Journal o f General Virology (1992), 73, 1499-1504.
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Printed in Great Britain
Nucleotide sequence of the polyhedron envelope protein gene region of the Lymantria dispar nuclear polyhedrosis virus Rebecca M. Bjornson and George F. Rohrmann* Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331-6502, U.S.A.
A 6.4 kb region from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome was sequenced and found to contain open reading frames (ORFs) homologous to the polyhedron envelope (PE) protein coding sequence, and the C-terminal half of O R F 1, which is a gene located upstream of the PE
protein gene in other baculoviruses. The proteins predicted from the L d M N P V genes encoding the PE protein, and O R F 1 demonstrated 27 and 34% amino acid sequence identity, respectively, with the corresponding genes in the Autographa californica multicapsid nuclear polyhedrosis virus.
The Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) is pathogenic for the gypsy moth (L. dispar) which is a major defoliator of forest and shade trees in the Northeastern United States. Although LdMNPV can contribute significantly to the collapse of gypsy moth populations and an LdMNPV virus preparation has been registered as an insecticide for use against the insect, the virus is not well characterized. Recently, restriction maps have been reported for two isolates of the virus (Smith et al., 1988; S. Hiremath, personal communication) and these indicate that the genome is about 163 kbp which is almost 30% larger than the better characterized Autographa californica MNPV (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV) genomes. In addition, the LdMNPV genome has a G + C content of about 60 % (McCarthy et al., 1979) which is substantially higher than that of other baculoviruses, e.g. 54% for OpMNPV (Rohrmann et al., 1977) and 40 to 53% for Spodoptera sp. NPVs (Harrap et al., 1977). Both the high G + C content and the large genome size suggest that the LdMNPV genome may differ substantially in its gene content and organization from other baculovirus genomes. In order to begin a more detailed characterization of the LdMNPV genome, we used D N A probes from representative OpMNPV genes to locate their homologues in the LdMNPV genome. In this report we describe the identification and characterization of the polyhedron envelope (PE) protein gene region. This gene encodes a protein that appears to be a major component of the polyhedron envelope (Gombart et al., 1989a;
Russell & Rohrmann, 1990). This region has been well characterized in both the AcMNPV and OpMNPV genomes and, in addition to the PE protein gene, contains genes for gp64 (a major envelope glycoprotein of the extracellular form of the virus); four open reading frames (ORFs 1, 2, 4 and 5) surrounding the PE protein gene, and the p26-p10-p74 gene region downstream of the PE protein gene (Blissard & Rohrmann, 1990). In AcMNPV there are two additional ORFs (35K and 94K) plus an enhancer element (hr5) located in this region (Friesen & Miller, 1987; Blissard & Rohrmann, 1990). To locate genes on the LdMNPV genome we used a set of cosmids that were supplied by the U.S. Forest Service Laboratory in Delaware, Ohio and derived from clonal isolate CI 5-6 (Slavicek, 1991). They are cloned into the cosmid vector pHC79 (Hohn & Collins, 1980) as partial digests of PstI or ClaI. All plasmid subcloning was done in pBlueScribe (Stratagene) modified by the addition of a BgllI site. Plasmids were grown in Escherichia coli host strains HBI01 and DH5~. To locate the PE gene region, a 1-2 kb PstI-SalI pl:obe containing the PE ORF and part of ORF 4 from OpMNPV (Gombart et al., 1989b) was labelled using the method of Feinberg & Vogdstein (1984) and hybridized to blots (Gene-Screen Plus; Dupont) of the LdMNPV cosmids. Non-stringent hybridization conditions (30~o formamide) were utilized as described by Sambrook et al. (1989). Hybridization to the BgllI D fragment (Fig. 1a), and a 1.3 kb BamHI-EcoRI fragment was observed. A 6.4 kb SstI fragment from this region was subcloned, exonuclease III deletion clones were produced (Henikoff, 1987), and sequenced (Fig. 2) using Taq polymerase and related reagents (Promega) by a modified dideoxynucleotide chain termination method following
