Download Sequence, Transcription and Translation of a Late Gene of the

J.
gen. Virol. (1989), 70, 2449-2459. Printedin Great Britain
2449
Key words: AcMNPV/transcriptionalmapping/codon usage bias
Sequence, Transcription and Translation of a Late Gene of the Autographa
californica Nuclear Polyhedrosis Virus Encoding a 34.8K Polypeptide
By J I A N G U O
W U AND L O I S K. M I L L E R *
Departments of Entomology and Genetics,
The University of Georgia, Athens, Georgia 30602, U.S.A.
(Accepted 4 May 1989)
SUMMARY
A 1"4 kb region downstream of the D N A polymerase gene of Autographa californica
nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were
identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as
determined by in vitro translation of both hybrid-selected R N A and R N A synthesized
in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K
polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation
sites, and potential sites for phosphorylation by casein kinase I and protein kinase C.
The p34.8 gene has a strong codon usage bias which is strikingly different from that of
the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an
A T A A G sequence and a G T A A G sequence 11 and 87 bases upstream of the A T G
codon respectively. A short upstream reading frame is present in the leader sequence of
the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint
correlates with a polyadenylation signal overlapping the translational termination
codon of the 927 base ORF. Transcripts of the latter were not observed early in the
infection cycle but appeared 6 h after infection and were maximally expressed at 12 to
24 h post-infection. The late nature of these transcripts was confirmed by their
sensitivity to aphidicolin and cycloheximide, inhibitors of D N A replication and
protein synthesis respectively. Attempts to construct viral mutants carrying a deletion
of the p34.8 gene and fusion with the fl-galactosidase gene suggest that the former gene
is essential for viral replication.
INTRODUCTION
Autographa californica nuclear polyhedrosis virus (AcMNPV) serves as a model system for
molecular biological studies of baculoviruses (Doerfler & B6hm, 1986) which have been used as
both insect pest control agents (Granados & Federici, 1986) and vectors for the high level
expression of foreign genes (reviewed in Miller, 1988). Approximately 20 of the genes encoded
by the 128 kb circular dsDNA genome of AcMNPV have been sequenced and transcriptionally
characterized to date. The genes can be divided into three basic transcriptional classes (Friesen
& Miller, 1986): early genes which are expressed before viral D N A replication, late genes which
are dependent on D N A replication and are maximally expressed between 12 and 30 h postinfection (p.i.) and very late genes which are maximally expressed after 30 h p.i. Members of all
three classes of genes appear to be dispersed throughout the AcMNPV genome without any
obvious organizational basis. Transcripts of late nucleocapsid genes (Wilson et al., 1987; Thiem
& Miller, 1989) and very late (Rohrmann, 1986) genes are initiated within a (G/A)TAAG
sequence which also serves as the major determinant in high level expression of the polyhedrin
gene (Rankin et al., 1988). A novel virus-induced ct-amanitin-resistant R N A polymerase activity
appears to be responsible for late and very late gene expression (Fuchs et al., 1983 ; Grula et al.,
1981).
To gain further insight into baculovirus gene organization, we have defined a region
downstream of the D N A polymerase gene (dnapol) of AcMNPV. We have found a late gene
0000-8847 © 1989 SGM
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2450
J. WU AND L. K. MILLER
encoding a 34.8K protein (p34.8) that appears to be essential for viral replication in cell culture
and have determined the map location and regulation of transcripts of this gene. The first of two
initiation signals for late p34.8 transcription is located 50 nucleotides downstream of the
transcriptional polyadenylation signal ofdnapoltranscripts. A short open reading frame (ORF),
18 codons long, lies within the leader sequence of the larger, 927 base ORF transcripts.
METHODS
Cells and viruses. AcMNPV L1 (Lee & Miller, 1978; Miller et al., 1983) was propagated in the Spodoptera
frugiperda IPLB-SF-21 (SF) cell line (Vaughn et al., 1977). The cells were grown at 27 °C in supplemented TC100
medium as described previously (Lee & Miller, 1978; Miller et al., 1986). For RNA and protein studies the cells
were inoculated at a multiplicity of 20 p.f.u, per cell. Viruses were allowed to adsorb to the cells for 1 h at room
temperature. Inocula were replaced with fresh medium and the infected cells were incubated at 27 °C. Time zero
p.i. corresponds to the start of incubation at 27 °C. In experiments requiring the inhibition of protein synthesis,
cycloheximide (final concentration of 100 gg/ml) was included in the medium 1 h before inoculation, in the viral
inocula, and also in the subsequent incubation medium. In experiments requiring inhibition of DNA synthesis,
aphidicolin (final concentration of 5 gg/ml) was included in the medium which was added following the viral
adsorption period.
Nudeotide sequencing. The PstI H fragment of AcMNPV DNA from 37-8 to 42.0 map units (m.u.) was cloned
into a Bluescript phagemid vector [pBSKS(- ), Stratagene] using standard procedures (Maniatis et al., 1982). The
restriction fragment resulting from cleavage at the PstI site at 37.8 m.u. and at the NruI site at 40.9 m.u. was cloned
into the same vector in the opposite orientation. A series of unidirectional deletions through the inserts of both
plasmids were constructed using the exonuclease IIl/mung bean nuclease deletion method described by Stratagene
and based on the original method of Henikoff (1984). Selected inserts in the recombinant phage ssDNAs were
sequenced by the dideoxyribonucleoside chain termination method (Sanger et al., 1977) using the T3 primer
(Stratagene), [~-35S]dATP (500 Ci/mmol; New England Nuclear, NEN) and a sequencing kit (Sequenase).
