Conservation of polyhedrin gene promoter function between Autographa californica and Mamestra brassicae nuclear polyhedrosis viruses

183

Virus Research, 12 (1989) 183-200 Elsevier

VRR 00471

Conservation of polyhedrin gene promoter function between Autographa californica and Mamestra brassicae nuclear polyhedrosis viruses Iain R. Cameron and Robert D. Possee NERC Institute of ViroIogy, Mansfield Road Oxford, U.K. (Accepted 25 October 1988)

The DNA sequence of the polyhedrin gene of the Mamestra brassicae multiple nucleocapsid nuclear polyhedrosis virus (MbMNPV) was determined and compared with the polyhedrin genes of Autographa californica (AC) and Panolis jlammea (Pf) MNPVs. Using this information, a transfer vector was constructed based on the EcoRI I fragment of AcMNPV in which the polyhedrin promoter was replaced by the homologous region extending 481 nucleotides upstream from the MbMNPV polyhedrin coding sequence. The Escherichia coli 1acZ gene was also included downstream from the putative MbMNPV promoter. Cotransfection of this transfer vector with wild-type AcMNPV DNA produced stable recombinant viruses expressing the 1acZ gene under the control of the MbMNPV polyhedrin promoter. The levels of beta-galactosidase produced by these recombinants in infected cells were 30% lower than the expression level obtained from viruses with the authentic AcMNPV promoter in front of the 1acZ gene. The MbMNPV promoter has thus been shown to function efficiently in the genetic environment of AcMNPV. The implications of this finding for the release of genetically manipulated baculovirus insecticides and for the construction of baculovirus multiple expression vectors are discussed. Baculovirus;

Correspondence

AcMNPV;

MbMNPV;

Polyhedrin sequence; 1acZ

to: I.R. Cameron, NERC Institute of Virology, Mansfield Road, Oxford, OX13SR, U.K.

0168-1702/89/%03.50

0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

184

Introduction There is considerable interest in the design of genetically modified baculovirus insecticides incorporating foreign genes (e.g. bacterial toxins or insect hormones) which enhance their efficacy and speed of action. A model system to assess the potential of genetic engineering to improve the potency of such viruses has used controlled experimental field releases of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) in the United Kingdom (Bishop, 1986; Bishop et al., 1988). Two other unengineered baculoviruses, Punolis flummeu (Pf) MNPV and Mumestru brussicue (Mb) MNPV, which are closely related to each other (Possee and Kelly, 1988) have been used in spray trials to control the pine beauty moth (Punolis flummeu, Lepidoptera:Noctuidae) on lodgepole pine in Scotland (Entwistle and Evans, 1987; P.F. Entwistle personal communication). MbMNPV has also been used to control M. brussicue on cabbages (Langenbruch et al., 1986). The biochemical and biological properties of MbMNPV have been documented (Kelly and Brown, 1980; Brown et al., 1981; Evans et al., 1981, Allaway and Payne, 1984). Such work raises important questions regarding the exchange of genetic material between an engineered baculovirus insecticide and other viruses coexisting in the environment or susceptible higher organisms. Homologous recombination between AcMNPV and the closely related Ruchiplusiu ou MNPV has been demonstrated in vivo (Croizier et al., 1988), AcMNPV has been shown to acquire a copia-like transposeable element during serial passage in culture (Miller and Miller, 1982) and both AcMNPV and Gulleriu mellonella (Gm) MNPV have also been shown to acquire middle repetitive DNA during cell culture (Fraser et al., 1983). In this paper we address the question of whether transferred genes can function in their new genetic environment. The polyhedrin gene promoter is currently favoured to drive foreign gene expression in recombinant baculoviruses (reviewed by Liickow and Summers, 1988). This promoter is also relatively well conserved between otherwise distantly related baculoviruses (Rohrmann, 1986). Thus it is possible that a foreign gene under the control of the polyhedrin promoter could be expressed if transferred to a heterologous baculovirus. MbMNPV and AcMNPV were chosen as suitable candidates for such an experimental exchange. MbMNPV is currently used as a biological control agent but it does not grow well in tissue culture. AcMNPV grows well in tissue culture and detailed knowledge of the function of its polyhedrin promoter is available (Matsuura et al., 1987; Possee and Howard, 1987). AcMNPV and MbMNPV have covalently closed circular genomes of approximately 130 and 150 kilobase pairs (kbp), respectively. However, with the exception of the polyhedrin gene these viruses show little DNA homology (Possee and Kelly, 1988). We report here the cloning and sequencing of the polyhedrin gene, promoter and flanking sequences from MbMNPV. Using this information we have constructed a transfer vector to introduce the MbMNPV polyhedrin promoter coupled to a marker gene (E. coli 1acZ) into AcMNPV. Recombinants have been isolated and characterised to show that the MbMNPV promoter is functional in its new location.