The nucleotide sequence data reported have been assigned accession number D10836 by EMBL, GenBank and DDBJ. 0001-0921 © 1992 SGM
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F (P-288) Cosmid ] - A (P-430) clones
BgIII I
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D (C-544) C (P-940)
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TCAGCTAGGTGGAAATAAGTTGAATAACA~AATTCACAAAACGAGTATAATATTTAGCCGCCATGTCCGCGCCTCACAACGTGCTCACGAAAAAGAATCCACGAC M S A P H N V L T K K N R
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GTGGGC GTTT TGCA 3360 G R F A 19
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3720 139
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CCCAACGACTCGGAGC TGCTGGACCGCATAGTGCGCCAGAACGACC TGATAC TGAACGGGC TCAAC CAGC TG TG TC TCAATCACAGCAATCAC CAC T TC ~ P N D S E L L D R I V R Q N D L I L N G N Q L C L N H S N H H F TCGATCAA~CGTGAACATCATCAATCAAC S I K L Q N V N I I N
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3840 179 3960 219
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GCCGACTTTGGCAGCGCGCTCGACGCCGCGCTCGCGCAGCTGCAGGACTCGCTGCGCAACGACC A D F G S A L D A A L A Q Q D S L R N Psi I
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5040
T G C TCAGACGGGAC-GAC T G C T A C A A G C C G C C C A A C T G T T C C A A G A T G T C G C A A G A G T C T T T G T G T TTCAAGAGCG 5160 SP L V A V L R G V T G L H R L L R Q E L A XbaI ~ >Poly(A) > . TGC-CCGCGCCCGACACGGAGAG TC TGGTGACCGAAAAGAT GAAGCGGCGCAACAACC.C.C~TCGATTGTATG.AACGAGCAG TACGTGC TC-C~GCAAAATAAACAT TTTAAAATT TC--GA~G 5 7 6 0 A A P D T E S L V E K M K R R N N G I D C M N E Q Y V L A Q N K H F K I W S 160 •
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6368
Fig. 2. Nucleotide sequence of the LdMNPV PE gene region. Late promoters (LP) and early promoter (EP) sequences and their direction are underlined and overlined. Major restriction sites, poly(A) sites and start codons of the major ORFs are also indicated. Ac Op Ld
MKPTNNVMFDDASVLW ID TDY IYQNLI~,IPLQAFQQLL ....... FTIPSKHRKMINDA-G .T.N .......... M...A ...... S .... ST ..... - ...... .S ........... I-. .SAPH.. LTKKNPRRGRF~IFPRRRV~GSIDEVV...RLPANIAIqG.HTR.K.CW..FR.
52 52 60
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A¢ Op Ld
GSCHN ........... TVKYMVD XYGAAVLVLRTPCS FADQ LLS TF IANNYLCYFYRRRR NPAC.PPSCSFPPSNS .............. A..C.SL,S .... T..T ..... S.CN.Q.,GGGGGGS ..... RVDGTKAF.. L.. LGY. CN.. N~TL.. Y. CTL. V. ET. KDA~CAQ--
I01 Itl 113
AC Op Ld
60 NAAP SHKL I K N N K N Y I H V F G L F K Y L S N Y N L N N K K R P K E Y Y T L K S I ISDLLMGAQGKVFDP 58 S ....... V R ..... L ......... Q . . . F . A . P H . P .... V, , V . C . . I A ..... T... I M S G C W .... QAV~.
Ac Op Ld
SRSRSRSRSRSRSPHCI~RSRSPHC R/'RSRSRSRSRSRSRS$SPRRGRR-QIFDALEKI 159 ............. .CPQ,----.CP Q.- ............... .FDCAQT..L ..... L 137 PPAPFPDYNA. -P. D.., ----. L•PO. - ............... .CKPNDSEL~. - --R. 149
Ac Op Ld
121 L C E V K T Q L C A I Q E S LNEAI S I L N V H S N D A A A N P P A P -D INKL . . . . . . Q E L - - - I Q D L - I]8 .. ,I . . . . . . . . . . . . . . . V T . . S H .... . T A E . . . G A P C D A . . . . . . R . . - - - V E T . - 15 ~ A D . I . S . . . T L . . C . - G . S . G S Q I - - - Y . P . T . . . SLYGLE•GLESMRA..CALT.R.KS
AC Op Ld
RHQNDMLMS NVNQ INLNQTNQFLE L SNMMTGVRNQNV- - - -QL .... LAALETAKDV ILT 211 AR.S.LVVNSL...$, . .S ........ TLNT..A, .A.... .I .... . ..... T..A... 189 vR...LILNGL..LC..H~..HHF .... ILNSIKL,.V]gIIN..SQIFDDGVLSGL ..... 205
AC Op Ld
RLNTLLAEITD ........ S L P D L T S M L D K L A E Q L L D A I N T V Q Q T C K N E L N N T N S I L T N L 263 . . .A.VDD,KA ......... A, . .QSAQ.QE..DK ...... $.A..L.G.M ......... 240 DEKLSRLIADLDGHFADFG. AL. A A L ~ L Q .............. DSLR.D.T. I .... A. 250
AC Op Ld
169 163 71
Ac
ASSVTN INGTLNNLL--AAIE~N LVGGGGG-GNFNEA-DRQKLDLVYTLVNE IKN ILTGT LTKK
Op
Ld
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.... I .... S ...... --.•.
Fig. 3. Comparisonofthe aminoacid sequenceoftheAcMNPV tothe OpMNPV and LdMNPV PE protein genes. Amino acid identities are indicated by the dots below the AcMNPV sequence, gaps inserted to facilitate alignment are indicated by ( - ) , underlined amino acids indicate identity between OpMNPV and LdMNPV. The AcMNPV and OpMNPV data are from Oellig et al. (1987) and Gombart et al. (1989a), respectively• Underlined amino acids are shared between LdMNPV and OpMNPV but not with AcMNPV.
