VIROLOGY

185, 229-241

(1991)

Mucleotide Sequence of the Autographa cafhbmica fW&ar PO 9.4 kbp EcoRI-I and -R (Polyhedrin Gene) Regian ROBERT D. POSSEE,’ TAI-PING SUN,2 STEPHEN C. HOWARD, MARTIN D. AYRES, MlCHiLE HILL-PERKINS3 AND KATHARINE L. GEARING’ NERC Institute of Virology and Environmental

Microbiology,

Mansfield Road, Oxford, OX1 3SR, Unired Kingdom

Received May 9, 1990; accepted July 11, 199 1 The nucleotide sequence of a 9.4-kbp region including the polyhedrin gene of the CX strain californica nucleer polyhedrosis virus (AcMNPV) genome was determined. Theee dope provide a co of the EcoRI-I fragment, which is used to produce transfer vectors for inserting foreign genes into potential o@en reeding frames (ORFs) were identified in the complete sequence, on either strand ofDh& The these &#s 1629 nucleotides in length and was located downstream from the pak/hedrin coding sequences, but on the opposite strand of DNA. Northern blot hybridization analysis of ORF 8 (1629) identified en RNA of Zoo0 which was produced in infected cells from 12 hr p.i. and remained until at lesst 46 analysis of COMA clones located the 3’ end of the mRNA at a site 16 nucleotides dew sequences. The 5’ end of the mRNA was mapped using primer extension aneiysis of start site was positioned within a late/very late consensus trenscriptii in&Won upstream from the potential ATG translation initiation codon. The biological signifiia wes aseessed by inserting a synthetic digonucieotide in the carboxyl terminal coding sequences o prematurely terminate translation. Recombinant viruses containing this mutation were not isolated, suggesting that the ORF 1629 gene product is essential for virus replication. 0 1991 Academic Press, Inc.

INTRODUCTION

producing a polyhedrin-negative virus which can still replicate efficiently in infected cells. To date the sequence of the f&RI-l region, which includes the polyhedrin gene, has onfy been partialfy determined (Hooft van lddekinge et al., 1983; Gearing and Possee, 1990; Tilakaratne er al., 19191). The complete sequence would assist investigators in their application of the baculovirus expression system by permitting accurate analysis of the pofyhedpin flanking regions. It would also provide fundamental knowledge of the genetic organization of this region of the virus genome. In this report we present the nucteotide sequence of the AcMNPV 7.3-kbp EcoRl-f region, which is the basis for most baculovirus transfer vectors, and that of the EcoRI-R 2.1 -kbp region. We have afso analyzed the expression and biological significance of a probable late gene which is located just downstream from the polyhedrin.

Baculovirus expression vectors derived from the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) have facilitated the high level synthesis of foreign gene products in insect cells (Smith et a/., 1983b; Pennock et a/., 1984; reviewed by Luckow and Summers, 1988; Miller, 1988). The system is based on the use of the very strong polyhedrin promoter which is active in the very late (6) phase (1872 hr p.i.) of viral gene expression, after the sequential expression of other virus genes in the immediate early ((Y),early (B), and late (y) phases (Carstens et a/., 1979; Dobos and Cochran, 1980; Wood, 1980; Kelly and LeScott, 1981; Maruniak and Summers, 1981; reviewed by Blissard and Rohrmann, 1990). The polyhedrin protein forms a proteinaceous cystalline matrix (polyhedron) around virus particles but is not essential for virus replication (Smith et a/., 1983a). In baculovirus expression systems, the polyhedrin coding sequences are replaced with those of the desired foreign gene, thus

METHODS Virus and cells

’ To whom requests for reprints should be addressed. ’ Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 021 14. 3 Present address: British Biotechnology Ltd., Brook House, Watlington Road, Cowley, Oxford OX4 5LY. 4 Present address: Department of Medical Nutrition, Huddinge University Hospital, Novum, S14186 Huddinge, Sweden.

Spodoptera frugiperda (IPLB-SF2 1) ceils (Vaughn et al., 1977) and AcMNPV (C6) were ted 8s described previously (Possee, 1986). AcMNPV C6 was derived from an original, insect-grawn stock of wildtype AcMNPV (supplied by Dr P. V. Vail in 1974) using 229

0042-6822/g

1 $3.00

Copyright 0 1991 by Aca&ma Press. Inc. All rights of reproduction in any form reserved

POSSEE ET AL.

230

a

b EcoR

I

Analysis of virus DNA with restriction

Hindlll

endonucleases

Virus DNA was digested with EcoRl or HindIll restriction endonucleases using conditions recommended by the supplier (Amersham International) and fractionated in 0.8% agarose gels stained with ethidium bromide (0.5 pg/ml). Construction vectors

DNA manipulations on ORF 8 (1629) were performed essentially as described by Maniatis et a/. (1982). The plasmid pUC8/6/8 contains the AcMNPV EcoRI-I fragment inserted at the fcoRl site‘of pUC8/6 (a derivative of pUC8 lacking all other restriction enzyme sites from the polylinker) (Possee, 1986). This was digested with SnaBI (position 553 1, Fig. 3) treated with calf intestinal phosphatase (CIP), and ligated with a synthetic oligonucleotide &/II linker (Pharmacia). After amplification in fscherichia co/i JM 105, this vector was digested with Bglll, treated with CIP, and ligated with a synthetic 91bp synthetic oligonucleotide (synthesised by Applied Biosystems) having BarnHI-compatible ends. Recombinant plasmids were isolated with the oligonucleotide in either orientation and designated pOLlGO or pOLIG02. The inserted oligonucleotide and the flanking regions were sequenced using the chemical method (Maxam and Gilbert, 1980).

FIG. 1. Agarose gel electrophoresis of AcMNPV C6, Li, and E2 DNA digested with EcoRl or HindIll restriction endonucleases. Fragments with differing electrophoretic mobilities are (a) EcoRI, L’of C6, (b) HindIll, Band H’of C6 comigrate with similar fragments in Ll , but not E2; L of C6 is larger than the same fragment in Ll or E2. The positions of molecular weight markers are indicated on the left of the gel.

plaque purification. The AcMNPV Ll and E2 strains were generously provided by Dr Lois Miller and Dr Max Summers, respectively. The derivation of the recombinant virus AcRPl .HA and the isolation of infectious virus DNA were reported by Possee (1986) and Matsuura et al. (1987). Hrl C

4 Ev

II

of ORF 8 (1629) plasmid transfer

Preparation

of ORF 8 (1629) recombinant

S. frugiperda cells were cotransfected with virus DNA from polyhedrin-negative AcRPl .PR8.HA (Mat-

I P

C

X

s

virus

*C--R-

s

EvB

CK

C

P

BS

III

C

E I

I

E

E

E Polyhedrin ORF 1 (504)

ORF 4 (327) ----)

+ ORF ’ (g84)

ORF 7 (735)

ORF 5 (630)

ORF 9 (588) +

*

ORz3)

ORF 11(>366) -

4

4 ORF 10 (1020)

ORF 8 (1629)

kb 0

1

2

3

4

5

6

7

8

9

Map units (%)

-0.5

0

1

2

3

4

5

6

6.9

FIG. 2. Partial restriction map and summary of the coding strategy in the DNA sequence presented in Fig. 3. The hrl and EcoRI-I and -R regions are indicated above the map. Restriction enzyme sites on top line: B, BernHI; C, C/al; E, EcoRI; Ev, EcoRV, K. Kpnl; P, Pvull; S, Se/l; X, Xhol. The positions of the predicted ORFs and the direction of transcription are shown. The two scales represent the size of the region in map units (% virus genome) and kilobase pairs.