185

Materials and Methods Viruses and cells MbMNPV (Oxford strain) (Brown et al., 1981) was used throughout. Polyhedra and virus particles were purified from infected laboratory reared iW. brassicae insects as described by Harrap et al., 1977. AcMNPV (C6 strain), AcMNPV.lacZ (AcRP23.lacZ of Possee and Howard, 1987) and recombinant viruses were grown and titrated (Brown and Faulkner, 1977) on monolayers of Spodoptera frugiperda cells (IPLB-Sf-21) (Vaughn et al., 1977) at 28’ C using TClOO medium supplemented with 10% foetal calf serum (TClOO-10F). Suspension cultures of S. frugiperda cells were also used to obtain virus stocks. Purification

of virus DNA

Virus DNA was purified from virus particles of MbMNPV, AcMNPV or recombinants as described previously (Brown et al., 1977). DNA was stored at 4 o C and digested with restriction endonucleases (Gibco-BRL or Pharmacia) using the manufacturers recommended conditions. For transfection experiments the DNA was used within 2 weeks of purification. Southern blotting and DNA hybridisation Restriction endonuclease fragments of virus DNA were fractionated on 0.6% agarose gels in Tris-borate buffer (pH 8.3) containing 0.5 pg/ml of ethidium bromide (Maniatis et al., 1982). DNA was blotted onto nitrocellulose filters (Schleicher and Schuell BA85) as described by Southern (1975). Filters carrying immobilised DNA were pretreated by boiling for 5 min in 5 mM EDTA (pH 7.5) prior to hybridisation with nick-translated DNA probes (Rigby et al., 1977) for 18 h at 37 o C in a solution containing 50% formamide, 6 x SSC, 50 mM Hepes-NaOH (pH 7.0), 0.1% each of BSA, ficoll and polyvinylpyrrolidone (Denhardt, 1966) and 100 pg/ml of sheared denatured salmon sperm DNA. Filters were washed for 5 min each in 4 changes of 2 X SSC at room temperature then for 1 h in 2 changes of 2 x SSC at 65 o C. The filters were air dried and exposed to x-ray film. Molecular cloning of DNA Hind111 or Asp718 restriction endonuclease fragments of MbMNPV were inserted into pUC19 vectors using standard protocols (Maniatis et al., 1982). The Hind111 and Asp718 clones containing the MbMNPV polyhedrin gene were identified by colony hybridisation; the probe was a 1 kb Sal1 fragment of the closely related virus PfMNPV known to contain the polyhedrin gene coding sequence (Oakey et al., 1988). These plasmids are referred to as pMbH0 and pMbA1 respectively.

186 !

ORFI

Ikb

Okb I..

POLYHEDRlN

.

.

.

.

.

1..

)

~

ORF2

2kb .

*.

‘.

.

3kb

1.

”

-

--

*

1.

*

1..

-

c

*

f--

-

--

*.

-

--

---_)

--Fig. 1. Physical map and sequencing strategy of plasmid pMbA1. The arrows in the figure indicate the size and direction of the DNA fragments sequenced. The arrows indicate the coding sequence for polyhedrin and the two large open reading frames revealed by the sequence. ‘c’ is the 12 bp consensus sequence 5’-TGTAAGTAATTT-3’ polyhedrin gene. Restriction sites are indicated as follows A = Asp718, X = XfioI, M = E = EcoRV, Hd-Hind111 and D = DdeI. A scale in kilobases is given.

lower part of the in the upper part ORFl and ORF2 upstream of the MluI, Hf = HinfI,

Purification of DNA from agarose gels DNA was purified from low gelling temperature by Possee and Kelly (1988).

(LGT)

agarose

gels as described

Construction of plasmid pMbA 1. 1acZ The 0.65 kb Hind111 fragment mapping between 1.33 kb and 1.98 kb on pMbA1 (Fig. 1) was excised from this plasmid and inserted into pUC19. This construct was partially digested with the restriction endonuclease AccI which cuts at positions equivalent to nucleotides 826 and 869 in pMbA1 (Fig. 2) and within the pUC19 polylinker. Linear DNA was isolated from a 0.6% LGT agarose gel and treated with Klenow enzyme at 37 o C for 1 min in the presence of 0.2 mM dATP to delete the coding strand of DNA back to the first nucleotide of the ATG. A flush ended molecule was produced by further treatment with Sl nuclease; the DNA was dephosphorylated with calf intestinal phosphatase and religated with BamHI linkers. After transformation of E. coli JM105 the recombinant plasmids were isolated with a BamHI site in lieu of the MbMNPV polyhedrin gene ATG codon (pMbH-B). The pMbA1 construct was digested with BarnHI, treated with Klenow enzyme and