-QSEYNKKITFTTDTILENLKNIKDLMCLNK -H...BQ.L..A ..... D.V,S .... V .... IHMDLTS.LA,SN,AM.DTF,~L...V-.R.KQ
198 191 102
22-IK 21.2K II,0K
Fig. 4. Comparison of the amino acid sequence of the AcMNPV to the OpMNPV and LdMNPV ORF 1 genes• For details see Fig• 3. The AcMNPV and OpMNPV sequences are from Gombart et aL (1989b). The solid triangle indicates the location of the transposable element in the ORF l gene in the AcMNPV E strain. number preceded show no database.
of LdMNPV O R F s w e r e f o u n d that are by the b a c u l o v i r u s late p r o m o t e r e l e m e n t s but h o m o l o g y to a n y b a c u l o v i r u s g e n e s in the A l t h o u g h s u c h a s u r v e y is i n c o m p l e t e b e c a u s e
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of the lack of a complete baculovirus genome sequence, it may indicate that much of the additional D N A in the LdMNPV genome codes for genes not present in AcMNPV or OpMNPV. We thank Christian Gross and Doug Leisy for critically reading this manuscript, Shiv Hiremath and Jim Slavicek for the LdMNPV cosmids and B. Glocker for doing the in vitro transcription of ORF 1 and 6. This project was supported by a grant from the USDA (9137302-6310). This is Technical Report No. 9805 from the Oregon State University Agricultural Experiment Station.
References BJORNSON,R. M. & ROHnMANN,G. F. (1992). Nucleotide sequence of the p39-capsid gene region of the Lymantria dispar nuclear polyhedrosis virus. Journal of General Virology 72, 1505-1508. BLISSARD, G. W. & ROHRMANN, G. F. (1989). Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 170, 537-555. BLtSSARD,G. W. & ROrmMANN,G. F. (1990). Baculovirus diversity and molecular biology. Annual Review of Entomology 35, 127-155. BRtrrLAG, D. L., DAUTRICOURT,J.-P., MAULIK,S. & RELPH, J. (1990). Sensitive similarity searches of biological sequence databases. Computer Applications in the Biosciences 6, 237-245. FEINBERG, A. P. & VO~ELSTEIN, B. (1984). A technique for radiolabeling DNA restriction fragments to high specific activity. Analytical Biochemistry 132, 6-13. FRIESEN, P. D. & MILLER, L. K. (1987). Divergent transcription of early 35- and 94-kilodalton protein genes encoded by the HindlII K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus. Journal of Virology 61, 2264-2272. GOMBART,A. F., PEARSON,M. N., ROrmMANN, G. F. & BEAUDREAU, G. S. (19890). A baculovirus polyhedral envelope-associated protein: genetic location, nucleotide sequence and immunocytochemical characterization. Virology 169, 182-193. GOMBART, A. F., BLISSARD, G. W. & RormMAr~, O. F. (1989/,). Characterization of the genetic organization of the HindlII M region of the multicapsid nuclear polyhedrosis virus of Orgyiapseudotsugata reveals major differences among baculoviruses. Journal of General Virology 70, 1815-1828. HARRAP, K. A., PAYNE, C. C. & ROBERTSON, J. S. (1977). The properties of three baculoviruses from closely related hosts. Virology 79, 14-31. HENIKOFF,S. (1987). Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods in Enzymology 155, 156-165.
HOITN, B. & COLLINS,J. (1980). A small cosmid for efficient cloning of large D N A fragments. Gene 11, 291-298. HOOPES, R. R., JR & ROH~MANN,G. F. (1991). In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells. Proceedings of the National Academy of Sciences, U.S.A. 88, 4513-4517. Koz.~, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292. McCARTHY, W. J., MURPHY, T. F. & LANGRIDGE, W. (1979). Characteristics of the DNA from Lymantria dispar nuclear polyhedrosis virus. Virology 95, 593-597. OELLIG, C., HAPP, B., MULLER, T. & DOERFLER, W. (1987). Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81.2 to 85.0) of the Autographa californicanuclear polyhedrosis virus. Journal of Virology 61, 3048-3057. [Ahthors' correction 63, 1494 (1989).] ROrmMANN, G. F., CARNEGIE, J. W., MARTIGNONI, M. E. & BEAUOREAU,G. S. (1977). Characterization of the genome of the nucleopolyhedrosis bundle virus pathogenic for Orgyia pseudotsugata. Virology 80, 421-425. ROHR~I~, G. F. (1986). Polyhedrin structure. Journal of General Virology 67, 1499-1513. RUSSELL, R. L. Q. & ROrmMANN, G. F. (1990). A baculovirus polyhedron envelope protein: immunogold localization in infected ceils and mature polyhedra. Virology 174, 177-184. SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. SLAVIeEK, J. M. (1991). Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation products. Virus Research 20, 223-236. SMITH,I. R. L., VANBEEK, N. A. M., PODGWAITE,J. D. & WooD, H. A. (1988). Physical map and polyhedrin gene sequence of Lymantria dispar nuclear polyhedrosis virus. Gene 71, 97-105. THIEM,S. M. & MILLER,L. K. (1989b). A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper. Journal of Virology 63, 4489-4497. TJIA, S., CARSTENS, E. B. ,& DOERFLER, W. (1979). Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus. II. The viral DNA and kinetics of its replication. Virology 99, 399-409. WHITFORD, M., STEWART, S., KUZIO, J. & FAULKNER, P. (1989). Identification and sequence analysis of a gene encoding gp67 an abundant envelope glycoprotein of the baculovirus, Autographa californica nuclear polyhedrosis virus. Journal of Virology 63, 1393-1399.