AcMNPV

EcoRI-I

suura et al., 1987) and pOLlGO or 2 using protocols described previously (Smith et a/., 1983a). Progeny virus was titrated in a plaque assay and plaques were screened for the presence of polyhedra. These viruses were placed into cell culture medium and retitrated in further rounds of plaque purification. On occasion, the resuspended virus from a plaque was used to infect 1O6 insect cells in a 35-mm-diameter culture dish. DNA was extracted by disrupting cells in lysis buffer (25 mM Tris-HCI, pH 7.8, 5 mn/l EDTA, 0.2% SDS, 2.5% 2mercaptoethanol), incubating with proteinase K (250 pg/mI) for 30 min at 37“, and then extracting twice with phenol/chloroform before ethanol precipitation. The DNA was resuspended in water and about 5 @I of the material was applied to a nitrocellulose filter (Possee and Kelly, 1988) and hybridized with the synthetic 9 lbp oligonucleotide (see above) end-labeled with [y32P]ATP and polynucleotide kinase. The virus DNA was also analyzed using Southern hybridization. In these experiments the AcMNPVHindIII V region was used as a probe after nick-translation (Possee and Kelly, 1988). DNA sequencing The sequence data presented in this report were produced using the following strategies. Nucleotides l-2527; overlapping DNA fragments generated by a number of restriction enzymes were subcloned into M 13mpl8 or 19 and sequenced using the dideoxynucleotide chain termination method (Sanger et al., 1977). Nucleotides 2528-4933; the derivation of this information was described previously (Gearing and Possee, 1990). The remaining sequence was obtained using subclones of virus DNA inserted into Ml 3 as described above or chemical degradation of radiolabeled DNA (Maxam and Gilbert, 1980). In the latter stages of this study an Applied Biosystems automated sequencer became available and was used to confirm regions of difficulty with Taq polymerase. Raw sequence data were analyzed using the computer programmes of Staden (1980). Each nucleotide on both strands of DNA was sequenced an average of five times. Transcriptional

analysis of ORF 8 (1629)

Total RNA was extracted from infected and uninfected cells (Possee and Howard, 1987), further processed to yield polyadenylated (poly(A)) RNA (Aviv and Leder, 1972), and analyzed using Northern blot hybridization (Possee, 1986). Radiolabeled probes specific for polyhedrin or ORF 8 (1629) RNA transcripts were prepared by subcloning the Asp71 8-HindIll fragment (nucleotides 5349-5856; Fig. 3) into pSPT18 (Pharmacia). RNA transcripts for each strand of DNA were pre-

AND

-R SEQUENCE

231

pared using T7 (ORF 8 (1629)-specific) or SP6 (polyhedrin-specific) polymerases, in the presence of [32P]UTP as recommended by the supplier. S 1 nuclease protection experiments to define the 3’ end of ORF 8 (1629) RNA were performed as described by Weaver and Weissman (1979) and Gearing and Possee (1990). The 3’ end was mapped with an Asp7 1El-HindIll fragment (nucleotides 5349-5856. Fig. 3) radiolabeled at the HindIll site with [32PJdATP and the f. m/i DNA polymerase I Klenow fragment. RNA was annealed with each probe at 45” for 18 hr before treatment with Sl nuclease. The 5’ end of the RNA was mapped using primer extension analysis, exactly as described by Blissard et al., (1989). cDNA cloning of ORF 8 (1629) mRNA Polyadenylated RNA from AcMNPV-infected cells (13 hr p.i.) was used as a template for producing cDNA clones in XgtlO, using commercially available kits (Amersham International). Inserts were sized using agarose gels and those specific for ORF 8 (1629) were identified using Southern hybridization analysis. Positive clones were further characterized using DNA sequence analysis as described above to determine the 3’ end of the ORF 8 (1629) mRNA. RESULTS Restriction

endonuclease

patterns of AcMNPV

DNA

The two most commonly used strains of AcMNPV are the E2 (Smith and Summers 1978, 1979) and Ll (Miller and Dawes, 1979). The HR3 strain of Cochran et a/. (1982) has also been studied. The virus used in this study was a plaque-purified strain, designated C6, derived in our laboratory from a wild-type AcMNPV stock. Therefore, we have compared this strain of AcMNPV with the E2 and Ll prototypes using restriction endonuclease digestion of virus DNA (Fig. 1). The profiles for fcoRl and HindIll are presented. Digestion with EcoRl revealed only one difference, namely an increase in size of the L fragment from 3.89 to 4.12 kbp. The remaining fragments comigrated with their counterparts in E2 and Ll . The profile of fragments from /-findIll digestion showed that C6 had the extra site within the B fragment as observed in Ll , thus producing B’ (15.14 kbp) and H’ (7.94 kbp). Furthermore, the L fragment was slightly larger (2.82 kbp) than the corresponding band (2.70) in the E2 and Ll strains. DNA sequence gene

of the region flanking the polyhedrin

The DNA sequence of 9430 nucleotides of the AcMNPV genome was determined as described under

POSSEE

232

ET AL.

ATCGATGTTGACCCCAACAGATTTATAATTAATCATA '. Cla I ACATTCATAAATGACACAGCAACATACATA~~CTTGCAT~T~TTT~TGA~TCATATTTGAG~T~C~TGACATTATCCCT

90 180 270

CGATTGn;TTTTACMGTAGAATTCTACCCGTAAAGCGRGTCCCCT ECOR I GATTGTGTTTTACAAGTAGATCTATCCGTAAAGCGAGr ECOR I ~GCTTATGACTCAAGTTATGAGCCGTGTGCAAAACATG BCOR v ~TACTCGTAAAGCCAGTTCGGTTATGAGCCGTGTGCAAAACATGACATCA

