Download Analysis of Transcription Initiation in the Panolisflammea Nuclear

J. gen. ViroL (1989), 70, 769-775.
769
Printed in Great Britain
Key words: nuclear polyhedrosis virus/Panolis flammea/polyhedrin
Analysis of Transcription Initiation in the Panolisflammea Nuclear
Polyhedrosis Virus Polyhedrin Gene
By R E B E C C A O A K E Y , t I A I N R. C A M E R O N , B E N J A M I N
E L I Z A B E T H D A V I S AND R O B E R T D. P O S S E E *
DAVIS,
NERC Institute of Virology, Mansfield Road, Oxford OX1 3SR, U.K.
(Accepted 15 November 1988)
SUMMARY
The nucleotide sequence of the polyhedrin gene of Pano/is flammea multiple
nucleocapsid polyhedrosis virus (PfMNPV) has been determined. The coding
sequences of this gene shared 82% similarity at the D N A level and 88% similarity at
the protein level with the polyhedrin gene from Autographa califomica (Ac) MNPV. A
single nucleotide deviation from the consensus transcription initiation sequence for
baculovirus very late genes was identified in the PfMNPV polyhedrin gene. R N A was
prepared from Mamestra brassicae larvae infected with PfMNPV and compared with
RNA harvested at 24 h post-infection from AcMNPV-infected Spodopterafrugiperda
cells using Northern blotting with an AcMNPV polyhedrin gene-specific probe. The
PfMNPV m R N A was estimated to be 1.0 kb compared with a larger size of 1-15 kb for
the AcMNPV polyhedrin m R N A . A c D N A copy of the 5' end of the PfMNPV
polyhedrin mR NA was made using the technique of primer extension and sequenced to
demonstrate that the point of transcription initiation was similar to that of AcMNPV
polyhedrin mRNA.
The very late phase of gene expression in baculovirus-infected cells is characterized by a
prolonged burst of transcriptional and translational activity which results in the production of
large amounts of two proteins, polyhedrin and p 10. It has been estimated that up to 90 % of the
polyadenylated virus R N A present at 48 h post-infection (p.i.) consists of transcripts specific for
these two genes (Smith et al., 1983). Polyhedrin is responsible for occluding enveloped virus
particles into crystalline structures to form polyhedra. These act as the agents of dispersal for the
virus in the natural environment. The p 10 is a non-structural protein of uncertain function but it
is thought to play a role in polyhedron morphogenesis in the infected cell (van der Wilk et al.,
1987; Vlak et al., 1988).
The mechanism by which the infected cell is able to produce such large amounts of these
proteins remains to be fully elucidated. For the Autographa californica nuclear polyhedrosis virus
(AcMNPV) polyhedrin gene it has been ascertained that a sequence extending 69 nucleotides
upstream from the ATG is essential for efficient promoter activity (Matsuura et al., 1987; Possee
& Howard, 1987). This sequence consists of a 20 nucleotide region upstream from the m R N A
transcription initiation (start) site (Possee & Howard, 1987) and a 49 nucleotide 5' non-coding
(leader) sequence before the polyhedrin ATG codon (Matsuura et al., 1987). Deletions in either
of these two sequences reduced the polyhedrin promoter activity. Analysis of the AcMNPV p l 0
5' leader sequence has also shown that it is required for high level expression of this gene (Weyer
& Possee, 1988).
Comparison of a number of polyhedrin genes from different baculoviruses has highlighted
the presence of a 12 nucleotide consensus sequence spanning the m R N A start site [(A/T)"~Present address: Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU,
U.K.
0000-8577
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Fig. 1. Physical map and sequencing strategy of the polyhedrin gene and flanking regions. H, HinfI;
Hp, HpalI ; F, Fok I ; S, Sail; X, Xho I ; D, DdeI ; Sc, ScaI. The arrows indicate the direction and length of
the sequenced DNA. The thick line shows the extent of the protein-codingsequencesof the DNA. The
size of the region sequenced is indicated.