Fig. 2. Nucleotide sequence of the MbMNPV polyhedrin gene and flanking open reading frames. The polyhedrin gene coding sequence and the open reading frames ORFl and 0RF2 (complementary sequence shown) are indicated together with the corresponding protein sequence. The polyhedrin transcription initiation site inferred from previous studies of PfMNPV (Oakey et al., 1988) and AcMNPV (Howard et al., 1986) is shown in the 12 bp consensus sequence (underlined). The EcoRV sites used in constructing plasmids (Fig. 4) are shown as is the Asp718 site in ORF2. Putative polyadenylation signals approximating to the eukaryotic AATAAA (Bimstiel et al., 1985) are indicated, along with their orientation, downstream of ORFl (base 42), 0RF2 (BASE 1602) and polyhedrin (base 1790). The major polyproline tract in 0RF2 is underlined (bases 2285 to 2317). The imperfect direct repeat is boxed between bases 2411 and 2548. Five complete and 3 partial copies of the 21 bp sequence are indicated.

POLYHEDRIN Y\ID,<

188

then religated to remove the BamHI site in the polylinker of pUC19, producing pMbAl-1. The Hind111 fragment spanning the polyhedrin promoter was then excised from pMbAl-1 using a partial Hind111 digest and replaced with the homologous region from pMbH-B to produce a transfer vector with a unique BamHI cloning site. The 1acZ gene from pCHllO-&$I1 (Possee and Howard, 1987) was then inserted in the correct orientation at this site to produce pMbAl.lacZ. DNA transfection and selection of recombinant

viruses

AcMNPV DNA (1 pg) and plasmid DNA (5 pg) were co-transfected onto monolayers of S. frugiperda cells using the calcium phosphate procedure of Graham and van der Eb (1973). After two days at 28” C the cells were harvested and virus was titrated under LGT agarose on monolayers of S. frugiperda cells. Recombinant viruses expressing the beta-galactosidase gene were identified as described by Possee and Howard (1987). Virus stocks were grown up as described above to titres > = lo8 PFU/ml after infection at low multiplicity (0.1 PFU/cell). PAGE analysis of infected cell proteins Recombinant viruses were used to infect S. frugiperda cells (lo6 per 35mrn Petri dish) at a multiplicity of 10 PFU/cell. The inoculum was removed after 1 h at room temperature (21°C), the cells were washed once with TClOO-10F and the dishes were incubated with 2 ml of TClOO-10F at 28OC. After 15, 18, 21, 24 or 30 h the cells were harvested, pelleted by low speed centrifugation and washed in 100 ~1 of TE. A 25 ~1 aliquot from each time point was analysed by polyacrylamide gel electrophoresis as described by Possee and Howard (1987). Assay of beta-galactosidase

activity in infected cell extracts

Equal numbers of cells from the time courses of infection with AcMbPl.lacZ and AcMNPV.lacZ were resuspended in 100 ~1 of TE and subjected to 3 cycles of freezing and thawing. After centrifugation the supernatant was assayed for. betagalactosidase activity by mixing 5 ~1 of extract with 106 ~1 of 2.5 PM Onitrophenyl-beta-D-galactopyranoside and 895 ~1 of 0.08 M sodium phosphate buffer (pH 7.7). Absorbance readings were taken at 420 nm over several minutes and the initial rates of increase were calculated from the slope of the resulting graph. DNA sequencing DNA was digested with an appropriate restriction endonuclease and labeled using the Klenow fragment of DNA polymerase (Pharmacia) and [ 32P]dNTPs. After redigestion DNA fragments were separated on a 4% non-denaturing polyacrylamide gel. Labeled fragments were eluted in 2 M ammonium acetate and precipitated with ethanol. Sequencing was by the chemical degradation method (Maxam and Gilbert 1980).

189

Result8 Cloning and sequencing of the MbMNPV polyhedrin

gene

The location of the MbMNPV polyhedrin gene was determined by Possee and Kelly (1988) and assigned to the Hind111 0 fragment. This fragment was inserted into pUC19 and the resulting plasmid (pMbH0) was used as a source of DNA for sequencing the MbMNPV polyhedrin gene. A preliminary analysis of the sequence data revealed that only the 3’ end of the polyhedrin gene was contained in pMbH0. Therefore MbMNPV DNA was digested with restriction endonuclease Asp718, the resulting fragments were separated electrophoretically and transferred to nitrocellulose (Southern, 1975). This filter was hybridised with a radiolabelled 2 kbp Sal1 fragment of PfMNPV which spans the entire polyhedrin gene (Oakey et al., 1988). This probe hybridised to a 3.5 kbp fragment (data not shown) which was subsequently isolated from a preparative LGT gel and cloned into pUC19. This clone (pMbA1) was mapped with several restriction endonucleases by analysis of the products of double digestion on agarose gels (Fig. 1) and then sequenced to secure the data for the remainder of the polyhedrin gene (Fig. 2). Comparison of the PfMNPV (Oakey et al., 1988) and MbMNPV polyhedrin genes shows them to be very closely related, the coding sequences share 92% identity at the DNA level and 98% identity at the protein level. The 61 bp of DNA immediately 5’ to the polyhedrin ATG codon is identical to that of PfMNPV, this sequence spans the 12 bp consensus sequence (boxed in Fig. 3) found upstream of all hyper-expressed very late NPV genes sequenced to date (Rohrmann, 1986). The initiation sites for polyhedrin mRNA from both PfMNPV (Oakey et al., 1988) and