360 TTTATGACATCATCCACTGATCGTGCGTTACAAGTAe

450 I 540 ECOR I 630 ECOR I

EcoR

ACTCATACTTGATTGTGTTTTACGCGTAW GAGTCATAATTMTCGTGCGTTACAAGTA~ ORF lb MFPARW

~TACTCGTAAAGCGAGTTCGGTTATGAGCCGTGTGCAAAACATGACATC TCTACTCGTAAAGCGAGTTGAAGGATCATATTTAGTn;CGTGTTTCCCGCGCGTTGGC

720

BNYLQCGQVIKDSNLICFKTPLRPELFAYV ACAACTATTTACAATGCGGCCARGTTATRRAAGATTCTAATTTGCGTACGTGA

810

TSEEDVWTAEQIVKQNPSIGAIIDLTNTSK CTAGCGAAGAAGATGTGTGGACCGCAGAACI\GATAGTAA

Cla

YYDGVBFLRAGLLYKKIQVPGQTLPPESIV ATTATGATGGTGTGCATTTTTTGCGGGCGGGCCTGTTATAC-

900

I

TTCAAGTACCTGGCCAGACTTTGCCGCCTGAAAGCATAGTTC

990

QEFIDTVKEFTE KCPGMLVGVBCTHGINRT AAGAATTTATTGACACGGTAG~TTTACAGARAAGTGTCCCGGCATGTTGGTGGGCGTGCACTGCACACACGGTATT~TCGCACCG

1080

GYMVCRYLMHTLGIAPQEAIDRFEKARGHK GTTACATGGTGTGCAGATATTTAATGCACACACCCTGGGTATTGCGCCG~GG~GCCATAGATAGATTCG~GC~GAGGTCAC~

1170

IERQNYVQDLLI* TTGARAGACARRATTACGTTCAAGATTTATTTATT~TTT~TT~TATTATT~CATTCTTT~C~TACTTTATCCTATTTTC~TTGTT

l

GIKLNN

1260

RKKWRVLVISRKSRKAQLRDTANNWDVTPN GCGCTTCTTCCAGCGAACCAAAACTATGCTTCGCTTGCTCCGTTTAGCTTGTAGCCGATCAGTGGCGTTGTTCC~TCGACGGTAGGATT

1350

PRINEVVINAVNINRKHKQNEVYTKQGTIA AGGCCGGATATTCTCCACCACAATGTTG~GC~CG~GATGTTACGT~ATGCTTTT~TTTTCCACGTACGTCTTTTGGCCGGT~TAGC

1440

TFTTGDRTVCLVPHKRKDAPYQVARDSLDV CGTARACGTAGTGCCGTCGCGCCTCACOCACAACACCGGATCCAC

1530

VKAVLDTVQADKKQMIEDKKQMTEQFGTYM CACTTTGGCAACTARATCGGn;ACCTGCGCGTCTGCGCGTCTTTTTTCTGCATTATTTCGTCTTTCTTTTGCATGGTTTCCTGG~GCCGGTGTACAT

1620

RNLDTMVRTVQLDKAEIQKDKIAVIREIFE GCGGTTTAGATCAGTCATGACGCGCGTGACCn;CAIULTCTTTGGCCTCGATCTGCTTGTCCTTGATGGC~CGATGCGTTC~T~CTC

1710

QKKVLEETKQAVVAQLANTHETFTAILKTV Tn;TTTTTTAACAAGTTCCTCGGTTTTTTGCGCCACCACCACCGCTTGCAGCGCGTTTGTGTGCTC~~~~TCGC~TCAGCTTAGTCAC

1800

QKIAPDYKGTCLVQ Q C~CTGT~TG~TC~CC~CC~CC~G~~~ATCGCGGGATCGTACT~CCGG~CAGAG~CT~AGG*TTACTTCTTCT-GC~

1890

PIVEELLW

EQLEIAYPLKSKMILQIVGSKTILVTHPQL TTCTn;TMTTCTATGGCGTMGGCMTTTGGACTTCATA

1980 Pvu

II

YLPDGRKVVSNSTPGGNQITQEFLSKYKND ATACAGCGGGTCGCCCCTTACGACGC~TTAGAGGTAGGGCCCCCATTT~GATGGTC~~T~CGA~TAT~AT~~

2070

VHVRIAKDCVTYKLSNAVDKAVFRVQQDRE TACATGAACACGTATAGCTTTATCACAAACTGTATATTTT~C~TTAGCGACGTCCT~GCCACG~CCGGACCTGTTGGTCGCGCTC 4oRF2 LVYRLNFTDEGFKFEGIKVRAM TAGCACGTACCGCAGGTTGACGTATCTTCTCCAAATTTAACACGTGTGTCGATTTTG

2250

CAACAACTATTGTTTTTTMCGCARACTAAACTTATTGTGCACTCGTCG

2340

TTATGAACGCAGACGGCGCCGGTCTCGGCGCAAGCGGCTATAGTACA

2430

FIG. 3. Nucleotide sequence of the hrl and predicted coding sequences of potential ORFs strand of DNA are underlined. The 5’end of the equivalent data for the polyhedrin gene (Howard published by Guarino et al. (1986) except for the a C after position 715. The following nucleotides Selected restriction enzyme sites are underlined scription initiation sites (ATAAG or its complement) analysis of ORF 8 (1629) RNA are also shown.

2160

EcoRI-I and -R regions. The strand of DNA encoding the polyhedrin gene is shown with the above the DNA sequence. The predicted protein coding sequences derived from the opposite ORF 8 (1629) was mapped to position 754617, and the 3’end to position 5470 (see Fig. 4). The et a/., 1986) are also shown. The data presented for nucleotides 1-181 agree with that following nucleotides which were missing in our sequence, an A after positions 7 and 688. and were not present in the data of Guarino et a/. (1986) CAC after position 691 and GC after 710. and named. Polyadenylation signals are underlined; potential consensus late/very late tranare double underlined. The positions of the oligonucleotides used for the primer extension

AcMNPV

EcoRI-I AND -R SEQUENCE

233

*LEGGANVR GTTTTGATTTGCATATTARCGGCGATrPTTTAAATTATCTTA~~T~TAGTTATGACGCCTAC~CTCCCCGCCCGCG~ACTCG

2520

QVELLENVGEETHGFVDLPHDIVLPTGCAV

2610

CTGCAC~~~ACfiCC'IXEGACGC~TCCGnC lmo L

I CYQCIRFYICN

SBDDPSPVDAEL

GE

2700

PQKRMDITANPMYTRVNIQMCASILDNRNT GGGTTGTTn;CGCATATCTATCGTGGCGTTGGGCAn;TACGTCCG~CGT~AT~~~C~GCCG~TT~TCATTGCGA~AGT

2790

RNFRQVDESKIMGDILDRLRTLHDFHPIIN GCGATTAARACGTTGTACATCCTCGCTTTTAATCATGCCGTCGATT~TCGCGC~TCGAGTC~GTGATC~GTG~G~T~TGTT 4oRF 3 EKYERTLRLAYKLLSAMKYTLKM TTCTTTG~ATTCCCGAGTCAAGCGCAGCAGCGCGTATTTT~C~CTAGCCATC~T~GTTAGTTTCATTT~~C~CTTTATCC~TA ORF 4, MYRTSRINNAPVVASQHDYDRDQIKREL ATATATTAn;TATCGCACGTCAAGAATTAACAATGCGCCCGTTGTCGCATCTC~CACGACTATGATAGAGATC~T~GCGCG~TT

2880 2970 3060

NSLRRNVHDLCTRSGTSFDCNKFLRSDDMT AAATAGCTTGCGACGCAACGTGCACGATCTGTGCACGCGTTCCGGCACGAGCTTTGATTGT~T~GTTTTTACG~GCGATGACATGAC PVVTTI TPKRTADYKI TEYVGDVKTI CCCCGTAGTGACAACGATCACGCCCAAAAGRACTGCCGACTAC~TTACCGAGTA~TCGGTGACGTT~CTATT~GCCATCC~

3150 K

P

S

N

3240

SGPLVREAAKYGECIV*

R P L V E ORFSbMANASYNVWSPLIR TCGACCGTTAGTCGAATCAGGACCGCTGGTGCGAGAAGCCCGTGTGGAGTCCGCTCATTAGAG

3330

ASCLDKKATYLIDPDDFIDKLTLTPYTVFY CGTCATGTTTAGACAAGAAAGCTACATATTTAATTGATCCCGATGATTTTATTGAT~TTGACCCT~CTCCATACACGGTATTCTACA

3420

NGGVLVKISGLRLYMLLTAPPTINEIKNSN ATGGCGGGGTTTn;GTCTTTCCGGACTGCGATTGTACATGCTGTT~CGGCTCCGCCCACTATT~TG~TT~TTCC~TT

3510

FKKRSKRNICMKECVEGKKNVVDMLNNKIN TTAAAAAACGCAGCARGAGACATTTGTATGARRGAATGTA

3600 Sal

I

MPPCIKKILNDLKENNVPRGGMYRKRFILN TGCCTCCGTGTATRARAAAAA TATTGAACGATTTGAAAGAC~TGTACCGCGCGGCGGTATGTACAGG~GAGGTTTATACT~CT

3690

CYIANVVSCAKCENRCLIKALTHFYNHDSK GTTACATTGCARRCGTGGTTTCGTGTGCCAAGTGTGAAAAGT

3780

CVGEVMHLLI KSQDVYKPPNCQKXKTVDKL GTGTGGGTGAAGTCATGCATCTTTTAATCAAATCCCAAGA

3870

TGAUACTGTCGACAAGCTCT Sal I

CPFAGNCKGLNPICNY* GTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGT~TTATTG~T~T~C~TTAT~TGCT~TTTGTTT~TA~A RYLGFAVLVNTTNDIISFAYNYDSLYYKF ACGATACAAACCRAACGCAACAAGAACATTTGTAGTATTAC

l

3960 4050

NEFSECNIQSLIIKNECLSSAKDDEEEYEK 4140

ATTTTCAIIRn;ATTCACAGTTAATTTTGCGAC~TAAT

4230 YIYIDKKIPBITGSRMTKRYNVVTSNEPLE ATAAATATATATGTCTTTTTTAATGGGGTGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGT~TTTAC~CAGTGCTATTTTCTGGTAGTTC

