N N M S C I L G I N 1860 TAGGT~CCGTAAATA~AGTAGTTTGC~GG~GGGT~CAC~CACG~G~A~TT~GAT~GG~GTG~AGAG~AGAG~T~GTAAATCA~GTGATCTTT~A I G N S V N I S S L P G W V T P H E A K I L R S G C A R V R E F C K S F C
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1980 AT~GAGA~ATG~ATGG~AGAGAT~CGT~GTTTA~ATTTATGTGT~ATG~TA~GA~CG~GCAGTATGCG~TATC~GGATA~TCATA~A~CGC~ N K R F Y A M A R D L V S L L F M C N Y V N I E I N E A V C E Y p G Y V I
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ATGGTGGTGTTGAT~GAAATTTAAATAT~ATTCTTAAAG~TAAA~AAAAGAT~TGCGTGATGCTGAT~TGTCC~TCCATTATATATTTCTACTTA~TTTAG~CTTTAT Y
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2460 CATC~GTGTTATTGGATATGTTTATG~ATG~GG~ATCAG~CATA~GGTT~AG~GCA~GTGT~u~TGG~TAAAG~GCATCTAGA~AGGAAAT~AGGTTTAGT~ A S S V I G Y V Y E P C G T S E H K I G S E A L C K M A K E A S R L G N L G L V 2580 ATCGTA~TGAAAGT~AC~CAAATGT~TAAATATGG~ATAG'LGGAGTATACGAAAAT~CAAA~AAAAACAAAATA~ATAGAG~TAT~GA~GT~TC~T~TA N R I N E S N Y N K C N K Y G Y G V Y E N N K L K T K Y Y R E I F D C N P
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2640 N
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2700 AT~TG~TT~TATCCAGATATGGATATAG~T~TGGATTTACATAAAATTGGAGAAATT~F~TGCAAA~ACGATGAAAGTG~T~CC~GCG~CG~GATGTCA~ACTTGG~G
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N N E L I S R Y G Y R I M D L H K I G E I F A N Y D E S E S P C E R R C H Y L E 2820 ATAGAGGTCTTTTATATGGTc~G~TATGTAcATcACAGATATC~G~TCATGTACGc~TAcGTTTGGA~T~CACAAA~GTGT~CAAGAAATGGTG~C~CACGTATACG D R G L L Y G P E Y V H H R Y Q E S C T P N T F G N N T N C V T R N G E Q H V ¥
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2940 A~TAGTTGTGGAGAT~TGC~CATGTGG~G~G~CAGGATATGG~G~G~GTAGGGATG~TGG~TGA~ATAGAAAACCCCACGT~ATGAC~GTGCCGATGC~Du%ATA E N S C G D N A T C G R R T G Y G R R S R D E W N D Y R K P H V Y D N C A D A N
3000
3060 G~CATCTTCAGATAG~CAGACAGTAGTAGTAGTAGTG~T~G~T~GA~CAGATGGA~GCGACACAGATG~AGTTTAGA~CTGATA~GAAAA~G~ATCAAAATC S S S S D S C S D S S S S S E S E S D S D G C C D T D A S L D S D I E N C Y Q N
3120
3180 CATCA~TGTGATGCAGGATG~AAATGAAATTT~TA~ATAT~TRT~CTTAC~Gy1AT~TCA~u%-~LATGA1~F~%AATGATA~ATCGATAG~G~AT~TG~ P E K C D A G C
3240
3300
3360
~C~AT~A~GCGATGA~AT~TA~ATCT~AGATATATTT~TA~AT~TCG~AC~T~TATTTA~C~A~AT~T~T~ATC~ATATATA
Fig. 3. Nucleotide sequence of the AmEPV spheroidin gene and its predicted protein product. Peptides derived directly by N-terminal sequencing are underlined once. A putative leucine repeat (leucine zipper) region is underlined twice. The nucleotides of the gene and its immediate flanking sequences are numbered from 1 to 3360.