Sequences were compiled and analysed using programs of Pustell & Kafatos (1984; International
Biotechnologies).
R N A preparation and nuelease protection assay. Total cell RNA was isolated by guanidinium isothiocyanate and
CsCI cushion methods (Chirgwin et al., 1979; Davis et al., 1986) from mock- or AcMNPV-infected monolayers
(10 T cells per 100 mm plate) at designated times p.i.
The 5' and 3' ends of the gene were mapped by a nuclease protection assay using a formamide-based
hybridization buffer and S1 nuclease digestion of non-hybrid ssDNA (Friesen & Miller, 1985). The 5' end probe
was made by digestion of plasmid pBSMVI/BS with NotI, dephosphorylation with calf intestinal phosphatase
(Boehringer-Mannheim) and radiolabelling with T4 polynucleotide kinase (Bethesda Research Laboratories,
BRL) and [y-3zp]ATP (3000 Ci/mmol, NEN) at the 5' end. The DNA was further cleaved at the HindlII site in the
multicloning site of the vector. The 1556 bp fragment was electrophoretically isolated and used as a probe. The 3'
end probe was generated by digestion of the same plasmid with JfbaI and radiolabelling the 3' end with T4 DNA
polymerase (BRL), [g-32p]dCTP (3000 Ci/mmol; NEN) and unlabelled dGTP, dTTP and dATP. The labelled
DNA was further cleaved at the Sphl site in the insert. This 674 bp fragment was electrophoretically isolated and
used as a 3"-terminal probe. D N A - R N A hybridization mixtures contained approx. 100 to 500 ng of labelled DNA
and about 200 gg of total cell RNA. Protected fragments were denatured and electrophoresed on 7 ~
polyacrylamide-8 M-urea-Tris-boric acid-EDTA wedge sequencing gets. MspI-digested pUC19 DNA was
radiolabelled and used as size markers.
In vitro translation. For isolation of RNA by hybrid selection, 30 gg of a single-stranded plasmid DNA (pBSPAF-NX) containing antisense viral DNA between the XbaI and NotI sites (39.2 and 39.55 m.u. respectively) was
heated at 100 °C for 2 rain, snap-cooled and absorbed onto a 1 cm 2 section of a nitrocellulose filter (Schleicher &
Schuell). The filter was washed with 6 x SSC and then baked for 2 h at 80 °C in a vacuum (Esche & Siegmann,
1982; Friesen & Miller, 1987). Hybridization was conducted for 12 h at 42 °C in 50~ formamide, 10 raM-PIPES
pH 6.4, 0-4 M-NaCI, 1 mM-EDTA with 3 mg of total RNA isolated from cells at 12 h after infection. The filters with
bound RNA were washed 20 times with 1 x SSC, 0.1 ~ SDS, 2 mM-EDTA at 58 °C. The RNA was released from
the filters by boiling for 2 min in 1 mM-EDTA and 10 lag of calf liver tRNA (Boehringer-Mannheim).
RNA for translation was also prepared by in vitro transcription of a fragment of AcMNPV DNA from the PstI
site at 37.8 m.u. to the SnaBI site at 40.0 m.u. ( - 171 from the ATG of the 927 base ORF) that was subcloned into
pBSKS(-). The plasmid template was linearized with BglII which cleaves within the vector 'downstream' of the
insert. The solution of linear DNA was treated with 200 lag/ml proteinase K (Boehringer-Mannheim) extracted
twice with phenol-chloroform (1 : 1). The DNA was precipitated with ethanol before the transcription reaction.
Transcription was carried out in a 25 p.1 reaction volume consisting of 5 gl of x 5 transcription buffer (200 mMTris-HCl pH 8.0, 40 m~l-MgC12, 250 mM-NaC1, l0 mM-spermidine), 1 lag of the linear DNA template, 10 mM each
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The p34.8 gene o f A c M N P V
2451
of rATP, rCTP, rGTP and rUTP, 1 ~tl of 0-75 M-dithiothreitol, 25 units RNasin (Promega Biotec) and 10 units T7
RNA polymerase (BRL). The reaction was incubated at 37 °C for 30 rain, diluted 10-fold with 225 p.1 of 40 mMTris-HCl pH 7.5, 6 mM-MgCI2 and 10 mM-NaC1, treated with 1 unit RNase-free DNase I (BRL) and extracted
with phenol-chloroform (1:1). The RNA was purified in a Sephadex G50-80 spun column and then precipitated
with ethanol (Stratagene method).
In vitro translation of hybrid-selected or in vitro synthesized RNA was carried in a reaction containing 35 ~tl
nuclease-treated rabbit reticulocyte lysate (Promega Biotec), 1 rtl of an amino acid mixture (containing 1 mM of
each amino acid except methionine; Promega Biotec), 6 txl RNA template in H20 (approx. 1 p.g), 5.5 ~tl [35S]methionine (1129 Ci/mmol; NEN) at 10 mCi/ml, 2.5 ~tl RNasin (23000 units/ml; Promega Biotec). The reaction
was incubated at 30 °C for 60 rain. Samples were frozen in liquid nitrogen and stored at - 7 0 °C until use.