4320

ESHTAKI ILNIYDIFKPDDTKYLINGAYYA TTCGGAGTGTGTTGCTTTAATTATTAAATTTTATAATCATACGC

4410

AEELEIVGNKLLVPNVYSELINYSGNFLFL AGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGA~~CAT~C~TCC~TGTTGTACG~CCGTT~C~CAG 4ORF 6 EGGKEISNDALLQKNNFIVAM TTCACCTCCCTTTTCTATACTATTGTCTGCGAGTTGTTATCA

4500 4590

FIG. 3-Continued

Materials and Methods. A partial restriction map of this part of the AcMNPV genome is shown in Fig. 2. The precise locations of each of these restriction sites are indicated on the nucleotide sequence (Fig. 3). The approximate map units for this region are -0.5 to +6.9% and thus encompass the complete EcoRI-I and -R fragments, together with the hrl immediately upstream from the EcoRI-I fragment. The sequence for the hrl

was reported by Guarino er al. (1986). Our data for this region are included because of a potential coding sequence which initiates within the region (see belovu). Our data for hrl agree with that previously pubtiahed, except for those positions described in the legend to Fig. 3. Other regions which have previously been reported include ORF 7 (735) or polyhedrin (l-&oft van lddenkinge eta/., 1983) ORF 6 (603) (Gearing and Pos-

POSSEE ET AL.

234

+5'

end

pol.

CAAACTGGAAATGTCTATCATATATAOTTGCTGCTGATATCAT~AGAT~TT~TGAT~CCATCTCGC~T~T~GTATTTTACT EcoR V ORF 7 IPolvhedrin) MPD-YgYR-PTIGRTYVYD GTTTTCGTAACAGTTTTGTAATAAAUAA CCTATAAATATGCCGGATTATTCATACCGTCCCACCATCGGGCGTACCTACGTGTACGACA

4680 4770

NKYYKNLGAVIKNAKRKKHFAEHEIEEATL ACAAGTACTACAAAARTTTAGGTGCCGTTATCAAGRACGCT~GCGC~G~G~CTTCGCCG~CATGAGATCG~GAGGCTACCCTCG

4860

DPLDNYLVAEDPFLGPGKNQKLTLFKEIRN ACCCCCTAGACAACTACCTAGTGGCTGA~~TCCTGGGACCC~C~G~CC~C~ACTCTCTTC~GG~TCCGT~~ BamB I VKPDTMKLVVGWKGKEFYRETWTRFMEDSF TTAAACCCGACACGATGAAGCTTGTCGTTGGATGGAAAGCCTTCC Bind11 PIVNDQEVMDVFLVVNMRPTRPNRCYKFLA CCATTGTTAACGACCAAGAAGTGATGGATGGATGTTTTCCTTGTTGTC~CATGCGTCCCACTAGACC~CCGTTGTTAC~TTCCTGGCCC

4950 5040 5130

QHALRCDPDYVPHDVIRIVEPSWVGSNNEY AACACGCTCTGCGTTGCGACCCCGACTATGTACCTCATGACGTGATTAGGATCGTCGAGCCTTCATGGG~GG~GC~~CGAGTACC

5220

RISLAKKGGGCPIMNLHSEYTNSFEQFIDR GCATCAGCCTGGCTAAGAAGGGCGGCGGCTGCCCAATAATCAGTTCATCGATCGTG

Cla

5310

I

VIWENFYKPIVYIGTDSAEEEEILLEVSLV TCATCTGGGAGMCTTCTACACCCATCGTTTA~TC~GACTCTGCTG~GAGGAGG~TTCTCCT~~GTTTCCCTGGTGT Rpn I (Asp 718) FKVKEFAPDAPLFTGPAY* TCRAAGTAAAGGAGTTTGCACCAGACGCACCTCTGTTCACTGGTCCGGCGTATT~CACGATACAT

5400

r TTATTAGTACATTTATTAAG -. 5490 3' end ORF 8 (162Y) P DKPHYTL SSETRQQ NVSLQQVYKLLENFK 5580 CGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTTGTACGTATTTT~T~TTCATT~TTTAT~TCTTTA~TGGTATGTTA SnaB I AFILHNEADKYRFKFILDEISKYFLNRNTE GAGCGAARATCAAATGATTTTCAGCGTCTTTATATCTGAAGATTAGTTT 5670 FLPQKESGIAPSDLPNESLAVFSALDQLLL CARACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCT~TGGATTTTCGCTC~CGCCAC~CTTGCC~TCTTGTAG~GCA

R A K D I N T Q T K N Q L L P E V D N L I t ATCTAGCTTTGTCGATATTCGTTTGTGTTTTTGTTTTGT~T~GGTTCGACGTCGTTC~TATTA~GCTT~TATTTCTTTCAT

5760 H%

=ld

SDNTYLQSSTFVNFLAQVYKVDPTNAKNPR CACTGTCGTTAGTGTACAATTGACTCGACTCGACGTTGCC

+?l:heri;

D 5850 5940

Bind11 NDDDDWGEDNSTAESSSKAMAVRRRNITDA GATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTn;CTTGTGTCGG

6030

IESQPRKQAPKLRVTG LVDAILNTTSSKPI CTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTC~TCT~CTGTGC

6120

SKLESLVNSLSPAPPPLMESPLDVLPPPPP CCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGn;C

6210

ASSLDVMPPPAPPAPPPPPPPPPPPSASPP GAGCTGATGATARATCTACCATCGGTGGAGGCGCAGGCGGGGCT~CGGC~AGGC~AGGCGGA~~GTGGCGGTGA~~GACGGCG

6300

KPEFTEKPLVTPVEVITSTEPATKPKVPRL GTTTAGGCTCAAAn;TCTCTTTAGGCAACACAGTCGGCACCCGGTCTGA

6390

RTRNKENRIAELLQQRDDLPAAPQPETPMP GACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAAGGTTGAGGTTCCGTCGGCATTG

6480

PAPPLESMSPPPPPPPAPINPVPSPPPPPA GTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGG~TGTTAGGCACGGGAG~GGTGGTGGCGGCGGTG Cla I APIIQEPKTQERVITPVPAPAAPQVVSPRR CCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTG~GGCACCGGCGCAGGCGCCGCTGGCTGCAC~CGG~GGTCGTC

6660

SRPLAQPPPLEINYNSYLDYFDAILMTIES TGCTTCGAGGCAGCGCTn;GGGTGGTGGCAATTCAATATTC~TATTAT~TTGG~TAC~TCGT~TCTGCTAT~GCATTGT~TTTCGC

6750

6570

FIG. 3-Continued

see, 1990) ORFs 4 and 5 by Carstens (1987), and the HindIll F fragment (Tilakaratne er al., 1991). Potential gene products sequence

encoded

by the DNA

The DNA sequence described above was analyzed to determine the potential coding capacity. ‘The predieted protein sequences of 10 open reading frames

are shown in Fig. 3, together with the first 122 amino acids of ORF 11 which probably extends beyond map units 6.9. The information is summarized in Fig. 2 and Table 1. The latter presents a list of the ORFs greater than 100 amino acids which were detected by computer analysis. ORF 4 (327) and ORF 5 (630) occur on the same strand of DNA with an overlap of 17 nucleotides.