ORFl

MbMNPV AcMNPV

MbMNPV AcMNPV

1 TTACCACGTA CATGATTACG ** * t * AAACTGGAAA TGTCTATCAA 11 1

61 CGTTTTTTGG * tt CCATCTCGCA 61

11 GAA * AAT 71

tttt

CACAATGTGA ** tt TATATAGTTG 21

8: St

31 41 TGGCTTCGGT ATAACCGACG * tt * * CTGATATCAT GGAGATAATT 31 41

CTC CTTTCGTAGA ttt*:*t

51 CGTATTTATA St : AAAATGATAA 51

111 101 AGATTGTGAA AAATAAAATA tt tttt t* tt tt AGTTTTGTAA TAAAAAAACC 111 101

P$XTHEDRIN MbMNPV AcMNPV

151 171 141 161 121 ATA CCCG-TTACA GTTACAATCC GTCGTTGGGA CGTACCTACG TCTACGACAA TA:t*t:*t*** * **tt**** ::t t : :t *: : tt: t:: t: t t:: TATAAATATG CCGGATTATT CATACCGTCC CACCATCGGG CGTACCTACG TGTACGACAA 121 -L 131 141 151 161 171

POLYHEDRIN Fig. 3. Comparison of the DNA sequences of the MbMNPV polyhedrin gene promoter and the AcMNPV promoter. The 12 bp consensus sequence is boxed and the transcriptional start sites are indicated. The alignment of sequences shown maximises the sequence homology around the initiation codons (indicated on the figure). The initiation codon of ORFl is also indicated.

190

AcMNPV (Howard et al., 1986) lie within this sequence. The sequence ATTAAA approximating to the eukaryotic polyadenylation signal AATAAA (Bimstiel et al., 1985) lies 229 bp downstream of the MbMNPV polyhedrin coding sequence. DNA sequences flanking

the polyhedrin

gene

The DNA sequence upstream of the polyhedrin gene contains a significant open reading frame (ORFl) on the opposite strand to the polyhedrin gene which could encode a protein of 216 amino acids (Fig. 2; positions 67 to 714). The first ATG codon of ORFl lies in the sequence ANNATGT which is favourable for an initiation methionine (Kozak, 1983). It is separated from the polyhedrin ATG codon by 110 bp. Downstream from the polyhedrin gene coding sequence and on the opposing strand the DNA contains a large open reading frame (ORF2). The polyhedrin and 0RF2 translation stop codons are separated by 53 bp, which also contains a polyadenylation signal (AATAAA) 14 bp downstream of the ORF2 (Fig. 2). Only the 3’ end of 0RF2 (capable of encoding 319 amino acids) has been sequenced so far. This contains several points of interest, it is GC-rich between positions 2285 and 2318 in the DNA sequence potentially encoding a continuous stretch of 11 proline residues, it also contains an imperfect direct repeat arrangement comprising 5 complete and 3 partial copies of a 21 bp sequence (Fig. 2). No function has yet been ascribed to these elements. Construction of AcMNPV

transfer vectors carrying the MbMNPVpolyhedrin

promoter

Since MbMNPV does not grow sufficiently well in M. brassicae cells for the easy generation and selection of recombinant viruses we decided to study the ability of the MbMNPV polyhedrin promoter to function in the genetic environment of AcMNPV. Considering that the promoter of the AcMNPV polyhedrin is located within 69 bp upstream of the ATG codon (Possee and Howard, 1987) and the consensus RNA initiation site of MbMNPV lies slightly closer to the ATG than does that of AcMNPV it was concluded that the MbMNPV promoter probably lies between ORFl and polyhedrin. Transfer vectors have been described which provide a convenient and efficient method for the introduction of novel DNA sequences into AcMNPV (Liickow and Summers, 1988; Possee and Howard, 1987). We describe below the construction of such a plasmid. The first step in the production of a transfer vector used the plasmid pMbAl.lacZ derived from pMbA1 which had been engineered to introduce the 1acZ gene of E. coli in place of the 5’ end of the polyhedrin coding sequence whilst retaining the entire polyhedrin gene leader sequence. An intact 5’ leader sequence has been shown to be important for maximum expression levels to be obtained in AcMNPV expression vectors (Matsuura et al., 1987). Digestion of the plasrnid pMbAl.lacZ with EcoRV generates 4 fragments of 4.2 kbp, 2.05 kbp, 1.7 kbp and 0.92 kbp. The 1.7 kbp fragment comprises 481 bp upstream of the RNA initiation site for polyhedrin, the polyhedrin leader sequence and 1.2 kbp from the 5’ end of the 1acZ gene (Fig. 4); this was purified after electrophoresis on a 0.6% low gelling tempera-