ATAAGNA(T/A/C)T(T/A)T; Rohrmann, 1986]. This consensus sequence is also present in
both of the pl0 genes which have been analysed [AcMNPV, Kuzio et al., 1984; Liibbert &
Doerfter, 1984: Orgyia pseudotsugata (Op) MNPV, Leisy et al., 1986c]. The subgroup B
baculoviruses (granulosis viruses; GVs) also have the same consensus sequence upstream of the
granulin gene, which encodes an occlusion body protein of similar function to polyhedrin
(Akiyoshi et al., 1985; Chakerian et al., 1985). The polyhedrin m R N A start sites have been
accurately determined for AcMNPV (Howard et al., 1986) and OpMNPV (Leisy et al., 1986a).
The same two viruses have been used to map the m R N A start site of the pl0 gene (AcMNPV,
Kuzio et al., 1984; L/Jbbert & Doerfler, 1984; OpMNPV, Leisy et al., 1986c). The data indicate
that transcription of the very late genes always initiates within the 12 nucleotide consensus. In
this study we report the cloning and sequencing of the polyhedrin gene from Panolisflammea
(Pf) MNPV and the determination of its m R N A start site.
The polyhedrin gene of PfMNPV has been mapped to an 8.08 kb D N A fragment (designated
F) produced after digestion with H i n d l I I (Possee & Kelly, 1988). This fragment was isolated
from a low gelling temperature agarose gel as described previously (Possee & Kelly, 1988) and
inserted into the H i n d l I I site of pAT153, using techniques described by Maniatis et al. (1982), to
produce the clone pPfHF. Further subcloning of SalI digest products from pPfHF produced a
plasmid with a 1.2 kb insert more convenient for D N A sequencing. The virus D N A in this
plasmid was sequenced by performing chemical degradation analysis (Maxam & Gilbert, 1980)
on fragments radiolabelled at one end with [32p]dNTP using the Klenow fragment of
Escherichia coli D N A polymerase I for 'fill-in' reactions, or [V-32p]ATP and T4 D N A
polynucleotide kinase for the Sca! digest. The sequencing strategy is outlined in Fig. 1.
Preliminary analysis of the data revealed that a SalI site was only 79 nucleotides upstream from
the polyhedrin ATG codon (Fig. 2). Therefore the sequence was further extended by returning
to the pPfHF clone and isolating the regions of interest for sequence determination.
In Fig. 2 the nucleotide sequence data for PfMNPV are presented; the polyhedrin gene
consists of a 738 nucleotide open reading frame encoding a 246 amino acid polypeptide of
predicted Mr 28 935. When the sequence data for the PfMNPV polyhedrin gene-coding region
were compared with the sequence of the homologous gene from AcMNPV (Hooft van
Iddekinge et al., 1983) they were found to share 82~o similarity at the D N A level and 88~o
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CCTTTCGGCAACTTTTTCGTTAATTTTTATCAGATAGCCATTTTT•AGCGTTTGATATCCAAAATA•TTGTAATCCAGGTCTAGTTTCATATTAATGTTCTCCTCTATGATTTTGCT•TG
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Fig. 2. Nucleotide sequence of the PfMNPV polyhedrin gene and flanking regions. The coding
sequence for the polyhedrin begins at position 581 and terminates at 1318. The predicted amino acid
sequence is shown above that of the DNA. The 12 nucleotide consensus sequence spanning the mRNA
transcription initiation site (T) is underlined from 533 to 544, with a gap representing the position of the
A normally present (Rohrmann, 1986). The sequence complementary to the primer used in the cDNA
extension of the mRNA is underlined from positions 605 to 621 and labelled C. The positions of the Sail
sites bounding the initial fragment used for DNA sequencing are indicated.
similarity at the amino acid level. A computer-assisted search of the Y non-coding region of the
P f M N P V polyhedrin gene did not reveal the presence of any potential polyadenylation signals
(Birnstiel et al., 1985). When the 344 nucleotide 3' non-coding sequence of the A c M N P V
polyhedrin gene (Howard et al., 1986) was compared with a similar length from the P f M N P V
polyhedrin gene only 28 ~o homology was calculated. Conversely, the 5' untranslated ends of each
virus polyhedrin m R N A were 70~o homologous. It was noted that the t2 nucleotide sequence
spanning the putative m R N A start site of the P f M N P V polyhedrin gene was not a perfect
match with the consensus proposed by Rohrmann (1986). The second nucleotide in this example
was a G, whereas in all other very late genes an A nucleotide has been recorded. The other 11
nucleotides agree with the remainder of the consensus.