AcMNPV

EcoRl-I

AND

235

-R SEQUENCE

YAITNYLSQRLSPDRVGI DNVTGINLLREI TATCGTTTACCGn;CCGATATTTAACAACCGCTCAATGTGCTTGTAT~T~GAGA~GTCTC~GCTC~~C~GCACGC~GA VLRKMKVVANYHRSVESDDVYMYAKNPPVD T~CAAGCCTTTTCATTTTTACAGCATTGTAGTGGCGAGACAC~~CTGTCG~~TACA~TA~~

6840

BUBI 6930

Sal

I 7020 7110 J

MATTNATLQTLVQFYENCKNVKTRYKII TCCTCATGGCCACCACAAATGCTACGCTGCAAACGCTGGTACAATTTTACGUAACTG NGRFGKISIL ACGGGCGCTTTGGCAAAATATCTATTTTA

CAAAAACGTCRAAACTCGGTATAAAATAATCA

7200

LYLQKTISAHNFN TGTATTTGCAGAAAACAATTTCGGCGCACAATTTTAACG

7290

KIYFNAGSINNQV TTTTATAUAATCTATTTTAATCACGGTTCCATCAACAACCAAGTGA

ADEIKVH CTGACGAAATAMAGTTCA

7380

IVMDYIDCPDLFETLQIKGELSYQLVSNII TCGTGATGGACTACATn;ACTGTCCCGATTTATTTGARACACT

7470

RQLCEALNDLHKHNF IHNDIKLENVLYFEA GACAGCTGTGTGAAGCGCTCAACGATTTTGCACkAC v pvu LDRVYVCDYGLCKHENSLSVHDGTLEYFSP TTGATCGCGTGTATGTTTGCGATTACGGATTGTGCAAACACGAARRCTCACTTAGCGTGCACGACGGCACGTTGGAGTATTTTAGTCCGG

5'

end

7560 ORP 8 (1629) 7650

EKIRHTTMHVSFDWYAAC* ARAAAATTCGACACACAACTATGCACGTTTCGTTTGACTGGTACGCGGCGTGTT~CATAC~GTTGCT~CCGGCGGCCGACACCCATT

7740

TGAAAAAAGCGAAGACGAAATGTTGGACTTGAATAGCATG

7830

CGTTAACGCTCGTGACTTTGTGTACTGCCTAACAAGATACAGTT

7920 L.-Q

TTTGTCGTAAAAATGCCACTTGTTTTACGAGTAGAATTCTTTATT

8010

EcoR

LBKNNLFSEFIYYFCDVDIEECDMVCLLDM GTAAATGTTTATTATTTACGATTCAAATATATAATATCCA

8100

c

R

LSNKDKDSMSHAHLLDFVDKLNKYVDKCQ TCAATGAGTTTTTGTCTTTATCCGACATACTAn;TGCATC

8190

GFlKIVRYCPFSKEEQTTKGRLYQFLlHMC TACCARRA_:TTTARTARCCCTATAACAAGGAAAAGACTTTTCTTTTCTTCTTGCGTGGTTTTGCCGCGCAGATATTGRRATAAAAn;TGCAn;C

8ZfiO

SLEHKSFHEKGIGCFRGYMEAIVRPCNYSE ACGACAAC~N;TGTTTACTAARATGCTCCTTGCCTATACCGC~CC~CCATACATTTCGGCGATTACAC~GGAC~T~TA~A~

8370

DVELRDDYDSKRLRIPKKVAESLRFVLEA r

8460

VSBYMIRHVYTVVCFKNYWWGVVDNNPNFV GCACG~ATACA~ATTCn;TGCACATkM;TTACC

8550

NAIRLLKGNRLFYEF'RGLIIQLCDLMDPS CG~GATGCGCAGCT~CC~T~~T~A~~TATGAkR E.REFNNLSFIQWGNQ TTTCTCGTT~TTGTTCARAGAGAATATCTGCCATCC

8640

VRVSVPVVADIQ

c c1a

8730

I

ERVNDDIGMTESTSFWHSLSKISDGADIMK ~TTCACGGA~GTTATCGTCGATGCCCATCGTTTCGCTGGTGCTG~CC~TG~~GGCTC~ATGG~~G~~G~GT~TAT~T~T

8820

~AEDFTCSGSKLRRHATVDREICSMGNVYS TGACCGCTTCGTCAAA~~~TGCAACTGCCGCTCTTCARRCGCCGCATAGCGGTCACGTCCCGCTCTA~CACGA~ATACCGTTTACGTACG

8910

KSLYEQVIRYHHYSEGYVDHVENYKAY~W ATTCTGATAGGTATTCCTGM~ATACGGTAA~;G~;ATACGACTC~CATACACGTCG~CAC~~~TAT~AGCATMTMT~ l oNF 10 IILKAALSM AARTTATTM~~PPGCAGCGAGAGACA~;T~;TCAGTAAA

Y OIV 11, MYRHACYLY

IISQGGELLSVAIIKEWPSVFITsLAAsTf4 TATM~PTCGCAAGGAAGCTGTTATCGGTTGCTATTAT

Hind

pooo pogo

111

9180

KRRRCKLNSLPQELFDKIVEYLSLSDYCNL GAAGCGTCGTCATTGTAAATTAAATTCTC~;CCTCAAGIVT

9270

VLVCKRPSSRYNVIFDSTNHQHLKGVYKKT GGTG~T~;TC~;TAAAAGACCTT~AGTAAATATAACGTGATA~TGATAGTACT~TCACC~~TTTG~~CGTGTAC~GAC

9360

DVQITSYNEYINCICNELRQDEF AGACGTGCAAATMCMGCTACMCGMTACATCMC~TAT~GC~CG~CTGAGACMGACG~TTC

9430 EcoR

FIG. 3-Continued

236

POSSEE ET AL. TABLE 1 SUMMARY

OF POTENTIAL

OPEN READING FRAMES’

Open reading frames No.

Coding strand

1 2 3 4 5

+ + +

;I

;

8 9 10 11*

+ +

Position

Size

702 2226 2949 2978 3288 4563 4719 7117 7116 9028 9065

504 984 453 327 630 603 735 1629 588 1020 >366

Polypeptides Kozak consensusb

Amino acids

AAAATGT AAAiiG TAAATGA A-I-TATGT AGTATGG ACAATGG AATATGC GCCATGA GTCATGT AACATGC CTGATGT PuNNATGPu

168 328 151 109 210 201 245 543 196 340 >122

a Summarized from Fig. 3. bThose sequences which agree with the consensus 1986) are underlined. c ORF 603 (Gearing and Possee, 1990). d Polyhedrin gene. ’ Incomplete ORF at end of the sequence.