(Received 22 October 1991; Accepted 17 February 1992)
1499
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Nucleotide sequence of the polyhedron envelope protein gene region of the Lymantria dispar nuclear polyhedrosis virus Rebecca M. Bjornson and George F. Rohrmann* Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331-6502, U.S.A.
A 6.4 kb region from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome was sequenced and found to contain open reading frames (ORFs) homologous to the polyhedron envelope (PE) protein coding sequence, and the C-terminal half of O R F 1, which is a gene located upstream of the PE
protein gene in other baculoviruses. The proteins predicted from the L d M N P V genes encoding the PE protein, and O R F 1 demonstrated 27 and 34% amino acid sequence identity, respectively, with the corresponding genes in the Autographa californica multicapsid nuclear polyhedrosis virus.
The Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) is pathogenic for the gypsy moth (L. dispar) which is a major defoliator of forest and shade trees in the Northeastern United States. Although LdMNPV can contribute significantly to the collapse of gypsy moth populations and an LdMNPV virus preparation has been registered as an insecticide for use against the insect, the virus is not well characterized. Recently, restriction maps have been reported for two isolates of the virus (Smith et al., 1988; S. Hiremath, personal communication) and these indicate that the genome is about 163 kbp which is almost 30% larger than the better characterized Autographa californica MNPV (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV) genomes. In addition, the LdMNPV genome has a G + C content of about 60 % (McCarthy et al., 1979) which is substantially higher than that of other baculoviruses, e.g. 54% for OpMNPV (Rohrmann et al., 1977) and 40 to 53% for Spodoptera sp. NPVs (Harrap et al., 1977). Both the high G + C content and the large genome size suggest that the LdMNPV genome may differ substantially in its gene content and organization from other baculovirus genomes. In order to begin a more detailed characterization of the LdMNPV genome, we used D N A probes from representative OpMNPV genes to locate their homologues in the LdMNPV genome. In this report we describe the identification and characterization of the polyhedron envelope (PE) protein gene region. This gene encodes a protein that appears to be a major component of the polyhedron envelope (Gombart et al., 1989a;
Russell & Rohrmann, 1990). This region has been well characterized in both the AcMNPV and OpMNPV genomes and, in addition to the PE protein gene, contains genes for gp64 (a major envelope glycoprotein of the extracellular form of the virus); four open reading frames (ORFs 1, 2, 4 and 5) surrounding the PE protein gene, and the p26-p10-p74 gene region downstream of the PE protein gene (Blissard & Rohrmann, 1990). In AcMNPV there are two additional ORFs (35K and 94K) plus an enhancer element (hr5) located in this region (Friesen & Miller, 1987; Blissard & Rohrmann, 1990). To locate genes on the LdMNPV genome we used a set of cosmids that were supplied by the U.S. Forest Service Laboratory in Delaware, Ohio and derived from clonal isolate CI 5-6 (Slavicek, 1991). They are cloned into the cosmid vector pHC79 (Hohn & Collins, 1980) as partial digests of PstI or ClaI. All plasmid subcloning was done in pBlueScribe (Stratagene) modified by the addition of a BgllI site. Plasmids were grown in Escherichia coli host strains HBI01 and DH5~. To locate the PE gene region, a 1-2 kb PstI-SalI pl:obe containing the PE ORF and part of ORF 4 from OpMNPV (Gombart et al., 1989b) was labelled using the method of Feinberg & Vogdstein (1984) and hybridized to blots (Gene-Screen Plus; Dupont) of the LdMNPV cosmids. Non-stringent hybridization conditions (30~o formamide) were utilized as described by Sambrook et al. (1989). Hybridization to the BgllI D fragment (Fig. 1a), and a 1.3 kb BamHI-EcoRI fragment was observed. A 6.4 kb SstI fragment from this region was subcloned, exonuclease III deletion clones were produced (Henikoff, 1987), and sequenced (Fig. 2) using Taq polymerase and related reagents (Promega) by a modified dideoxynucleotide chain termination method following
The nucleotide sequence data reported have been assigned accession number D10836 by EMBL, GenBank and DDBJ. 0001-0921 © 1992 SGM
1500
Short communication
F (P-288) Cosmid ] - A (P-430) clones
BgIII I
II
D (C-544) C (P-940)
U F OSLMKT
EQG (a)
B (P-291)
I
I II1 I
II I I
137.5
ORF A
I I
J RP I
B
... ..