Viral proteins were also labelled in vivo by pulse-labelling with [35S]methionine. Cells (106 per 35 mm plate) were
infected with 20 p.f.u, per cell. At designated times p.i., the medium was replaced with methionine-deficient
medium lacking supplements and ceils were incubated for 1.5 h. This medium was then replaced with 0.5 ml of
methionine-deficient medium containing 50 ~tCi [35S]methionine (see above) and the ceils were incubated for 1 h
to radiolabel the proteins. The cells were then collected, washed with phosphate-buffered saline (Lee & Miller,
1978), and lysed in 1~o Nonidet P-40, 50 mM-Tris-HCl pH 8-0 and 150 mM-NaC1. In vitro translation products and
intracellular proteins were subjected to SDS-PA G E (Laemmli, 1970) followed by autoradiography using a Kodak
XAR5 film.
fl-Galactosidase gene insertion into the 927 base ORF. The Escherichia coil fl-galactosidase gene (lacZ) was
obtained from plasmid pSKSI06 (Casadaban, 1983) by digestion with Sail, BamHI and then PstI. The cohesive
ends were removed with mung bean nuclease. The largest fragment, containing lacZ, was purified by agarose gel
electrophoresis and inserted between blunt-ended SstI and Xbal restriction sites in a Bluescript minus-sense
plasmid containing AcMNPV DNA from the PstI site at 37-8 m.u. to the Nrul site at 40.9 m.u. to construct the
transplacement plasmid pBSKS106. Ten ~tg of pBSKS106 DNA and 1 ~tg of AcMNPV L1 DNA were
cotransfected into SF cells (Miller et al., 1986). The recombinant viruses were selected as blue plaques on SF
monolayers in the presence of X-gal (Pennock et al., 1984). Purified DNAs from the putative recombinant viruses
were compared with that of wild-type virus by restriction endonuclease digestion (Pstl and SstI) and Southern blot
analysis.
RESULTS
ORFs downstream of the AcMNP V DNA polymerase gene
Both strands of A c M N P V D N A extending from the SmaI site at 40.3 m.u. (see Fig. 1, centre)
to a SphI site (38.6 m.u. approximately 1.4 kb downstream of the carboxy terminus of dnapol
were sequenced using the strategy shown in Fig. 1 (top section). A major ORF, 927 bases in
length, was observed on the same strand downstream from but in a different reading frame than
that of dnapol (see Fig. 1, bottom). A second smaller ORF, 474 bases in length, was observed on
the opposite strand, downstream of the 927 base ORF (Fig. 1, bottom section).
The D N A sequence of this region and the predicted amino acid sequence of the 34.8K
polypeptide product (p34.8) of the 927 base O RF are both presented in Fig. 2. The 12 N-terminal
amino acids of p34.8 are hydrophobic and two potential glycosylation sites (Asn-X-Ser) are
present at amino acid residues 195 and 295. There is a potential protein kinase C
phosphorylation site (Arg-Lys-Ser) at amino acid 292 and a potential casein kinase I
phosphorylation site (Glu-Glu-Ser) at amino acid 285. There are three potential tyrosine kinase
sites (Glu-Xz-Tyr) but as the X residues are not acidic, it is unlikely that these sites are utilized.
The polypeptide p34.8 is rich in basic and aromatic amino acids. The D N A sequence and the
two polypeptide sequences were screened for nucleotide or amino acid sequence homology
against GenBank release 55.0 using the IBI/Pustell 'Cyborg' software which employs a
modification of the FASTP algorithm of Lipman & Pearson (1985). N o significant homology
was observed for either ORF.
Mapping of 927 base ORF transcripts
The 3' and 5' ends of transcripts of the 927 base ORF were mapped using two probes derived
from plasmid pBSMVI/BS which is schematically presented in Fig. 3 (a). A 674 bp probe (probe
A) was labelled at the 3' end at the XbaI site at 39.2 m.u. R N A isolated from infected cells at 6, 12
and 24 h p.i. in the absence of inhibitors protected D N A fragments of approximately 187, 337
and 674 bases (Fig. 3 b). These fragments were not protected by R N A from mock-infected cells
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J. WU AND L. K. MILLER
2452
D
4
4
BclI XbaI
Sphl
~;1(386)
II If llIII
II|I
II Ill
II
I
IIIllgIlll~
474 base ORF II
I i gill
II II I
II
i 1[
SnaBI
NotI SstI
11(39-6
I (39-2)
I
PvulI
I
I l
I II
I fl gill ii
II1111
II II
1
0
I
[ II g i l
II
llI
II II illll I!
Ill 11 II ill I I
1 lift III II li II
1
I
927 base ORF
!