In order to determine what effect the substitution of a G nucleotide at the second position of
the consensus sequence might have on the site of transcription initiation from the P f M N P V
polyhedrin gene it was necessary to extract m R N A transcripts specific for this gene from
infected cells. In c o m m o n with many other N P V s this example does not replicate in cell culture,
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772
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Fig. 3
Fig. 4
Fig. 3. Northern blot analysis of polyhedrin mRNA. Total RNA extracted from M. brassicaelarvae 5
days after infection with PfMNPV (lane 1) or from AcMNPV-infected S.frugiperda cells (24 h p.i.)
(lane 2) was fractionated in a 1~ agarose-glyoxal gel and transferred to Genescreen membrane. The
filter was hybridized with a nick-translated probe specific for the AcMNPV polyhedrin gene (pAcHV;
Possee, 1986). The sizes of the respective mRNA species identified are indicated on each side of the
autoradiograph.
Fig. 4. Sequence analysis of cDNA prepared using primer extension of PfMNPV polyhedrin mRNA.
The oligonucleotide polyhedrin primer was radiolabelled at its 5' end with [?-3zP]ATP and
polynucleotide kinase before extension on the mRNA template with reverse transcriptase. The cDNA
was fractionated in a polyacrylamide urea gel, purified and sequenced using the chemical method. The
sequence complementary to the polyhedrin AUG is indicated by the vertical line and arrowhead at the
side of the gel.
e v e n w h e n using P. flammea cell lines (C. J. A l l e n & R. D. Possee, u n p u b l i s h e d data). A n
a d d i t i o n a l p r o b l e m is that P.flammea insects c a n n o t easily be m a i n t a i n e d in l a b o r a t o r y culture.
T h e r e f o r e R N A was isolated f r o m Mamestra brassicae larvae, w h i c h are p e r m i s s i v e for
P f M N P V , 5 days after infection w i t h P f M N P V using the C s C l - g u a n i d i n i u m i s o t h i o c y a n a t e
m e t h o d ( M a n i a t i s et al., 1982). N o r t h e r n blot analysis o f this R N A and A c M N P V m R N A
extracted f r o m Spodoptera frugiperda cells at 24 h p.i. was carried out as d e s c r i b e d previously
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773
(Possee, 1986), using a nick-translated (Rigby et al., 1977), cloned copy of the 3' end of the
AcMNPV polyhedrin gene (pAcHV; Possee, 1986) to detect homologous sequences (Fig. 3).
The size of the AcMNPV polyhedrin mRNA was estimated to be 1-15 kb which agrees closely
with the previously published value of 1.2 kb (Smith et al., 1983). However, the PfMNPV
polyhedrin mRNA, with a size of 1.0 kb, was significantly smaller. To determine the
transcription initiation site for the PfMNPV polyhedrin mRNA an approach similar to that
employed for the accurate 5' mapping of the AcMNPV polyhedrin mRNA was used (Howard et
al., 1986). An oligonucleotide primer (5"-GTACGTCCCAACGACGG-3') was synthesized
which corresponded to the complement of the coding sequence from +25 to +41 of the
PfMNPV polyhedrin gene (Fig. 2; positions 605 to 621). This was radiolabelled at the 5' end
with [~-32p]ATP and polynucleotide kinase (Howard et al., 1986) and used to prime copying of
the mRNA with reverse transcriptase. Sufficient cDNA was produced to permit sequencing of
the transcript and thus determine the 5' end of the PfMNPV polyhedrin RNA. The sequencing
gel is shown in Fig. 4 and is clearly readable up to a T nucleotide at the 3' end of the cDNA,
followed by two unreadable bands in all four lanes. If the T nucleotide is taken as the point of
transcription initiation then the mRNA start site is located at a position 44 nucleotides upstream
of the PfMNPV polyhedrin ATG (Fig. 2). This is in the central region of the consensus
transcription initiation motif highlighted by Rohrmann (1986) and is only one nucleotide
upstream from the site previously determined for AcMNPV polyhedrin mRNA (Howard et al.,
1986).