M 19,289 37,638 17,446 12,435 23,927 23,481 28,642 60,583 22,864 39,962

(Kozak,

The region of DNA of particular interest in this study was ORF 8 (1629) which is located 35 nucleotides downstream and in the opposite orientation of the polyhedrin gene (nucleotide 7 117-5489, Fig. 3). One of our goals in developing improved baculovirus expression vectors is to identify regions of the virus DNA which may be deleted without affecting virus replication, thus facilitating their replacement with foreign coding sequences. Therefore, we investigated expression of ORF 8 (1629). Transcription

from ORF 8 (1629)

The coding region of ORF 8 (1629) overlapped with the 3’ noncoding end of the polyhedrin gene. To determine whether a transcript was produced from this potential gene, Northern blot hybridization analysis was carried out with poly(A) RNA extracted from cells at varying times after infection (Fig. 4). Duplicate filters were incubated with single-stranded RNA probes specific for ORF 8 (1629) or polyhedrin. The ORF 8 (1629)-specific probe hybridized to a transcript of about 2000 nucleotides between 12 and 48 hr p.i. Low concentrations of polyhedrin-specific RNAwere detectable at 12 hr p-i., increased in abundance by 18 hr p.i., and remained at high levels until at least 48 hr p.i. The 3’ end of the 2000-nucleotide ORF 8 (1629)-specific RNA was mapped using Sl nuclease protection (Fig. 5). A 3’ end-labeled probe consisting of a 548-nucleo-

tide#indlll-Asp718 fragment protected a 41 O-nucleotide fragment when annealed with RNAfrom cells at 24 hr p.i. This positioned the end of the RNA at about nucleotide 5487 in Fig. 3. This result was confirmed by sequencing several XGTlO cDNA clones which contained inserts that hybridized to ORF 8 (1629). The 3’ end of the ORF 8 (1629) RNA could be identified after a run of poly(A) in the cDNA at position 5470 in Fig. 3. Therefore, the 3’ end of the RNA terminates at a point 16 nucleotides downstream from the end of the polyhedrin coding sequences and 12 nucleotides downstream from a polyadenylation signal (AATAAA) which overlaps the ORF 8 (1629) termination codon. Preliminary studies using Sl nuclease analysis of the same RNA indicated that the 5’ noncoding region of the RNA consisted of approximately 400 nucleotides (data not shown). To confirm the result, three primers (2 1 -mers) were synthesized, beginning at positions 7095 (primer 38) 7231 (primer 39) and 7310 (primer 40) (see Figs. 2 and 4). Primer 38 corresponded to a position within the ORF 8 (1629) coding region, and primers 39 and 40 to positions within the 5’ noncoding region. Primer extension analysis of the poly(A) RNA from AcMNPV-infected cells mapped the 5’ end of the RNA to nucleotides 7546/7 (Fig. 2) which coincide with a late/very late gene consensus transcription initiation motif (ATAAG; Rohrmann, 1986). Functional

analysis of ORF 8 (1629)

The role of ORF 8 (1629) in AcMNPV replication was studied by inserting a short synthetic oligonucleotide at a SnaB I site (position 5533, Fig. 3) within the 3’ coding sequences of the gene. This sequence (99 bp) served to insert translation stop codons in all six reading frames (data not shown), without adding methionine codons. One orientation of this insertion (pOLIG02) relative to the polyhedrin and ORF 8 (1629) genes and the consequences for coding capacity are shown in Fig. 6. The extra DNA and predicted protein sequences are shown as lowercase letters. This caused an insertion within the 3’ noncoding region of the polyhedrin mRNA which was expected to have little effect on gene expression. However, the position of the oligonucleotide meant that the coding sequence of ORF 8 (1629) was disrupted by translation stop codons, thus preventing the synthesis of the full-length protein. The predicted amino acid sequence was reduced from 543 to 535 residues (Air 59,894), of which 7 amino acids were donated by the synthetic oligonucleotide. The plasmids (pOLlGO or 2) containing this insertion within the entire EcoRI-I region were coprecipitated with infectious DNA from a polyhedrin-nega-

AcMNPV

EcoRI-l AND -R SEQUENCE

237

Hind Ill 1

Asp 718

1 5349

5856

POLYHEDRIN ORF 1629

PC&Y(A) ORF PROBE

POLYHEDRIN PROBE

FIG. 4. Analysis of transcription from the polyhedrin gene region. The top panel shows the strategy for producing strand-specific ra&oJabeled @RF 8 (16291) or 5P6 RNA probes from the Asp71 Wiindlll fragment (nucleotides 5349-5856, Fig. 3) inserted in pSPTl8 with T7 polymef@w polymerase (polyhedrin). The bottom panel shows the Northern hybridization analysis of poly(A) RNA from uninfected S. frug@er& cells (Sri and AcMNPV-infected cells (4-48 hr p.i.). The blot was probed with the ORF 8 (1629)qacific RNA transcript (left, ORF probe) or the polyhedrin-specific transcript (right). The 2000-nucleotide ORF 8 (1629) and the 1200-nucleotide polyhedrin transcripts are indicated. Size markers in nucleotides are shown.

tive virus (AcRPl .PR8.HA, Matsuura et a/,, 1987) and used to transfect insect cells, The progeny virus from this infection was titrated in a standard assay and plaques containing polyhedra were selected. These were sequentially titrated in an attempt to remove contaminating polyhedrin-negative virus. However, this procedure failed to derive genetically homogeneous virus stocks. Selection of poiyhedrin-positive virus was always accompanied by the presence of some polyhedrin-negative virus. Dot hybridization analysis of DNA from polyhedrin-negative or -positive virus revealed that only the latter retained the inserted oligonucleotide (data not shown). Southern hybridization of the same DNA, digested with HindIll and incubated with a HindIll-V region radiolabeled probe, identified the normat 928-nucleotide fragment, together with two submolar bands in each virus derived from pOLlGO and 2 (Fig. 7). The sizes of these bands (pOLIGO1, 596 and 43 1; pOLIGO2,633 and 394) correspond to those produced after digestion of the original plasmids with Hindlll.

DlSCUSSlON In this study we have determined the sewnce of a 9.4-kbp portion of the AcMNPV @nome surrounding the polyhedrin gene. The information providers a complete sequence of the region of the AcMNPV which is incorporated in the widely used polyhedrin gene promoter-based baculovirus transfer vectors and permits the derivation of accurate restriction maps for use in engineering and characterizing these plasmids. It will also facilitate the detailed analysis of transcription products from this portion of the virus genome. The virus used in this study and in a number of previous reports (Possee, 1986; Howard et a/., 1986; Posse@ and Howard, 1987; Weyer and Possee, 19@3, 7333; @Wing and Possee, 1990) was the C6 strain of AcM%lPV. We have provided a comparison of the virus genome, using restriction enzymes, with some other &rains of AcMNPV. These included AcMNPV E2 (Ssni% and Summers, 1978) and AcMNPV Ll (Miller and Dswes, 1979). The DNA profiles confirm that our virus is very similar to the other strains currently in use.