III
g
Sinai
t(403t:
II I 1
II 12
II 1 3
--dnapol--
/
III II I II
[ [ /llllllI I
II lilt I
n
1'
2'
3'
Fig. 1. Key restriction sites, sequencing strategy and distribution of ORFs for the AcMNPV region
between 38-6 and 40-3 m.u. In the centre, a restriction map of a 2106 bp region from 38.3 to 40.3 m.u. on
the AcMNPV physical map is schematically presented. Numbers in parentheses below selected
restriction endonucleases indicate the m.u. scale. At the top, the strategy employed for sequencing both
strands of DNA is presented. At the bottom of the figure, the distribution of ORFs on the top strand
(frames 1, 2 and 3) and on the bottom strand (1', 2' and 3') are presented. The dnapol ORF and an ORF
of 927 bases are read from right to left (counterclockwise with respect to the circular AcMNPV map)
whereas a 474 base ORF is read from left to right.
nor by R N A isolated 2 or 4 h p.i. (Fig. 3b). Significant levels of protection of the 674 bp probe
were observed with RNA from 6, 12 and 24 h p.i. but not with R N A from mock-infected cells or
infected cells at other times p.i. indicating that the appearance of this fragment is due to R N A
protection (i.e. some transcripts cross the entire length of this 674 bp fragment).
Aphidicolin, an inhibitor of both cellular and viral D N A polymerases, inhibited the synthesis
of transcripts through this region indicating that the transcripts are 'late' in the sense that they
depend on D N A replication for expression. Cycloheximide, an inhibitor of protein synthesis,
also inhibited synthesis of these RNAs. Thus protein synthesis during virus infection is required
for transcription; this suggests that one or more viral proteins are required for the activation of
the 927 base ORF transcription. The position of the ends of the protected 187 and 337 base
fragments are shown in Fig. 2 and are located just downstream of polyadenylation signals
( A z U A 3 ) , strongly suggesting that these ends represent polyadenylation sites.
Fig. 2. Nucleotide sequence of the region encompassing the 927 base ORF and the amino acid
sequence of its predicted polypeptide product. The nucleotide sequence of the region encompassing the
SnaBI to SphI region (Fig. l) is presented; the positions of the SnaBI and SphI sites are underlined and
identified. The NotI site (GCGGCCGC) between +217 and +224 and the XbaI site (CTCTAG) at
39.2 m.u. are underlined once and twice respectively. The end of the dnapol gene is indicated under the
top line of sequence (dnapol/) where the slash mark notes the TAA termination codon of the dnapol
ORF. The (G/A)TAAG boxes at the 5' ends of transcripts through the 927 ORF are indicated by
addition signs ( + ) under the boxes. Polyadenylation signals (A2TA3) near the 3' ends of transcripts are
indicated by carets (A) below the nucleotides. The exclamation marks below two nucleotides indicate
the approximate position of the polyadenylation or 3' ends of the RNAs as mapped by nuclease
protection experiments. Two potential sites for N-glycosylation (Asn-X-Ser) are noted in bold print.
Amino acids located at a potential casein kinase I site and a protein kinase C phosphorylation site are
singly and doubly underlined respectively. The nucleotides are numbered (along the right side)
according to + 1, + 2 and -I-3 in reference to the ATG of the 927 base ORF. Amino acids of the 927 base
ORF product are numbered in parentheses below the nucleotide numbers. The asterisks are above
every tenth nucleotide.
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2453
The p34.8 gene of AcMNPV
AAA TGA
CAC T C A
ATG
TGC
TAA
CAA
TAC
GTA TAA
AAT
GAA
CTG TAA
+ +++
GGT
+
GTT
TCC
GAT
TAG
TGT
-.19
GCG
Ala
ATT CAC
Ile His
GCG
Ala
+42
(14)
ATT
TTG
TTT
GTA TAA
TTA
SnaBl
*
TTT
ATA
ATT
ATA
TAC
GAT
ACT
ATG
TTT
TAT
TGT
ATG
ATG TTT
CAA
CAT
AAC
GTT
TTT
GAC
CAA GAT TAC
GAC
AGC
GGT TAT TAT
AAG
AGT
++
TTG
ATA ATC
ATA
ATG
Met
ATT GCA
Ile Ala
TTA TTA
Leu L e u
ATT
Ile
CGC
CAC
GGC
TAT
TCC
CCG ACG
Pro T h r
GCC
Ala
GAT
ATT
TCT
ATA
+++
CCG
Pro
GCA GTG
Ala Val
A r g S e r H i s G l y T y r L e u Ser V a l
AAA
GAT
AAC
GGA
TCT
TTT
TAT
TGG
CCC
TTG
GAC
AAT
GTG
GGC
GCA TTG
Ala Leu
AAC
L y s ASp G l y Asn Phe T y r T r p P r o Asp A s n G l y Asp Asn I l e
AAT
ASh
GCT
Ala
GCT
TCC
Ala Ser
TAC
AAA
TCA
GTC
TAT
TAT
AAA
TAT
GCT
CTC
ACA
GCG
CAA TAC
ATG
TTT
CAA
AAT TAT GAC GAT TTT GAC CTA ATC
A s h T y r Asp Asp Phe Asp L e u I l e
AAA
GGT
CAG TAT
TTT
AAC
GAT
CGC
AAT
TCT
GTG
CAA
ATG
GAA
AGG
GTT
GTG
Lys Gin Arg Val Val
GGT
TGG
AGG
CCA
AAT
ACG
CTA
TAT
TTA
AAT
TAC
GCG
GAT
AAA
AGC
CGC
TAT
CAA
--79
AAA
Lys
TGT TTT
Cys P h e
+102
(34)
GCC
GCG
TGC
+162
(54)
CCC G A C
TTA
--139
CAA TAT
Gln Tyr
CGC
P r o Asp A ! a A l a Cys A r g
T y r Met G l u T y r A l a
G l y S e r Asn Asp A r g Asn S e r V a l Phe G l y Asp L y s S e t
AAC
Ash
GAT
CGT
Arg
ATT
GAA
TCC
GGG
GCG
GCC
T y r L y s S e r V a l T y r T y r L y s T y r A r g A l a L e u Asp L e u G l u S e t G l y A l a A l a
T h r A l a G l n T y r Met P h e G i n G i n
TCA
CGA
TTT GCT
Phe Ala
AAA ATA
dnapol/
A l a Val Ala
GGT
GIy
CCT
Pro
.