II
D
C
N
____.---~p~~
[ I
I
I IIIII II I I
140.2
I
Ill
I
142.2 144 kb ORF 2 ORF 5 ORF 1 ORF 3 (PE) ORF 4 ORF 6 < [.-,
TATC TATTACGGCTAGGTGGAAATAAGTTGAATAGCAAAAAT
3240
ORF 3 (PE)-->
TCAGCTAGGTGGAAATAAGTTGAATAACA~AATTCACAAAACGAGTATAATATTTAGCCGCCATGTCCGCGCCTCACAACGTGCTCACGAAAAAGAATCCACGAC M S A P H N V L T K K N R
R
GTGGGC GTTT TGCA 3360 G R F A 19
TTTCCTCC~6GCCGCGTACGTGCTC~TTC6ATCGACGAGGTGGTC~C TC-CTGC GCCTGCC C G C C A A C A T C ~ C ~ ~ C ~ ~ T ~ C ~ C T T T A ~ 3480 F P R R R V R A G S I D E V V Q L L R L A N I A N G I H T R H K C W N D F R 59 Sall GGCGC.-CGG~GCC-GCGGCGGAGGC TC TCGTGTGGACGGGACGCGCGCG TTCG TCGACC TGTACGGTTTGGGC TATCT GTGCAACCGAAC GAAC TC CACCC TGGC CGAC TATC TGTGCACG 3600 G G G G G G G G S R V D G R A F V D L G L G Y L C N R T N S T A D Y L C T 99 CTGTTCGTCGCCGAGGCGTACCGCGA~GCGTGCTGTGCCCAGCCGCCGGCGCCCTTCCCCGACTACAACGCGTCGCCGCCCGACTGCCGGcCCCCGCTGCCGCCCCAGCCGCCGTGCAAG L F V A E A Y R D A C C A Q P P A P F P Y N A S P P D C R P L P Q P P C K
3720 139
EcoRl
CCCAACGACTCGGAGC TGCTGGACCGCATAGTGCGCCAGAACGACC TGATAC TGAACGGGC TCAAC CAGC TG TG TC TCAATCACAGCAATCAC CAC T TC ~ P N D S E L L D R I V R Q N D L I L N G N Q L C L N H S N H H F TCGATCAA~CGTGAACATCATCAATCAAC S I K L Q N V N I I N
Q
L
L
~ ~ ~CATAT ~ S N I L N
TGTCGCAAATCT TCGACGACGGAG TGCTGAGC~3GCTCGACGAGAAGCT GTCGCGGC TCATCGCCGACC TGGACGGACACTTC S Q I F D D G V L S G L D E K L $ R L I A D L D G H F
3840 179 3960 219
1503
Short communication
GCCGACTTTGGCAGCGCGCTCGACGCCGCGCTCGCGCAGCTGCAGGACTCGCTGCGCAACGACC A D F G S A L D A A L A Q Q D S L R N Psi I
D
L
TCACCAACATCAATTCGATCCTGGCCAATC T I N I L A N
.
.
.
L
TCACCTCCAGCCTGACCAACA CAAT T S S L T N I N
TCGACCATCAACAATTTGCTGCAGACGCTCCAGAA~CTGC~TCTGC~c~GAGGTGGGCGCC~AGCTCAACGACGTCCAA~CCGT~CCG~TACT~GT~AC~CC~ S T I N L L Q T L Q N L L G E V G A K L N V Q T V D R I L G V L T P E I Notl
.
Notl
4080 259
.
4200 299
LP>
GTGGCGC'CC-GC~CGC C-GCC C ~ G C G C G C T C A C T G A C G G C GT C G C C G G C G C G A A G G A A T A A G T C G A A T TAGT TTTTC~TT~TC TTTCG~TTATT TT~ ~ C C C C ~ V A A A A A A A A K R A H ORF 4--> • PstI ATGATCAACGAGGT TTGCGC CGAC GCGGCGC TC~CGCCACGAGGCGCGGCGAGATCC~GAGT TGC~C~ T~AG ~ ~CTGTAC~TGTGTC~C ~ ~ C ~ ~ C ~ C M I N V C A D A A L G R H E A R R D R E L R L Q Q L Y E V C R R S G V A D
'I~GTT T T ~ TI2CAGTC~ ' C ~ T ~ G T C C CGTC,GC~Gn2~C~3CGC42GCC~CGTC C/~GCCC TACT T T i ~ A ~ S F C S L M M S P v G V G G A T S K P Y F K •
~ G
G V
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R
~GT ~AG ~ GT~ ~ C~C~ACG~C V Q S C S A A M T
4320 312 •
4440 40
4560 80
S
XhoI.