238

POSSEE ET AL. Asp 718

Hind III

Cla I

5856

5349

6504

Hind 111

EcoR I 7954

6938

ORF8(1629)RNA 754617

5470 548 nts 410 nts

*

Probe

*

S 1 Protected fragment

38; 7095*

-

39; 7231 * 40; 7310*

Sf

-m--

Primer extension products

-

AC

-

Ml3

L48-

410-

237/8=

FIG. 5. Sl nuclease and primer extension analysis of ORF 8 (1629) transcription. (Top, left) The Asp71 8-HindIll fragment was radiolabeled (1) at the 3’ end to produce a probe of 548 nucleotides for mapping the 3’ end of the ORF 8 (1629) RNA. The probe was annealed in separate reactions with total RNA from AcMNPV-infected cells at 12 and 24 hr pi. before treating with Sl nuclease and analysis in polyactylamide(PAGE)-urea gels (bottom, left panel). P, probe incubated alone; -, probe incubated with yeast tRNA; M, molecular weight standards. (Top, right) Three, Pl-mer oligonucleotides (38-40) complementary to sequences within the ORF 8 (1629) coding region or the 5’ untranslated leader were 5’ end-labeled and used for primer extension analyses with poly(A) RNA from uninfected (Sf) or AcMNPV-infected (AC) cells at 24 hr pi. Lanes labeled M 13 represent an M 13 sequencing ladder of M 13mpl8 which was used to determine the size of the primer extension products in a PAGE-urea gel (bottom, right panel). The M 13 G band corresponding to a size of 2 10 nucleotides is included as a reference. The sizes of the primer extension products are shown on the left side of the gel.

240

POSSEE

of 1.25 and 3.5 kb were detected. This region of the virus genome is evidently rich in transcriptional activity and provides excellent scope for future studies. ACKNOWLEDGMENTS We thank Chris Hatton for photographic supported by a NERC CASE studentship Department of the Environment.

services. K. Gearing was and T. P. Sun by the UK

REFERENCES AVIV, H., and LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acidcellulose. Proc. Natl. Acad. Sci. USA 69, 1408-l 412. BLISSARD, G. W., and ROHRMANN, G. F. (1990). Baculovirus diversity and molecular biology. Annu. Rev. Enromol. 35, 127-l 55. BLISSARD, G. W., &ANT-RUSSELL, R. L., ROHRMANN, G. F.. and BEAUDREAU. G. S. (I 989). Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata. Virology 168, 354-362. CARSTENS, E. 8. (1987). Identification and nucleotide sequence of the regions of Autographa califomica nuclear polyhedrosis virus genome carrying insertion elements derived from Spodoprera frugiperda. Virology 16 1, 8- 17. CARSTENS, E. B., TJIA, S. T., and DOERFLER, W. (1979). Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus. Virology 99, 386-398. COCHRAN, M. A., CARSTENS, E. B., EATON, B. T., and FAULKNER, P. (1982). Molecular cloning and physical mapping of restriction endonuclease fragments of Aurographa californica nuclear polyhedrosis virus DNA. J. Virol. 45, 96 l-970. Doeos, P., and COCHRAN, M. A. (1980). Protein synthesis in cells infected by Autographa californica nuclear polyhedrosis virus (AcNPV): The effect of cytosine arabinoside. Virology 103, 446464. GEARING, K. L., and POSSEE, R. D. (1990). Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus. J. Gen. Virol. 71, 251-262. GOMBART, A. F., BLISSARD, G. W., and ROHRMANN, G. F. (1989). Characterization of the genetic organisation of the Hindlll M region of the multicapsid nuclear polyhedrosis virus of Orgyia pseudorsugala reveals major differences among baculoviruses. J. Gen. Viral. 70, 1815-1828. GUARINO, L. A., GONZALEZ, M. A., and SUMMERS, M. D. (1986). Complete sequence and enhancer function of the homologous DNA of Autographa californica nuclear polyhedrosis virus. J. Virol. 60, 224-229. HARDIN, S. E.. and WEAVER, R. F. (1990). Overlapping divergent transcripts mapping to the Hindlll F region of the Autographa californica nuclear polyhedrosis virus. J. Gen. Viral. 71, 225-229. HOOFT VAN IDDEKINGE, B. J. L., SMITH, G. E., and SUMMERS, M. D. (1983). Nucleotide sequence of the polyhedrin gene of Aufographa californica nuclear polyhedrosis virus. Virology 131, 561565. HOWARD, S. C., AYRES, M. D., and POSSEE, R. D. (1986). Mapping the 5’ and 3’ ends of Autographa californica polyhedrin mRNA. Virus Res. 5, 109-l 19. KELLY, D. C., and LESCOTT, T. (1981). Baculovirus replication: Protein synthesis in Spodopfera frugiperda cells infected with Trichoplusia ni nuclear polyhedrosis virus. Microbiologica 4, 35-47. KOZAK, M. (1986). Point mutations define a sequence flanking the

ET AL. AUG initiator codon that modulates translation by eukaryotic ribosome% Cell 44, 283-292. LEE, H. H., and MILLER, L. K. (1978). Isolation of genotypic variants of Autographa californica nuclear polyhedrosis virus. 1. Viral. 27, 754-767. LUCKOW, V. A., and SUMMERS, M. D. (1988). Trends in the developments of baculovirus expression vectors. BioTechnology 6, 4755. MAINPRIZE, T. H., LEE, L., and MILLER, L. K. (1986). Variation in the temporal expression of overlapping baculovirus transcripts. Virus Res. 6, 85-99. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MARUNIAK, J. W., and SUMMERS, M. D. (I 981). Autographa cakfornica nuclear polyhedrosis virus phosphoproteins and synthesis of intracellular proteins after virus infection. Virology 108, 25-34. MATSUURA, Y., POSSEE. R. D., OVERTON, H. A., and BISHOP, D. H. L. (1987). Baculovirus expression vectors: The requirements for high level expression of proteins, including glycoproteins. J. Gen. Wrol. 68, 1233-l 250. MAXAM, A. M., and GILBERT, W. (1980). Sequencing end-labelled DNA with base specific chemical cleavages. In “Methods in Enzymology” (L. Grossman and K. Moldave, Eds.). Vol. 65, pp. 499560. Academic Press, San Diego. MILLER, L. K. (1988). Baculoviruses as gene expression vectors. Annu. Rev. Microbial. 42, 177- 199. MILLER, L. K., and DAWES, K. P. (1979). Physical map of the DNA genome of Autographa californica nuclear polyhedrosis virus. J. Viral. 29, 1044-1055. 001, B. G., RANKIN, C., and MILLER. L. K. (1989). Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene. J. Mol. Viol. 210, 721-736. PENNOCK. G. D., SHOEMAKER, C., and MILLER, L. K. (1984). Strong and regulated expression of Escherichia co/i &gelactosidase in insect cells using a baculovirus vector. Mol. Cell. Biol. 4, 399-406. POSSEE. R. D. (1986). Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. tirus Res. 5, 43-59. POSSEE, R. D.. and HOWARD, S. C. (1987). Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus. Nucleic Acids Res. 15, 10,233-l 0,248. POSSEE, R. D.. and KELLY, D. C. (1988). Physical maps and comparative DNA hybridization of Mamesrfa brass&a and Panojis flammea nuclear polyhedrosis virus genomes. J. Gen. Viral. 69, 12851298. RANKIN, C., 001, B. G., and MILLER, L. K. (1988). Eight base pairs encompassing the transcriptional start point are the major determinant for baculovirus gene expression. Gene 70, 39-49. ROHRMANN. G. F. (1986). Polyhedrin structure. J. Gen. Viral. 67, 1499-1513. SANGER, F., NICKLEN, S.. and COULSON, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nat/. Acad. Sci. USA 74, 5463-5467. SMITH, G. E.. and SUMMERS, M. D. (1978). Analysis of baculovirus genomes with restriction endonucleases. Vifology 89, 517-527. SMITH, G. E., and SUMMERS, M. D. (1979). Restriction maps of five Autographa californica MNPV variants, Trichopksia ni MNPV, and Galleria me//one//a MNPV DNAs with restriction endonucleases Smal, Kpnl, BarnHI, Sacl, Xhol, and /%coRI. 1. Viral. 30, 828-838. SMITH, G. E., FRASER, M. J., and SUMMERS, M. D. (1983a). Molecular engineering of the Autographa californica nuclear polyhedrosis virus genome: Deletion mutations within the polyhedrin gene. J. Viral. 46, 584-593. SMITH, G. E., SUMMERS, M. D., and FRASER, M. J. (1983b). Production