+282
(94)
GCT
GTG
GCC
+222
(74)
CCG
Pro
CAC A C A
His Thr
CTT
Leu
TGC
Cys
GGT
Gly
GCC
Ala
-~342
(114)
GGA
ATG
GAA
CCT
TTT
AAC
+402
(134)
GAC
G l y Met Asp G l u P r o Phe A s n
,
CCG
GTT
TAT
CAG
ATG
T r p A r g P r o Asn T h r L e u T y r L e u A s n A r g T y r G l n F r o V a ! T y r G l n M e t
.
AAT GTT
ASh Val
+462
(154)
CAT TTT
H i s Phe
TGT
Cys
CCA
Pro
ACA
Thr
GCC
Ala
A T A CAC G A G
Ile H i s G l u
CCC
Pro
AGT TAT
Set T y r
TTT
Phe
GAA
Glu
GTG
Val
TTT
Phe
ATT
Ile
ACT
Thr
AAA TCA
Lys S e r
,
~522
(174)
AAC
Asn
GAT
CGT
CGC
AAT
CCA
ATA
ACT
TGG
AAC
TTA
T r p A s p A r g A r g ASh P r o
Iie
T h r T r p A s n GI,] L e u
G A A TAC
glu Tyr
ATA
ile
GGT
Gly
GGT AAC GAT
GIy Asn Asp
.
+582
(194)
GAT
Asp
TAT TCT ATA
T y r Ser Ile
~642
{214)
TGG
TCA AAT
Set Asn
TCA
Set
GAA
TTA TGT GAC AAC
L e u Cys Asp A s n
AGT
Ser
CTA GTC
Leu Val
ATG
Met
TAT
Tyr
GTG
Val
CGT
Arg
TGG
Trp
CAG
Gln
CGC
Arg
ATA
ile
+702
(234)
CTG
GTG
TTT
Phe
GAA
Glu
ACT
Thr
CTA GAC
Leu A~p
GAC
Asp
,
+762
(254)
TTA
Leu
ATT
Ile
CCA AAT
Pro A s h
CCG
Pro
GGC
GI¥
CCC GTA GTA
Pro Val Val
ATA
Ile
CCG
Pro
TAC
Tyr
AGG
Arg
TCA AAT
Set A s h
CAA TTT GTA
G l n Phe V a l
GAT
GGT
GAA
GGA TTT
TAT
TGC
CCC
GTG
AAT
GCC
GAT
Asp P r o V a l G l y G l u G l y P h e T y r Asn Cys A l a Asp L e u V a l
GAA TGC
AGA
TAC
GCT
CAA
ATG
GCT
AAA
GTG
GTC
G l u Cys A r g T y r A l a G l n Met A i a L y s V a l V a l
GAT
GCA
CGC
ATT
GAT
CAT
AAT
GAC
GAA
GAA
TCT
AGA
Arg
AGC
Ser
CAA TTA CAA
Gln Leu Gln
AAG
Lys
CAT
His
CTT
Leu
*
+822
(274)
TGT
TGG
CGT
AAA
TCA AAT TAT
Ser Ash Tyr
+882
(294)
AAT TAC
ATA TTA
TAT
!
~942
ATT TCA
AAC
ATG TCT
TCA
+1002
GCA
CGC
Asp A l a A r g I l e
Asp H i s Asn Asp G l u G l u Ser Cys T r p A r g A i a A r g L y s
TCG TCA TTT
Set Ser Phe
TTT
Phe
AAT
Asn
TTA
GCA
AGT AAT AAC
AAT
TCG TAG
TCT
GCT
CTG
TCA
TAA
TTA
TCA
TCG
TCA AAA
AGA
CCA TTT TTG
CAG
CCA
Pro
GGA
TTT
G l y Phe
TAA
TAA
AAC
AAA
CAA
AAT TTT
CTA
CAA TCA
ATA
CGA
ACA
AAA TAT
AAA
Lys
......