•
TCGTCG TCqZCCGCGCGCGTCCCTTAACTACAGC~CC GCCATGAAC.4~G T C G G A C G T ~ G G A C G C GGAGTATC~GGTC-CCGCTC GAC~-_ACTTT~ C G ~ T G T C ~ C G T A ~ C G S S C R A S L N Y R P A M K A D v D P D A Y A V F L E H F D V E V S P Y
T
4680 120
TGTTCGAGC C C S S G
T
C 4800 160
~ C G A
R
GTC-C G T G C G C G T G T C G G G T C G C C G G C TCC-CGTGC T T G T T C ~ G C A A C G G G A G T C A C A C R V A G S R A C C A T G V
GC ~ ~ A A
C
C A
C ~ R P
A
CC ~ C ~ C C C A ~ C ~ C ~ C R A ,A P Q R R P
G G G C C G C A A G A G G TC C T C - C ~ G A A C G T G T G T T T C A A G G G C G C G A C G A G C C G C C G C G A A C T G G A G C G G A C T C T G A C ~ ~ G T ~ C C T ~ C ~ C G T ~ A C C ~ G P Q E V L Q E R V F Q G R D E P P R T G A D S D G E S E P A A V A > P R 8 S G G R V P A S E S P S L S G A A T A H G
C T~ ~
A
Q
4920 194
Q
P
T C G ~ C ~ C G P A E D R R
G
H i n c l l
AGTTCGAGATTCGCAACCGGC~GATCGCTACCGCAAGCGGT L E L N A V P A I V A L P
TC G T G T ~ A A T T G C T A T C T C A T C A A C A C G A C G A C G T C ~ A C G G C G T ~ T ~ T E H E I A I E D V R R R A R R A V P
T G C TC T A C G A G C G C G A C G A G A A G T G C G T G C G C G A A A T G A T G T C G C H E V L A V L L A A F H H R Q
5040
T G C TCAGACGGGAC-GAC T G C T A C A A G C C G C C C A A C T G T T C C A A G A T G T C G C A A G A G T C T T T G T G T TTCAAGAGCG 5160 SP L V A V L R G V T G L H R L L R Q E L A XbaI ~ >Poly(A) > . TGC-CCGCGCCCGACACGGAGAG TC TGGTGACCGAAAAGAT GAAGCGGCGCAACAACC.C.C~TCGATTGTATG.AACGAGCAG TACGTGC TC-C~GCAAAATAAACAT TTTAAAATT TC--GA~G 5 7 6 0 A A P D T E S L V E K M K R R N N G I D C M N E Q Y V L A Q N K H F K I W S 160 •
X h o I
.
AAGTCATCd~CGCGCCCGCGGTCGTGATCGAC TGGCGT TT C~ACATGGAAGCGCATCACdkC.T~,.¢r2~TCATAAATC TGC~ C V M N A P A V V I W R L D M E A H Q T R V I N L L H .
TGTTGG~CTATTATCATTTGTTGCCCATATTGAGAAGGAGGTAC L V Y Y H L L P I R R R
Y
.
.
.
GACCTCGT T T A C A G A T ~ C ~ D L V Y R W E T R Clal
GTGG TGTGCGACAAAGGCGACTGCCC GGCCCC~ TGAGGGAGGC~~TTCGAC~TCG V V C D K G D C P A R L R E A I D Q G
.
C E
N
~CG D V
5880 200
•
S
T
V
V
~ T~GCT L R
W
6000 240
GGCCC GCCC~GC GGAC TTC-GAAGCCGCCAAACGGGAGGCGGTCGAGGC CTGCAAGG TTCCGC TGG ~ C G ~TC~C C ~ C ~ T ~ T C ~ C ~ C T T ~ T 6120 P A G A D L E A A K R E A V E A C K V P L V A V I A R D S E W L R S A D F E R Y 280 . . . > Poly(A) > . . . . ACGTCGTCGAC-CGCGTCT TGAATCGCGCCTTTGAT TGTTAAAATATGTAATAAATTAAAATTTTTATATATTGTAATAATTAATTTTCTAATATATAT TG TAATAATTAAT TTTCTAATA 6240 V V E R V L N R A F D C > 292 > Poly(A) > < Poly(A) < . TATATTGTATAGAATCATGTACATTT TAA~.~GTGTAATAAAAAG TGTGC~_.AACGGTAAAAGGTTTTTAT TGAT TCCCAATTAGAC TAAGTCC-CAGTATAAAAC TAAACTCAGCGAGCCG 6360 SstI
6368
Fig. 2. Nucleotide sequence of the LdMNPV PE gene region. Late promoters (LP) and early promoter (EP) sequences and their direction are underlined and overlined. Major restriction sites, poly(A) sites and start codons of the major ORFs are also indicated. Ac Op Ld
MKPTNNVMFDDASVLW ID TDY IYQNLI~,IPLQAFQQLL ....... FTIPSKHRKMINDA-G .T.N .......... M...A ...... S .... ST ..... - ...... .S ........... I-. .SAPH.. LTKKNPRRGRF~IFPRRRV~GSIDEVV...RLPANIAIqG.HTR.K.CW..FR.