EcoRl

EcoRl

EcoRl

Hindlll

Fig. 4. (A) Construction of an AchINPV transfer plasmid (pAcMbPl.lacZ) containing the MbMNPV polyhedrin gene promoter and E. co/i la&! gene. The plasmid pMbAl.lacZ was cleaved with EcoRV and the 1.7kb fragment (heavy arrow) spanning the polyhedrin promoter and the 5’ end of the 1acZ gene was purified after elution from an LGT agarose gel. This fragment was ligated to the 12kb EcoRV fragment of pAcMNPV.lacZ (heavy arrow) similarly purified. After ligation in the correct orientation the 1acZ gene is reconstituted. In the resulting transfer vector pAcMbPl.lacZ the MbMNPV polyhedrin promoter is substituted for that of AcMNPV. The plasmid vector (single line portion of the circle) for pMbAl.lacZ is pUC19 that for pAcMNPV.lacZ and pAcMbPl.lacZ is pUC8. Black portions of the circle represent MbMNPV sequences, open portions represent AcMNPV sequences. The 1acZ gene is denoted by the hatched area. The single line is the plasmid vector; pUC19 in the case of pMbAl.lacZ and pUC8 in the case of pAcMNPV.lacZ. (B) DNA sequence organisation of the MbMNPV polyhedrin promoter and flanking sequences in pAcMbPl.lacZ. The EcoRV site recreated by ligation of the AcMNPV and MbMNPV sequences is underlined as are the Hind111 site and the 12 bp consensus sequence. The MbhfNPV polyhedrin leader sequence is indicated up to the junction created between MbMNPV and 1acZ leader sequences.

192

ture (LGT) agarose gel. The AcMNPV transfer vector pAcMNPV.lacZ (Possee and Howard, 1987) contains the same 1acZ sequence as that in plasmid pMbAl.lacZ. It contains two EcoRV sites, the first lies 42 bp upstream of the RNA initiation site for polyhedrin, the second lies at the same site within the 1acZ coding sequence as that in pMbAl.lacZ. Cleavage of this plasmid with EcoRV generates two fragments of 1.2 kbp and 12 kbp the larger of which was purified after electrophoresis on a 0.5% LGT agarose gel. This fragment was ligated together with the 1.7 kbp fragment carrying the MbMNPV polyhedrin promoter from pMbAl.lacZ to produce a plasmid pAcMbPl.lacZ in which the 1acZ gene was reconstituted, preceded by 524bp of MbMNPV polyhedrin 5’ sequence replacing 91 bp of AcMNPV polyhedrin 5’ sequence (Fig. 4).

Transfer of the MbMNP V polyhedrin promoter into AcMNPV The transfer vector pAcMbPl.lacZ, together with infectious AcMNPV DNA was used to cotransfect S. frugiperda cells. Progeny virus was titrated on monolayers of

AcMNPV.lacZ AcMbPl.lacZ 24 30 15 18 21 24 30 MI

15 18 21

R-gal

>

Fig. 5. Temporal expression of the 1acZ gene under the control of the MbMNPV polyhedrin gene promoter. Spodoptera frugiperdu cells (106) were infected with 10 PFU/cell of either AcMNPV.lacZ (tracks 1 to 5) or AcMbPl.lacZ (tracks 6 to 10). Cells were incubated in TClOO-10F at 2S°C and harvested 15, 18, 21, 24 and 31 h post-infection, respectively. Infected cells were washed in PBS and dissolved in dissociation mix. Samples were electrophoresed on a 12% discontinuous polyacrylamide gel which was then stained with coomassie blue. AcMNPV.lacZ = AcMNPV with the 1acZ gene under the control of the AcMNPV polyhedrin promoter. AcMbPl.la&Z = AcMNPV with the 1acZ gene under the control of the MbMNPV polyhedrin promoter. MI = Mock-infected S. frugiperda cells. &gal = Betagalactosidase produced by the 1acZ gene.

193

cells and recombinant viruses were picked from plaques which stained blue in the presence of the chromogenic substrate X-gal. The levels of IacZ expression in such viruses could then be compared with that in viruses with the IacZ gene under the control of the AcMNPV polyhedrin promoter. Analysis