AG~
TCT
ATG
CTA
AAA
TTG
TTA
ATT
AAA
TCT
TCC
ATA
TTT
TCA
CAC
+1062
TGT AAT
AAA
AAT
AGT
TTA
AAG
CCA TCT
TGA
TCT
CGT
TTG
GAT
ATT
TCG
+1122
AGA TAT
TGC
AAA
GCA AAC
TGT
ACT
TCT
TTG
GCG TAA
GGA TTT
AAC
AGT
+1182
ACT
CTG
TCT
ACG
AGC
GTA TTG
AGA
CTT TCC
GCA
GTT
AAA GTG TTG
+1242
TCA
CAA AAC
CGC
TTG ATT
TCA
TCG
GCT
ACT
TGA
+1302
TAT
TTT
GCT TCT
CCG
TGC
AGC
CCA
+1362
ATG TTG
CTC
TAA AAT
TAA
TAA
AAC
ACT
+1422
ACG
CAA CTG
ATT
GCC
GGT
TTC
AGA
CGA
GTT
TTT TCA
AAA TCC
ATT
TGA
GAC
ATT
AAA TAT TTG
TTC
AAG
TGA
AGA TAG
ATC
ATA
AAT
TTA
GAT TGC
ATG
CAA TCA
AAT
G
TCG
TAC
TAG
SphI
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2454
J. WU AND L. K. MILLER
(a)
ATAAG-"-Iv U G T A A G
3'(~
v
-4--I5' RNA
~-304"~
674
~]
~-337"-t
927 base ORF
)
PstI
I
Hss,i
(40.9)/N
(39.6)
XbaI (39.2) NotI
SphI
(37-8)
]
HindlII
674 bases
1556 bases
[
Probe A
SphI
(3')
XbaI
Probe B
(5')
HindlII
NotI
(b)
(c)
M
0
2
4
6 6A 12 A C
24
M
2
6
12 A
C 24 48
P
1556~
,674
501N_
489--404 m
"337
331~
3091,
242
2331"
190~
4187
147~
Fig. 3. Nuclease protection mapping of transcripts of the 927 base ORF. (a) A schematic diagram of
the plasmid pBSMVI/BS from which DNA probes were prepared in order to map transcripts of the 927
base ORF. Vector sequences are denoted by shaded blocks ([]) labelled 'BS-'. AcMNPV sequences
between the PstI site at 37.8 m.u. and the NruI site at 40.9 m.u. are represented by the open blocks and
the position of the 927 base ORF is represented by the open arrow immediately above the region. Key
restriction sites and m.u. are noted immediately below the region. The NruI site was removed by fusing
this site and the EcoRV site of the vector; both sites are noted by a shaded block (~1). Below the
restriction sites are representations of the radiolabelled DNA probes (A and B) used for nuclease
protection studies. Probe A was 674 bases in length and was labelled exclusively at the 3' end of the Xba I
site as noted by the asterisk. Probe B was 1556 bases long and labelled exclusively at the 5' end of the
NotI site as indicated by the asterisk. A summary of the 5' and 3' ends determined from nuclease
protection experiments are shown at the very top of the figure. The thin open arrow shows the position
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)
The p34.8 gene of A c M N P V
2455
The 5' ends of the 927 base O R F transcripts were analysed using a 1556 bp probe which was 5'
end-labelled at the NotI site within the O R F (Fig. 3, probe B). R N A from 6, 12, 24 and 48 h p.i.
protected two fragments of approximately 233 and 309 nucleotides in size (Fig. 3b). The R N A
from mock-infected cells or infected cells at 2 h p.i. did not protect the probe (Fig. 3 b). Similarly
R N A from infected cells that were inhibited by aphidicolin did not protect the probe, indicating
that these are late transcripts. Cycloheximide also inhibited the synthesis of these transcripts.
The end of the protected 233 nucleotide fragment maps to an A T A A G sequence at - 11 relative
to the A T G of p34.8 and the protected 309 nucleotide fragment maps to a G T A A G sequence at
- 8 7 (Fig. 2).
Translation of the 927 base ORF transcripts
To determine the nature of the product of the transcripts through this ORF, R N A
homologous to the antisense strand of the O R F was hybrid-selected using a plasmid containing
the region spanning the XbaI to NotI sites (see Fig. 3). The selected R N A was translated in vitro
using a rabbit reticulocyte in vitro translation system including radiolabelled methionine. A
polypeptide product of 34K to 35K was observed by autoradiography (34.8K, Fig. 4, lane H);
controls with no added exogenous R N A or vector DNA-selected R N A showed that this 34.8K
protein was a specific product of the 927 base O R F selected R N A (control data not presented).
The same size product was observed when R N A synthesized in vitro from a template containing
the entire 927 O R F beginning at - 171 from the A T G (Fig. 4, lane S). The 34-8K polypeptide
migrated in SDS-polyacrylamide gels just above the polyhedrin protein (Fig. 4, 30 h lane).
Attempts to construct p34.8-~-galactosidase fusions
It has been possible to isolate mutant viruses containing deletions in genes which are nonessential for virus replication in cell culture by fusing the N-terminal portion of the O R F to the
E. coli /~-galactosidase gene and selecting recombinant virus plaques using a chromogenic
indicator of/~-galactosidase (Pennock et al., 1984; Crawford & Miller, 1988; Vlak et al., 1988).