52 52 60
Ac Op
I MNTDAQS TSNTRNFMYSPDS S LEWYI TNSDGDHDGYLELTAAAKVMS PFLSNGS SAVWT ] M A N A D S LDA. AFS. A.. A. F.. I I . . .APN ........ N, . .RLLA.. -QKNI.. LW.
A¢ Op Ld
GSCHN ........... TVKYMVD XYGAAVLVLRTPCS FADQ LLS TF IANNYLCYFYRRRR NPAC.PPSCSFPPSNS .............. A..C.SL,S .... T..T ..... S.CN.Q.,GGGGGGS ..... RVDGTKAF.. L.. LGY. CN.. N~TL.. Y. CTL. V. ET. KDA~CAQ--
I01 Itl 113
AC Op Ld
60 NAAP SHKL I K N N K N Y I H V F G L F K Y L S N Y N L N N K K R P K E Y Y T L K S I ISDLLMGAQGKVFDP 58 S ....... V R ..... L ......... Q . . . F . A . P H . P .... V, , V . C . . I A ..... T... I M S G C W .... QAV~.
Ac Op Ld
SRSRSRSRSRSRSPHCI~RSRSPHC R/'RSRSRSRSRSRSRS$SPRRGRR-QIFDALEKI 159 ............. .CPQ,----.CP Q.- ............... .FDCAQT..L ..... L 137 PPAPFPDYNA. -P. D.., ----. L•PO. - ............... .CKPNDSEL~. - --R. 149
Ac Op Ld
121 L C E V K T Q L C A I Q E S LNEAI S I L N V H S N D A A A N P P A P -D INKL . . . . . . Q E L - - - I Q D L - I]8 .. ,I . . . . . . . . . . . . . . . V T . . S H .... . T A E . . . G A P C D A . . . . . . R . . - - - V E T . - 15 ~ A D . I . S . . . T L . . C . - G . S . G S Q I - - - Y . P . T . . . SLYGLE•GLESMRA..CALT.R.KS
AC Op Ld
RHQNDMLMS NVNQ INLNQTNQFLE L SNMMTGVRNQNV- - - -QL .... LAALETAKDV ILT 211 AR.S.LVVNSL...$, . .S ........ TLNT..A, .A.... .I .... . ..... T..A... 189 vR...LILNGL..LC..H~..HHF .... ILNSIKL,.V]gIIN..SQIFDDGVLSGL ..... 205
AC Op Ld
RLNTLLAEITD ........ S L P D L T S M L D K L A E Q L L D A I N T V Q Q T C K N E L N N T N S I L T N L 263 . . .A.VDD,KA ......... A, . .QSAQ.QE..DK ...... $.A..L.G.M ......... 240 DEKLSRLIADLDGHFADFG. AL. A A L ~ L Q .............. DSLR.D.T. I .... A. 250
AC Op Ld
169 163 71
Ac
ASSVTN INGTLNNLL--AAIE~N LVGGGGG-GNFNEA-DRQKLDLVYTLVNE IKN ILTGT LTKK
Op
Ld
322 36"5K .GIG.D. ..L.D---.-.. .A.NE.LS. , T . . R . . .M. . A R . 297 32-4K .T..L .... S.~[ .... QT LQN L~..~EV. AK LN~2VQ$ TVDR I LGV .... .TF.. VAAAAAA4~A. P~AH 312 33"8K
.... I .... S ...... --.•.
Fig. 3. Comparisonofthe aminoacid sequenceoftheAcMNPV tothe OpMNPV and LdMNPV PE protein genes. Amino acid identities are indicated by the dots below the AcMNPV sequence, gaps inserted to facilitate alignment are indicated by ( - ) , underlined amino acids indicate identity between OpMNPV and LdMNPV. The AcMNPV and OpMNPV data are from Oellig et al. (1987) and Gombart et al. (1989a), respectively• Underlined amino acids are shared between LdMNPV and OpMNPV but not with AcMNPV.
-QSEYNKKITFTTDTILENLKNIKDLMCLNK -H...BQ.L..A ..... D.V,S .... V .... IHMDLTS.LA,SN,AM.DTF,~L...V-.R.KQ
198 191 102
22-IK 21.2K II,0K
Fig. 4. Comparison of the amino acid sequence of the AcMNPV to the OpMNPV and LdMNPV ORF 1 genes• For details see Fig• 3. The AcMNPV and OpMNPV sequences are from Gombart et aL (1989b). The solid triangle indicates the location of the transposable element in the ORF l gene in the AcMNPV E strain. number preceded show no database.
of LdMNPV O R F s w e r e f o u n d that are by the b a c u l o v i r u s late p r o m o t e r e l e m e n t s but h o m o l o g y to a n y b a c u l o v i r u s g e n e s in the A l t h o u g h s u c h a s u r v e y is i n c o m p l e t e b e c a u s e
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of the lack of a complete baculovirus genome sequence, it may indicate that much of the additional D N A in the LdMNPV genome codes for genes not present in AcMNPV or OpMNPV. We thank Christian Gross and Doug Leisy for critically reading this manuscript, Shiv Hiremath and Jim Slavicek for the LdMNPV cosmids and B. Glocker for doing the in vitro transcription of ORF 1 and 6. This project was supported by a grant from the USDA (9137302-6310). This is Technical Report No. 9805 from the Oregon State University Agricultural Experiment Station.