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body gene of CbEPV or that of AmEPV, and hybridized individually to Southern blots containing both CbEPV and AmEPV genomic fragments. Blots were washed at low stringency and exposed to X-ray film (data not shown). Only D N A specific for CbEPV was detected with the probe derived from the major occlusion body gene of CbEPV. Conversely, the probe specific for the spheroidin gene of AmEPV failed to hybridize with genomic fragments from CbEPV. The preceding results were the first indication that the CbEPV major occlusion body gene and the AmEPV spheroidin gene differed at both the level of nucleic acid and encoded protein• Cloning and predicted sequence of the AmEP V spheroidin protein Degenerate oligonocleotides were constructed from the partial protein sequence data derived by N-terminal sequencing as outlined in Methods. A cDNA library was synthesized from the m R N A of IPLB-LD-652 cells infected with AmEPV and probed with these 32p. labelled oligonucleotides. Several clones which contained inserts of 1900 to 2200 bases were identified and sequenced• The nucleotide sequence of one clone (Am93) was deduced to contain an open reading frame (ORF) which encoded the amino-terminal peptide forecast by N-terminal sequencing• Both strands of this clone were sequenced in their entirety and the predicted polypeptide contained two other peptide sequences corresponding to N-terminal sequences determined previously. This clone contained a unique internal XmnI site in the open reading frame. A genomic library consisting of XmnI fragments of AmEPV D N A cloned into pUC19 was probed with EcoRI-XmnI fragments from the eDNA •clone Am93. Genomic clones containing the 5' and 3' portions of the AmEPV spheroidin gene were identified and sequenced. An ORF of 3009 nucleotides was determined to encode spheroidin. The 5' flanking (1183 nucleotides) and 3' flanking (470 nucleotides) regions of the gene were also determined (data not shown). The sequences of the flanking and coding regions have been submitted to GenBank. The D N A sequence of the AmEPV spheroidin gene and its predicted polypeptide are also shown in Fig. 3. The predicted product of the AmEPV gene consists of 1003 amino acid residues which specify an unmodified protein of Mr 114881 with an isoelectric point of 6.3• AmEPV spheroidin contains 38 cysteine residues and 14 potential Asn-linked glycosylation sites. An interesting feature of the protein is a leucine zipper pattern located between amino acids 119 and 140, which could be important in forming protein aggregates• This leucine repeat pattern is indicated by the double underlining in Fig. 3. Six basic amino acid regions were located
between residues 9 and 13, 17 and 26, 320 and 345, 396 and 418, 675 and 697, and 927 and 946, with acidic regions between residues 380 and 394 and 470 and 478. These regions may be important for charge interactions during aggregation of the monomers. A potential hydrophobic membrane-spanning region exists between amino acids 631 and 647 but its significance is unclear. Overall, AmEPV spheroidin had the properties of a globular protein with a propensity to form short domains of 0t-helix and with scattered hydrophobic regions over the first 800 amino acids. The final 200 amino acids at the carboxy terminus are entirely hydrophilic. This spheroidin molecule showed no similarity to the analogous protein of CbEPV, the A-type inclusion (ATI) body protein of cowpox virus or the polyhedrin proteins of baculovirus. However, the major occlusion body proteins of poxviruses all have a large number of cysteine residues: 38 for AmEPV, nine for CbEPV and 26 for the ATI body protein of coxpox virus• The cysteines of AmEPV spheroidin were clustered mainly at the beginning, in the middle and at the very end of the protein• A search against sequences in the GenBank database revealed that the AmEPV sequence was unique. The late gene promoters of poxviruses are characteristically rich in A and T residues and contain a TAAAT element which overlaps the A U G initiation codon for the majority of late proteins (Davison & Moss, 1989)• This element specifies 5' polyadenylation of poxvirus mRNA. These late promoters usually extend about 27 nucleotides upstream from the TAAAT sequence. The 5' upstream region of the AmEPV spheroidin gene is a classic example of a poxvirus late promoter and bears a resemblance to many of the late gene promoters of vaccinia virus (Davison & Moss, 1989). It is A +T-rich and possesses a TAAAT element adjacent to its initiation codon. This promoter region extends 31 bases upstream before a C residue is encountered. Our laboratory has demonstrated previously that the CbEPV spheroidin promoter consists of 33 bases which could function as a strong late promoter in the vaccinia virus system (Pearson et al., 1991).