To determine whether p34.8 is non-essential in cell culture, a transplacement plasmid was
constructed based on pBSMHVI. The transplacement plasmid contained the E. coli ~galactosidase gene fused in frame with the p34.8 gene at the SstI site within the 927 base O R F
(39.6 m.u. ; Fig. 3). The ~-galactosidase gene replaced 927 base O R F sequences from the SstI site
down to the XbaI site at 39.2 m.u. and was flanked by approximately 1-5 kb of viral sequences on
both sides to permit allelic replacement. Cotransfection of the plasmid with wild-type viral
D N A resulted in virus stocks which contained a low proportion of blue plaques. Eight
representative blue plaques were picked and plaque-purified at least four times. Stable stocks of
pure blue plaque viruses could not be isolated; instead, clear (non-blue) plaques continued to
arise at a high frequency. Restriction endonuclease characterization of the viral D N A from
stocks of four of these isolates suggested that the plasmid had recombined by a single rather than
and direction of transcripts. The closed arrowheads show the positions of the two 5' ends whereas the
open arrowheads show the positions of the mapped 3' ends or polyadenylation sites. The dashed lines
and arrowhead indicate that some of these transcripts extend through the SphI site used in the probe
construction. The numbers between the bracket below the transcripts indicate the lengths (in bases) of
the protected fragments shown in (b) and (c). (b) An autoradiogram of DNA fragments protected from
S 1 nuclease digestion by RNA isolated from mock-infected cells (M) or from AcMNPV-infected cells at
time zero (0) and at 2, 4, 6, 12 and 24 h p.i. in the absence of inhibitors or in the presence of aphidicolin
at 6 and 12 h p.i. (6A and A respectively) or cycloheximide at 12 h p.i. (C). Mr markers are shown at the
far left. The sizes of the protected fragments are shown at the right of (b). (c) An autoradiogram of DNA
protected by RNA isolated from mock-infected cells (M) or AcMNPV-infected cells at 2, 6, 12, 24 and
48 h p.i. in the absence of inhibitors or at 12 h p.i. in the presence of aphidicolin (A) or cycloheximide
(C). Lane P contains the probe alone. Molecular size (bases) markers are on the far right. The positions
of the protected fragments of 233 and 309 bases and of the 1556 base probe are indicated by arrows at
the left of (c).
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2456
M
J. WU AND L. K. MILLER
3
6
12
30
S
H
--Endogenous Fig. 4. SDS-PAGE of products from in vitro translation of both hybrid-selected RNA and in vitro
synthesized RNA of the 927 base ORF, also of in vivo
labelled proteins from infected cells and their
analysis by SDS-PAGE. RNA selected by hybridization to antisense DNA of the 927 base ORF was
translated (H) and the products were compared by
p34.8
SDS-PAGE to those obtained from translation of
- - Polyhedrin RNA synthesized in vitro from the 927 ORF (S) by
SDS-PAGE. For additional comparison, proteins
were pulse-labelled in vivo at 3, 6, 12 and 30 h p.i. and
in mock-infected cells (M). The position of the major
background protein produced from RNA endogenous to the rabbit reticulocyte lysate is noted (endogenous) on the right. The solid arrowhead on the right
indicates the major 34.8K product of translation of
both the hybrid-selected RNA and in vitro synthesized RNA. The position of polyhedrin from
AcMNPV-infected cells (note 30 h p.i. lane) is also
indicated on the right.
a double crossover event (data not shown). Thus, wild-type viruses were generated upon passage
with the loss of the fl-galactosidase gene. These results strongly suggest, but do not prove because
of their negative nature, that the p34.8 gene is essential for virus replication.
DISCUSSION
We have found that a late gene, encoding a 34.8K polypeptide is located downstream of the
D N A polymerase gene of A c M N P V . Like dnapol, the p34-8 gene is transcribed in the
counterclockwise direction but the regulation of the two genes is distinctly different.
Transcription of dnapol is maximal at 6 h p.i. (Tomalski et aL, 1988) and occurs in the presence
of cycloheximide, indicating that it is an early gene. Normally, transcripts of dnapol are switched
off by 12 h p.i. but their presence is extended past 12 h p.i. in the presence of cycloheximide. In
contrast, the p34.8 gene transcripts are initiated around 6 h p.i., the beginning of viral D N A
replication, and are present maximally from 12 h to 48 h p.i. Transcription of the p34-8 gene does
not occur in the presence of cycloheximide suggesting that a viral gene product is required for
transcription. Transcription of the p34.8 gene also appears to be dependent on D N A replication
since transcription of the 927 base O R F is not observed in the presence of aphidicolin. These
properties place the p34.8 transcripts in the late class which persists through the very late
(occlusion) phase of viral infection. The organization of genes around p34.8 most closely
resembles the gene organization around the p 10 gene (Rankin et al., 1986; Kuzio et al., 1984). It
remains to be determined whether such gene organization has regulatory significance.
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The p34.8 gene of A c M N P V
2457
Similar to all other late and very late transcripts of AcMNPV that have been characterized to
date, p34.8 RNAs initiate within an (G/A)TAAG sequence which forms the nucleus of the
major determinant for high level expression from the polyhedrin promoter (Rankin et al., 1988).
The p34.8 RNAs initiate from two different (G/A)TAAG sequences, an ATAAG which is
located only 11 bases upstream of the ATG of p34.8 and a G T A A G which is 87 bases upstream.
Utilization of multiple (G/A)TAAG sites has been observed previously for transcripts of the
major capsid protein (Thiem & Miller, 1989). Three (G/A)TAAG sequences are utilized for
capsid gene transcriptional initiation; one is a GTAAG site located 330 bases upstream of the
ATG codon of the capsid ORF. Linker scan mutations of the polyhedrin promoter which
convert the ATAAG to GTAAG decrease expression fourfold (Rankin et al., 1988) suggesting
that the GTAAG start is less well utilized as an initiation site than ATAAG. Consistent with
this observation, the level of p34-8 transcripts initiating at the G T A A G site appears to be
approximately one-third of that of transcripts initiating at the A T A G G site (Fig. 3).