References BJORNSON,R. M. & ROHnMANN,G. F. (1992). Nucleotide sequence of the p39-capsid gene region of the Lymantria dispar nuclear polyhedrosis virus. Journal of General Virology 72, 1505-1508. BLISSARD, G. W. & ROHRMANN, G. F. (1989). Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 170, 537-555. BLtSSARD,G. W. & ROrmMANN,G. F. (1990). Baculovirus diversity and molecular biology. Annual Review of Entomology 35, 127-155. BRtrrLAG, D. L., DAUTRICOURT,J.-P., MAULIK,S. & RELPH, J. (1990). Sensitive similarity searches of biological sequence databases. Computer Applications in the Biosciences 6, 237-245. FEINBERG, A. P. & VO~ELSTEIN, B. (1984). A technique for radiolabeling DNA restriction fragments to high specific activity. Analytical Biochemistry 132, 6-13. FRIESEN, P. D. & MILLER, L. K. (1987). Divergent transcription of early 35- and 94-kilodalton protein genes encoded by the HindlII K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus. Journal of Virology 61, 2264-2272. GOMBART,A. F., PEARSON,M. N., ROrmMANN, G. F. & BEAUDREAU, G. S. (19890). A baculovirus polyhedral envelope-associated protein: genetic location, nucleotide sequence and immunocytochemical characterization. Virology 169, 182-193. GOMBART, A. F., BLISSARD, G. W. & RormMAr~, O. F. (1989/,). Characterization of the genetic organization of the HindlII M region of the multicapsid nuclear polyhedrosis virus of Orgyiapseudotsugata reveals major differences among baculoviruses. Journal of General Virology 70, 1815-1828. HARRAP, K. A., PAYNE, C. C. & ROBERTSON, J. S. (1977). The properties of three baculoviruses from closely related hosts. Virology 79, 14-31. HENIKOFF,S. (1987). Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods in Enzymology 155, 156-165.
HOITN, B. & COLLINS,J. (1980). A small cosmid for efficient cloning of large D N A fragments. Gene 11, 291-298. HOOPES, R. R., JR & ROH~MANN,G. F. (1991). In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells. Proceedings of the National Academy of Sciences, U.S.A. 88, 4513-4517. Koz.~, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292. McCARTHY, W. J., MURPHY, T. F. & LANGRIDGE, W. (1979). Characteristics of the DNA from Lymantria dispar nuclear polyhedrosis virus. Virology 95, 593-597. OELLIG, C., HAPP, B., MULLER, T. & DOERFLER, W. (1987). Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81.2 to 85.0) of the Autographa californicanuclear polyhedrosis virus. Journal of Virology 61, 3048-3057. [Ahthors' correction 63, 1494 (1989).] ROrmMANN, G. F., CARNEGIE, J. W., MARTIGNONI, M. E. & BEAUOREAU,G. S. (1977). Characterization of the genome of the nucleopolyhedrosis bundle virus pathogenic for Orgyia pseudotsugata. Virology 80, 421-425. ROHR~I~, G. F. (1986). Polyhedrin structure. Journal of General Virology 67, 1499-1513. RUSSELL, R. L. Q. & ROrmMANN, G. F. (1990). A baculovirus polyhedron envelope protein: immunogold localization in infected ceils and mature polyhedra. Virology 174, 177-184. SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. SLAVIeEK, J. M. (1991). Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation products. Virus Research 20, 223-236. SMITH,I. R. L., VANBEEK, N. A. M., PODGWAITE,J. D. & WooD, H. A. (1988). Physical map and polyhedrin gene sequence of Lymantria dispar nuclear polyhedrosis virus. Gene 71, 97-105. THIEM,S. M. & MILLER,L. K. (1989b). A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper. Journal of Virology 63, 4489-4497. TJIA, S., CARSTENS, E. B. ,& DOERFLER, W. (1979). Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus. II. The viral DNA and kinetics of its replication. Virology 99, 399-409. WHITFORD, M., STEWART, S., KUZIO, J. & FAULKNER, P. (1989). Identification and sequence analysis of a gene encoding gp67 an abundant envelope glycoprotein of the baculovirus, Autographa californica nuclear polyhedrosis virus. Journal of Virology 63, 1393-1399.
(Received 22 October 1991; Accepted 17 February 1992)