of recombinant

AcMNPV

carrying the ~b~~PV

pQ~~edri~ promoter

and

ILXZ

In order to quantify the levels of expression of 1acZ under the control of the MbMNPV promoter, 6 independently derived recombinant virus clones were studied. S. frugiperda cells were infected with virus at a high multiplicity of infection and the intra~llular proteins extracted at 24 h pi. were analysed by SDS-PAGE. The results demonstrated that beta-galactosidase was expressed to the same extent by all 6 clones (data not shown). One of these clones (AcMbPl.lacZ) was selected and used to study temporal aspects of beta-galactosidase expression during virus infection. S. frUgperda cells were inoculated with virus as before and harvested at 15, 18, 21, 24 and 30 h post-infection. As a control AcMNPVJacZ (Possee and Howard, 1987), carrying the 1acZ gene under the control of the authentic AcMNPV polyh~~ promoter, was used to produce a parallel time course under identical conditions of infection. Cells were harvested and the proteins were analysed on a 12% polyacrylamide gel (Fig. 5). It is clear that the temporal expression of 1acZ by AcMbPl.lacZ is the same as that by AcMNPV.lacZ indicating that the MbMNPV promoter is able to fun~tion~ly replace the AcMNPV promoter and produce high level expression. The overall level of expression of 1acZ by AcMbPl.lacZ was estimated by assaying infected cell extracts for beta-galactosidase activity (Table 1). This indicates that levels of beta-galactosidase produced by AcMbPl.lacZ are approximately two-thirds of those from AcMNPV.lacZ. ~o~~rrnat~~n that the ~b~~PVpromot~r

is present in AcMbPI.lacZ

The restriction fragment pattern of AcMbPl.lacZ with E=coRI confirmed the structure of the recombinant. The promoter region was cloned from AcMbPl.lacZ

TABLE

1

Temporal expression of beta-galactosidase by AchKNPV.lacZ and AcMbPlJacZ Time p.i. (hours)

15 18 21 24 30

AcMNPV.lacZ

AcMbPl.lacZ

OD+&nin

%

ODo/min

%

0.02 0.06 0.29 0.70 0.14

2.7 8.1 39.2 94.6 100

0.05 0.10 0.32

6.8 13.5 43.2 60.8 12.9

0.45 0.54

% = Activity expressed as a percentage of that produced by AcMNPV.lacZ

at 30 hours pi.

194 AcMNPV OpSNPV MbMNPV PfMNPV

-MPDY SYRPT IGRTY VYDNK MYTR. ..N.S I,.... . . .. . MYTR. ..N.S I,.... . . . . . MYTR. ..N.S I,.... .. . . .

YYKNL GAVIK NAKRK KHFAE HEIEE ... .. A.. . . . . . :A:::-.::;:: 1;:::: ::L.. . . . . . . . . . . ..N.. . ..I. ..L..

ATLDP H.... K K::::

50

AcMNPV LDNYL OpSNPV ..K.. MbMNPV ..R.. PfMNPV ..R..

VAEDP ... .. . .. .. .. ...

FLGPG ... .. ..... . ....

KNQKL . ... . . .... . . ...

TLFKE .. ... .... . .....

IRNVK ... .. .... . .. ...

PDTMK .... . ..... . ....

LVVGW . ..N. ...N. . ..N.

KGKEF S.... S.... S....

YRETW 100 L.... L.... L....

AcMNPV TRFME OpSNPV . . . . . MbMNPV . . . . . PfMNPV . . . . .

DSFPI ... .. . .... .. ...

VNDQE .. ... ... .. . ....

VMDVF I.... .. ... .... .

LVVNM ..I.. ..I.. ..I..

RPTRP .... . a.... . .. . .

NRCYK . ..FR . ..F. . . . ..

FLAQH ..... a.... .....

ALRCD ..... ..... .....

PDYVP 150 .E... .. . . . .E...

SFEQF

IDRVI

AoMNPV HDVIR IVEPS WVGSN NEYRI SLAKK GGGCP IMNLH SEYTN OpSNPV MbMNPV

.E... .E...

. . . . . ..a..

Y.... Y....

PfMNPV

.E... . . . . . Y....

AcMNPV WENFY KPIVY IGTDS OpSNPV . . . . . . . . . . V.... MbMNPV . . . . . . v.... PfMNPV a.... ::::. V....

. . . . . . . ..V

. . ..R . . ..R

. . . . . V.... . . . . . V....

. . ..V . . ..R . . . . . V....

200

A.... . . . . .

E. .N..H :::E. . N... . . . . . . ..E. .N...

AEEEE ILLEV SLVFK VKEFA PDAPL . . . . . . . . . . . . . . . I.... . . . . . .. . . . . . I.... . . . . . ..::: ::::: . . . . . I.... . . . . .

FTGPA YS... YN... YN...

Y

246

. .

.

Fig. 6.Comparisonof thepolyhedrin protein sequence of MbMNPV Iddekinge AcMNPV

with those of AcMNPV (Hooft van et al., 1983), OpSNPV (Leisy et al., 1986) and PfMNPV (Oakey et al., 1988). The prototype sequence is shown in full. Differences from AcMNPV are indicated. Dots indicate identity

withAcMNPV.

into pUC19

and sequenced to confirm the integrityof the MbMNPV

front of the 1acZ gene in AcMbPl.lacZ

promoter in

(Fig. 4B).