A number of baculovirus polyhedrin and granulin genes have been cloned and sequenced.
These include AcMNPV (Hooft van Iddekinge et al., 1983). Bombyx mori MNPV (Iatrou et al.,
1985), OpMNPV (Leisy et al., 1986a), Op single nucleocapsid NPV (Leisy et al., 1986b),
Trichoplusia ni GV (Akiyoshi et al., 1985) and Pieris brassicae GV (Chakerian et al., 1985). The
polyhedrin genes have a high degree of similarity as do the two granulin genes. It is also notable
that even though NPVs and GVs do not share significant sequence similarity as judged by
hybridization analyses (Smith & Summers, 1982), they do show similarity at the nucleotide and
amino acid level when sequence data are compared (Rohrmann, 1986). In this same study a 12
nucleotide consensus was highlighted at the 5' end of all the polyhedrin and granulin genes
which had been sequenced; it also features in the 5' region of the two pl0 genes sequenced
(AcMNPV, Kuzio et al., 1984; Ltibbert & Doerfler, 1984: OpMNPV, Leisy et al., 1986c). A
sequence very similar to the late consensus has also been found spanning the mRNA start site
used by the AcMNPV 39K gene at 18 h p.i. (Guarino & Summers, 1986). This is of particular
significance because transcription of the 39K gene early in infection is mediated by a different
site closer to the ATG codon. Therefore the very late consensus appears to be intimately
associated with the temporal regulation of both late and very late genes. Furthermore, a similar
settuence has been identified upstream of the arginine-rich (basic protein) gene of AcMNPV
(Wilson et al., 1987). Although the sequences found in the 39K and basic protein genes do not
agree precisely with the consensus proposed by Rohrmann (1986) it is noticeable that the central
ATAAG motif is preserved in both of these examples. When the 5' ends of late and very late
mRNAs have been accurately mapped using primer extension analysis or sizing of S 1-protected
probes against DNA sequencing ladders the conclusions have invariably been that the
transcripts initiate within this motif (AcMNPV polyhedrin, Howard et al., 1986; OpMNPV
polyhedrin, Leisy et al., 1986a; AcMNPV basic protein gene, Wilson et al., 1987; OpMNPV
pl0, Leisy et al., 1986c; PfMNPV polyhedrin, Fig. 2). Wilson et al. (1987) proposed that the
differences in the sequences flanking the ATAAG motif might account for the differential
temporal regulation of the basic protein and polyhedrin genes. However, in the PfMNPV
polyhedrin gene described here the first A of this pentanucleotide region is replaced with a G.
This was confirmed by sequencing both strands of DNA. The G nucleotide also occurs in a
similar position in the M. brassicae MNPV polyhedrin gene promoter (Cameron & Possee,
1989). This baculovirus is very similar to PfMNPV, sharing 70~o homology at the DNA level
when compared using dot blot hybridization analysis in 50~o formamide (Possee & Kelly, 1988).
The difference in the consensus did not significantly alter the position of transcription initiation
from the polyhedrin gene of PfMNPV; the mRNA was found to initiate only one nucleotide
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further upstream from the site utilized in AcMNPV polyhedrin (Howard et al., 1986). This
suggests that the central T A A G sequence of the consensus may be the most important feature
for transcription initiation of late and very late baculovirus genes. It will be interesting to
determine whether the other late and very late genes of PfMNPV have the same consensus
transcription initiation sequence and to investigate the effect of altering single nucleotides
within the central pentanucleotide.
The photographic skills of C. D. Hatton are gratefully acknowledged.
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