Discussion This publication reports the sequence of the gene for the major occlusion body protein (spheroidin) of AmEPV. We found the predicted protein to differ substantially from the major occlusion body protein of CbEPV, the sequence of which has been determined previously (Yuen et al., 1990). The CbEPV protein has an Mr of 47K and an isoelectric point of 5.01, but also possesses a large number of evenly spaced cysteine residues. Some
Entomopoxvirus occlusion body protein
mammalian viruses, such as cowpox virus, are also capable of forming occlusion bodies in their respective host cells (Patel et al., 1986). These occlusion bodies are irregular in shape and are called ATI bodies. These ATIs are composed of a single protein of 160K, the gene encoding which has been cloned and sequenced recently (Funahashi et al., 1988). The predicted cowpox virus occlusion body protein contains 26 cysteine residues and 11 potential sites for Noglycosylation, exhibits an abundance of basic and acidic amino acids, and has an isoelectric point of 4.87. With the exception of a large number of cysteine sulphydryl groups, no homology was apparent when the ATI protein was compared to AmEPV or CbEPV occlusion body proteins. However, the ATI protein gene promoter is a typical late poxvirus gene promoter and contains the TAAATG consensus sequence found in the AmEPV spheroidin gene promoter. The A-t-T-rich region of the ATI protein gene promoter is shorter; 23 nucleotides compared to 31 nucleotides for the AmEPV promoter. The extensive GAT repeats of the cowpox virus promoter are also not present in the region upstream of the AmEPV spheroidin promoter. Vaccinia virus contains an analogous protein (94K) which is a truncated version of the ATI protein (Patel et al., 1986). The 94K protein accumulates in large quantities late in infection, but is not capable of aggregating to form inclusion bodies. The deletions which have produced this smaller protein may be a result of evolutionary divergence. From these findings, we conclude that the major occlusion body proteins of three poxviruses are different. Their only similarity seems to be a high content of cysteine, and the physical ability of the protein to aggregate and include virions. Other investigators have demonstrated that the occlusion proteins of some insect cytoplasmic polyhedrosis viruses also differ substantially from one another (Fossiez et al., 1989). A previous publication from our laboratory (Yuen et al., 1991) has indicated that another CbEPV gene product, nucleoside triphosphate phosphohydrolase I, shares significant similarity with the analogous protein of vaccinia virus. Its gene promoter also exhibits the motifs characteristic of the late gene promoters for poxviruses. Despite the lack of homology between occlusion body proteins, these findings still support the classification of entomopoxviruses within the family of poxviruses. We initiated our work on entomopoxviruses with the ultimate intent of harnessing the strong promoters of the spheroidin gene for purposes of constructing a new family of insect virus expression vectors analogous to the baculovirus system (Luckow & Summers, 1988). This concept is presently being tested in our laboratory. In addition, the promoter from the spheroidin gene of
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CbEPV has already been inserted into a vaccinia virus expression vector and determined to function fivefold better than the 7.5K protein promoter normally used in many vaccinia virus vectors (Pearson et al., 1991). A similar approach was suggested and adopted using the cowpox virus ATI protein gene promoter described above to direct higher levels of expression from vaccinia virus recombinants in mammalian cells (Funahashi et al., 1988; Patel et al., 1988). Very little is known concerning the molecular biology of entomopoxviruses, and the viral proteins of AmEPV and CbEPV have not been clearly defined. The existence of permissive cell lines is enabling these studies to be pursued. The authors would like to thank Dr R. R. Granados and Ms Lynn Gallo for sending us the BTI-EAA cell line. We would also like to thank Dr R. H. Goodwin and Dr M. Shapiro for making the IPLB-LD-652 cell line available to us. Appreciation goes to Dr R. L. Hall and Dr W. F. Hink for sending us occlusion bodies, non-occluded AmEPV and E, acrea eggs to start us off. We are very grateful to Elaine Brabant and Marion Lalumi6re for helping us raise the insect larvae. This work was supported by the NRC of Canada and a Canadian MRC operating grant.
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(Received 22 August 1991; Accepted 29 October 1991)