Discussion The polyhedrin gene of NPVs and the related granulin gene of GVs comprise the most extensively studied region of the baculovirus genome (reviewed by Rohrmann, 1986). To date the polyhedrin genes of 5 nuclear polyhedrosis viruses AcMNPV (Hooft van Iddekinge et al., 1983), BmMNPV (Iatrou et al., 1985), OpMNPV (Leisy et al., 1986a), OpSNPV (Leisy et al., 1986b) and PfMNPV (Oakey et al., 1988), and 2 granulosis viruses PbGV (Chakerian et al., 1985) and TnGV (Akiyoshi et al., 1985) have been sequenced. In addition the polyhedrin proteins of BmMNPV, GmMNPV and Lymantria dispar MNPV have been sequenced directly (Kozlov et al., 1981). We have determined the DNA sequence of the polyhedrin gene and its flanking sequences from MbMNPV (Fig. 2). The predicted protein sequence for MbMNPV polyhedrin (Fig. 2) closely matches those of other polyhedrins; in particular it shares 98% identity with that of PfMNPV (Oakey et al., 1988). Of the 5 amino acids which differ between PfMNPV and MbMNPV polyhedrins, two (S,, and YJ8) are at sites known to be particularly variable between polyhedrins and granulins (Rohrmann, 1986), all 5 are functionally conservative changes (Fig. 6). The homology between polyhedrin proteins has been discussed in detail (Rohrmann, 1986). When

195 the sequences of MbMNPV and PfMNPV polyhedrins are included it is apparent that they are most closely related to the OpSNPV polyhedrin (94% and 95% identity respectively). This compares with between 85% and 89% identity to the polyhedrins of AcMNPV, BmMNPV and OpMNPV. In particular, unique to PfMNPV, MbMNPV and OpSNPV is the amino acid sequence MYTR at the N-terminal of the polyhedrin protein. Of the 32 other residues at which one or other of these three viruses differ from the prototype AcMNPV, 17 are common to all three, of these 6 and (N,,, S,,, I&z, Yi6i and I,,,) are common across the range of polyhedrins granulins, the remaining 11 (N,, Sic,, Lii, I,,, I,,,, R,,,, Visi, Pi,,, Nis,, V,ii and Yzw) seem generally characteristic of the PfMNPV/MbMNPV/OpSNPV group of viruses (see Fig. 6), a few of these 11 are shared with one or other of LdMNPV, BmMNPV, PbGV or TnGV but there is no consistent pattern. This seems to indicate that in an evolutionary context PfMNPV and MbMNPV may be most closely related to the singly encapsidated OpSNPV. This is supported at the DNA level, the sequence across the initiation codon is ATAATGTATAC in all three viruses. By comparison with the AcMNPV sequence (Fig. 3; Hooft van Iddekinge et al., 1983) it is likely that the creation of a new ATG codon upstream from the existing ATG along with a frame shift mutation downstream has occurred in the ancestor of these three viruses. It would be illuminating to perform cross-hybridisation studies between an SNPV and either MbMNPV or PfMNPV. The polyhedrin 5’ leader sequences of PfMNPV and MbMNPV are identical (Oakey et al., 1988, Fig. 2), and share closer homology to OpSNPV than to AcMNPV. However, the OpSNPV polyhedrin 5’ leader is the same length as that of AcMNPV (i.e. 5 bp longer than that of PfMNPV/MbMNPV). Where the polyhedrin mRNA CAP sites have been accurately determined (AcMNPV, Howard et al., 1986; OpMNPV, Leisy et al., 1986a; and PfMNPV, Oakey et al., 1988) transcription has been found to begin in or near the core TAAG of the 12 bp consensus sequence found upstream of all late hyperexpressed baculovirus genes so far sequenced (polyhedrin, granulin and ~10; Rohrmann, 1986). It is of interest to note that the nucleotide immediately upstream of the TAAG sequence is a G in both MbMNPV and PfMNPV (Oakey et al., 1988) whereas all other polyhedrin, granulin and p10 genes have an A at this site. The significance of this observation is unclear at present but it does not appear to affect the site of transcription initiation (Oakey et al., 1988). The putative polyadenylation signal (AATAAA; Bimstiel et al., 1985) of the polyhedrin gene lies downstream from the MbMNPV polyhedrin coding sequence. This position is close to that predicted by Sl mapping of the 3’ end of PfMNPV polyhedrin mRNA (Oakey et al., 1988; I.R. Cameron unpublished observations). In PfMNPV, however, the corresponding sequence is GTTAAA. This raises questions about the importance of different sequences in promoting polyadenylation in baculoviruses. A polyadenylation signal (AATAAA) occurs, located within 40 bp downstream of the polyhedrin gene and in the opposite orientation, in MbMNPV, PfMNPV, AcMNPV, OpSNPV and TnGV. This may be a feature preventing transcription of RNA complementary to the polyhedrin coding sequence which could interfere with high level expression of polyhedrin protein.