AcMNPV

EcoRI-I AND -R SEQUENCE

Poiyhedrin PAY l TCCGGCGTATTAAARCACGATACATTGTTATTAGTACATTAGTACATTTATT~GCGCTAGA~CTGT 10 20 30 40 50 60 AGGCCGCATAATTTTGTGCTATGTAACAATAATCATGTRRA *

l

AS

GCGTTGTTGATTTACAGACAATTGTTG~CagatcctaggtcgttaggagggCttatcc 70 80 so 100 110 CGCAACAACTAAATGTCTGTTAACAACATGgtCt~gg~tCc~gc~atcctcccgaatagg RQQNVSLQQVld*ttlla*g Bind111 aggccta~~~gggactc~:~ctattgacctaggtta~gcctagatccactgtgc 150 160 170 tccggatcggccctgaggatggataactggatccaatttcgaacggatctaggtgacacg P = apvgv*qglnfsa*iwqa ggggatc:;vAAW&ATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGC 210 220 230 CCCCtagaCCATARAATTATTARGTARTTTllRATATTAG~TCCCACCATAC~TCTCG P iqYKLLENFKYDKPHYTLA

FIG. 6. Nucleotide sequence of the pOLIG02. The inserted oligonucleotide ters (9 l-l 89) with the predicted coding cleotides of the disrupted SnaBl site are tains the entire EcoRI-I region.

SET

120

180

240 ORP 1629

modified ORF 8 (1629) in is shown in lowercase letsequence. Each three nuunderlined. pOLlGO con-

The ORFs detected in the sequence have not been well characterized by transcriptional analysis, apart from the well studied polyhedrin gene (e.g., Hooft van lddekinge et al., 1983; Smith et al., 1983c; Howard et al., 1986; Possee and Howard, 1987; Rankin et a/., 1988; Ooi et al., 1989) and ORF 6 (603) (Gearing and Possee, 1990). Therefore, some uncertainty must remain as to the authenticity of our computer predictions. However, for most of the ORFs identified in this study the Kozak rules for translation initiation (Kozak, 1986) appear to apply. The ORF 8 (1629) produced an RNA species of 2000 nucleotides with a 3’end mapping very close to the end of the polyhedrin gene coding sequence. The 5’ end of the RNA was mapped using primer extension analysis to a ATAAG motif implicated in the transcription of baculovirus late and vety late genes (Rohrmann, 1986; Possee and Howard, 1987; Rankin ef al., 1988; Ooi et al., 1989). This result, together with the time at which the ORF 8 (1629) RNA first appeared in virus-infected cells (12 hr p.i.), suggests that the gene should be classified as a late or possibly very late transcript. The RNA appeared to remain stable in infected cells up to 48 hr p.i., but it is not certain that transcription continues for this time. The biological role of a putative ORF 8 (1629) gene product was examined by interrupting the coding sequence at the 3’ end of the gene with a synthetic oligonucleotide that served to insert translation stop codons. This mutation resulted in the production of recombinant viruses that were unstable and appeared to only be able to replicate in the presence of a helper virus. While it is difficult to prove conclusively from such an approach that the gene product is an essential component of the virus life cycle, this is considered to

239

be the most likely explanation for our results. This suggests that it will not be possible to delete this region of the virus genome and insert foreign genes. Although we have not investigated other transcripts produced by this part of the virus genome, some data are available from other studies. Hardin and Weaver (1990) detected two overlapping transcripts in the Xhol-SalI (2528-3574, Fig. 3) region. The 5’ end of a 2.1 -kbp transcript was mapped to a position which we estimate would correspond with nucleotide 2954 (Fig. 3) at early times after infection (2 hr p.i.) and to nucleotide 2954 (Fig. 3) at late times (8-24 hr p.i.). This transcript could encode ORF 3 (453) which has two potential translation initiation sites. However, because of the length of this transcript it would also encompass ORF 2 (984). Mainprize et al. (1986) also mapped several transcripts using a 650-bp cDNA clone specific for the region spanning the two Sad sites (35 and 3859). Northern blot hybridization analysis revealed a 0.8-bp transcript at 2 hr p.i. and a 1.4-kb transcript at 6 hr p.i. At 12 hr p.i., transcripts of 0.5, 0.8, 1.1, 1.4, 1.75, 2.0, 3.05, 3.85 and 4.6 kb were evident. By 24 hr p.i., transcripts

FIG. 7. Hybridization analysis of ORF 8 (1629) recombinant virus DNA. -5’. frugiperda cells were cotransfected with AcRPl.PR8.HA and pOLIGO.1 (pl) or 2 (p2), the progeny virus was titrated in a plaque assay, and polyhedrin-positive (Pl+ and P2+) or -negative viruses (Pl- and P2-) were isolated. Total DNA was purified from cells infected with the plaque isolates, digested with HindIll, and analyzed using Southern hybridization with a nick-translated probe specific for the AcMNPV HindIll-V (V) region. Other samples digested with Hindlll include: AC, AcMNPV; pl, pOLIGO1; p2, pOLIG02. The sizes (nucleotides) of the bands are indicated on the right side of the blot. p, the backbone of the pOLlGO plasmids which hybridized to contaminating plasmid sequences in the probe.

AcMNPV

EcoRI-I AND -R SEQUENCE

df human &interferon in insect ceils infected with a baculovirus expression vector. Mol. Cell. Biol. 3, 2156-2165. SMITH, G. E., VU\K, J. M., and SUMMERS, M. D. (1983c). Physical analysis of Autographa californica nuclear polyhedrosis virus transcripts for polyhedrin and lO,OOO-molecular weight protein. /. viroi. 45, 215-225. STADEN. R. (1980). A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Res. 8, 3673-3694. TILAKARATNE,S., HARDIN,S. E., and WEAVER,R. F. (1991). Nucleotide sequence and transcript mapping of the HindIll F region of the Autographa californica nuclear polyhedrosis virus genome. J. Gen. Viral. 72, 285-291. VAUGHN,J. L., GOODWIN,R. H., TOMPKINS,G. J., and MCCAWLEY, P. (1977). The establishment of two cell lines from the insect Spodopfera frugiperda (Lepidoptera: Noctuidae). In vitro 13,213-217.

241

WEAVER, R. F., and WEISSMANN,C. (1979). Mapping of an RNA by a modification of the Berk-Sharp procedure: The 5’ termini of 15s p-globin mRNA precursor and mature @-globin mRNA have identical map coordinates. Nucleic Acids Res. 7, 1175-l 193. WEYER,U., and POSSEE,R. 0. (1988). Functional analysis of the ~‘10 gene 5’ leader sequence of the Autogmpha californica nuclear polyhedrosis virus. Nucleic Acids Res. 16, 3635-3653. WEYER,U., and POSSEE,R. D. (1989). Analysis of the promoter of the Autographa californica nuclear polyhedrosis virus ~10 gene. /. Gen. Viral. 70, 203-208. WHITFORD,M., STEWART,S., Kuz~o, J., and FAULKNER,P. (1989). Identification and sequence analysis of a gene encoding gp67. an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. f. Viral. 63, 1393- 1399. WOOD, H. A. (1980). Autographa californica nuclear polyhedrosis virus-induced proteins in tissue culture. Virology 102, 2 l-27.