An interesting feature of the GTAAG-initiated p34.8 transcript is that it contains a short
ORF which would encode an 18 amino acid peptide terminating at a UAG encoded upstream of
the ATAAG initiation site. Whether this peptide is synthesized and functional or whether this
ORF represents some type of translational regulation remains to be determined. The codon
usage of this upstream ORF is similar to that of the 927 base ORF. The GTAAG-initiated
transcript of the major capsid gene also has an upstream ORF that would encode a very short
peptide (six amino acids). The dnapol gene has two ATGs in the leader sequence of its RNAs
(Tomalski et al., 1988) but unlike the late RNA leader ATGs of the capsid and p34.80RFs,
translation products beginning at these 'upstream' ATGs in the polymerase RNA would
terminate downstream of the ATG of the polymerase ORF. Such an arrangement could
dramatically decrease levels of DNA polymerase expression. In the case of the capsid and p34.8
ORFs, reinitiation at the ATG of the major ORF could occur.
The positions of two 3' ends or polyadenylation sites for p34-8 transcripts have been identified
and map just downstream of consensus polyadenylation signals (AEUA3). One of these signals
overlaps with the double (tandem) UAA termination codons of p34.8. Such a tight signal
organization appears to be a common feature in AcMNPV. The polyadenylation sites, however,
do not appear to be utilized efficiently as transcripts crossing both sites and terminating further
downstream appear to be abundant, particularly late in infection (Fig. 3a, 674 base probe
protection at 12 and 24 h p.i.). The lack of efficient polyadenylation of late transcripts at
consensus polyadenylation sites seems to be a common characteristic of late gene transcription;
it has been observed for pl0, polyhedrin and 6.9K core protein gene transcription (Friesen &
Miller, 1985; Wilson et al., 1987) as well as the series of late transcripts in the EcoRI H and S
region (Friesen & Miller, 1987; Oellig et al., 1987). Transcripts continuing through the
polyadenylation site within the 927 base ORF could regulate expression of the downstream 474
base ORF because they would be antisense.
It is possible that the p34.8 protein is modified post-translationally. It contains signals for both
N-glycosylation and phosphorylation. The highly hydrophobic N terminus might serve as a
signal sequence for membrane transport or anchoring; however, the region lacks basic residues
at the N terminus which are usually found in signals for endoplasmic reticulum transport.
Glycosylated proteins of 34K and 37K (Stiles & Wood, 1983) and a prominent phosphorylated
protein of 34K have been observed in AcMNPV-infected cells (Maruniak & Summers, 1981 ;
O'ReiUy & Miller, 1988). A 34K 'calyx' protein which is phosphorylated was recently identified
as a component of the outer envelope of occlusion bodies (Whitt & Manning, 1988) but the gene
encoding this calyx protein is reported to be at 83 m.u. (Gombart et al., 1989) and would be
expected to be non-essential. A 35K early protein was previously identified (Friesen & Miller,
1986). If p34.8 is modified by either glycosylation or phosphorylation, its migration with respect
to in vivo labelled proteins might be altered. Thus, there is some difficulty in ascribing a
particular infected cell-specific protein to p34.8. The nature and function of p34.8 will need to be
further pursued using specific antibodies raised to p34.8 epitopes. The failure of our attempts to
delete this gene strongly suggests that p34.8 plays an essential role in viral replication.
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2458
I. W U AND L. K. M I L L E R
A rather unusual feature of the p34.8 gene is the codon bias utilized. Usually, strong codon
usage bias is correlated with high level gene expression. The p34.8 gene has a strong codon usage
bias which differs from that of the abundantly expressed very late polyhedrin gene. For
example, all 16 phenylalanines of p34-8 are encoded by TTT codons whereas 13 of the 14
phenylalanines of polyhedrin are encoded by the other codon for phenylalanine, TTC. Strong
and opposite codon biases between polyhedrin and p34-8 are also observed for tyrosine, lysine
and isoleucine. Some codon bias is also found in other highly expressed baculovirus genes. The
pl0 gene tends to utilize codons in a fashion similar to the polyhedrin gene. The major capsid
gene, however, has a codon usage bias that shares similarities with those of both polyhedrin and
p34-8. With the exception of the valine codon, the p34.8 codon usage favours A and T residues at
the wobble position. Codon bias may reflect specific aspects of gene evolution to regulate
expression or the acquisition of these genes from different sources during baculovirus evolution.
The emerging picture for the gene organization of the AcMNPV genome is one in which early
and late genes are intermixed; transcriptional, post-transcriptional and translational regulatory
signals are brief and closely spaced. Most of the nucleotides of the AcMNPV DNA genome are
devoted to information for protein synthesis rather than serving as cis-acting regulatory signals.
Overlap of RNAs could, by itself, serve some regulatory function. The presence of short
upstream reading frames within the leaders of two GTAAG-initiated late RNAs may be of
significance in terms of late gene regulation and this needs further analysis.
We thank Suzanne M. Thiem (University of Georgia) for advice and help in performing the hybrid selection
and in vitro translation reactions. We also thank Dr David O'Reilly (University of Georgia) for advice and help in
homology searches in GenBank and for many helpful suggestions concerning the manuscript. This research was
supported in part by Public Health Service grant AI 23719 from the National Institute of Allergy and Infectious
Diseases.
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