196

Possee and Kelly (1988) have estimated 70% homology between the DNAs of PfMNPV and MbMNPV by hybridisation in 50% formamide. The DNA sequence of PfMNPV (Oakey et al., 1988) and MbMNPV flanking the polyhedrin gene indicates that the actual level of homology may be higher. Excluding the polyhedrin gene coding sequences, which are highly conserved, the known sequence of the two viruses exhibits 88% homology. If this area is representative of the whole genome, then the 70% estimated by hyb~disation may underestimate the real level of homology between the two viruses. Comparison of the sequences flanking the MbMNPV polyhedrin gene with those flanking the AcMNPV polyhedrin gene (Hooft van Iddekinge et al., 1986, SC. Howard and R.D. Possee personal communication) shows no significant homology upstream, although both viruses possess an open reading frame at this position in the opposite orientation to the polyhedrin gene. In the downstream region of ~MNPV there is a GC-rich region in ORF2 which, if it were translated would be very rich in proline (underlined in Fig. 2). Chen et al. (1988) have reported a GC-rich region at a similar distance downstream from the polyhedrin gene of OpMNPV. In the AcMNPV sequence there is also such a region (SC. Howard and R:D. Possee personal communication). Apart from this high GC content, however, there is no obvious homology between the three viruses in this region. The DNA sequence of 0RF2 of MbMNPV reveals an imperfect direct repeat comprising 5 complete copies and 3 partial copies of a 21 bp sequence (boxed in Fig. 2). This part of ORF2 is also rich in proline although this could be a chance occurence and not related to any function which may be ascribed to the GC-rich/ pol~ro~ne tracts elsewhere in this region. Direct repeat sequences are present in the hrs regions of AcMNPV (Cochran and Faulkner, 1983; Guarino and Summers, 1986) and in OpMNPV (Chen et al., 1988), however, there is no evidence of common sequence or function between them in these viruses. The knowledge of the DNA sequence of the highly expressed baculovirus polyhedrin gene and its promoter has led to the development of expression vectors based on AcMNPV or BmMNPV. Many different foreign genes have been expressed in AcMNPV vectors using the AcMNPV polyhedrin promoter (reviewed by Liickow and Summers, 1988). The integrity of the 5’ leader sequence has been shown to be essential for maximum expression from the polyhedrin promoter (Matsuura et al., 1987) the limit of which has been defined to between 56 bp upstream of the ATG (Possee and Howard, 1987). The 1acZ gene of E.coli has been shown to be highly expressed under the control of the AcMNPV polyhedrin promoter (Pennock et al., 1984). The ease of detection of 1acZ means that it is a suitable marker gene to determine the efficiency of a heterologous virus promoter introduced into AcMNPV. We have constructed a plasmid in which 1acZ is coupled to DNA sequences encompassing the MbMNPV polyh~~n promoter, this cassette is bounded by DNA which normally flanks the AcMNPV polyhedrin gene. This plasmid was used as a transfer vector to introduce the MbMNPV promoter/lacZ cassette into AcMNPV in place of the polyhedrin gene. The level of expression from 1acZ in the recombinant virus approached 75% of that from 1acZ driven by the AcMNPV polyhedrin promoter (Table 1). Clearly,

197

essential elements are conserved between the two promoters. The 5’ leader sequence of MbMNPV polyhedrin is identical at 31 out of 45 positions to that of AcMNPV (Fig. 3) although AcMNPV and MbMNPV are only distantly related (2% by DNA hybridisation; Possee and Kelly, 1988). The MbMNPV polyhedrin gene promoter is able to function in a distantly related baculovirus. Since the polyhedrin promoter may be used to drive the expression of foreign genes in genetically engineered baculovirus insecticides, studies will be needed to determine the likely frequency of recombination between coinfecting baculoviruses. DNA sequencing and experiments such as those described here may determine whether other baculovirus promoters are sufficiently conserved for them to function in a heterologous virus. The recent development of multiple expression AcMNPV vectors {Emery and Bishop, 1988) involved duplicating the polyhedrin promoter region, thus allowing two genes, the N protein of lymphocytic choriomeningitis virus and polyhedrin, to be expressed at a high level. Future development of such vectors may require the expression of larger numbers of foreign proteins. The duplication of the AcMNPV poiyhedrin promoter could lead to instability of the recombinant virus. The availability of a highly active promoter which has less homology to the AcMNPV polyhedrin promoter may be useful in these vectors. Since the 5’ leader sequence of the MbMNPV polyhedrin promoter is 5 bp shorter than that of AcMNPV and identical at only 31 out of the 45 remaining positions (the longest stretch of identity being 7 bases) it would be possible to use it in a multiple expression vector in place of an AcMNPV promoter.

Acknowledgements We would like to thank Professor D.H.L. Bishop for his interest in this work and Chris Hatton for photography of the figures.

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