1. health and fitness
2. disease

Journal of General Virology (1994), 75, 1703-1711. Printedin Great Britain
1703
BICP22 of bovine herpesvirus 1 is encoded by a spliced 1.7 kb RNA which
exhibits immediate early and late transcription kinetics
M a r t i n S c h w y z e r , * U r s V. W i r t h , t Bernd V o g t and Cornel Fraefel
Institute of Virology, Faculty of Veterinary Medicine, University of Ziirich, Winterthurerstrasse 266a,
CH-8057 Ziirich, Switzerland
Kinetic analysis of the two divergent immediate early
(IE) transcription units of bovine herpesvirus 1 (BHV-1)
revealed an unexpected behaviour. The IE1.7 promoter
was not turned off at the end of the IE period but acted
as a late promoter, unlike the adjacent IE4.2/2.9
promoter which was active only under IE conditions.
The genome region specifying the IE1.7 gene was
sequenced (0-814 to 0-839 map units). The IE1.7
promoter was found to overlap with duplicated sequence
elements bearing close similarity to herpesvirus origins
of replication, which may explain the biphasic transcription kinetics. Exons 1 and 2 of the spliced IE1.7
transcript were non-coding. Exon 3 was found to contain
a single open reading frame encoding a protein of 300
amino acids that was designated BICP22 because of its
homology to ICP22 (Vmw68) of herpes simplex virus
type 1 and related proteins from other herpesviruses.
The protein probably represents IEP-55, the most
abundant BHV-1 phosphoprotein observed under IE
conditions.
Introduction
non-coding 0.35 kb leader sequence (exon 1). Exon 2 of
IER4.2, located in the inverted repeats, encodes the
homologue of infected cell protein (ICP) 4, also known
as Vmw175, of herpes simplex virus type 1 (HSV-1). This
BHV-1 protein, named BICP4 or p180, has been shown
to be capable of trans-activation as well as autorepression
(Schwyzer et al., 1993). Exon 2 oflER2.9 is Y-coterminal
with an unspliced early RNA (ER2.6) located in the
right-hand end of U L. Both IER2.9 and ER2.6 encode
the homologue of ICP0 (Vmwll0) of HSV-1 (a zinc
finger trans-activator protein) designated BICP0 or p 135
(Wirth et al., 1992). The third transcript, IER1.5, was
discovered recently (Fraefel et al., 1993). It contains the
same leader RNA as transcription unit 1 but extends into
the left-hand end of U L because it arises from the
covalently joined genome ends (TRs-UL). This observation led to the name circ for the encoded protein.
The present report concerns transcription unit 2 which
runs towards U s but is located entirely within the
inverted repeats, and therefore occurs in two copies (Fig.
1). It specifies IER1.7, a spliced IE RNA of 1-6 to 1.8 kb
depending on the virus strain (Wirth et al., 1989, 1991).
Nucleotide sequence analysis showed that exons 1 and 2
of IER1.7 were non-coding and that exon 3 encoded an
IE protein designated BICP22 or p55, the homologue of
ICP22 (Vmw68) ofHSV-1 (McGeoch et al., 1985). Genes
encoding ICP22 homologues have been identified for
several other alphaherpesviruses, including equine
herpesvirus type 1 (EHV-1) (Holden et al., 1992; Telford
The genome of bovine herpesvirus 1 (BHV-1) is a linear
double-stranded DNA molecule (138 kb) which may be
subdivided into a unique long segment (U L, 106 kb) and
a short segment containing a unique region (Us, 10 kb)
flanked by internal and terminal inverted repeats OR s
and TRs, 11 kb each). The structure and function of
about 20 BHV-1 genes are known (Schwyzer, 1993;
Wyler et al., 1989). As in the case with other herpesviruses, the BHV-1 genes are expressed in a temporally
regulated cascade. Therefore, they can be assigned to the
kinetic classes immediate early (IE), early or late (Misra
et al., 1981; Wirth et al., 1989).
The IE genes of BHV-1 belong to two divergent
transcription units starting approximately in the centres
of the inverted repeats (Wirth et al., 1991). Transcription
unit 1 specifies three alternatively spliced IE RNAs
designated IER4.2, IER2.9 and IER1.5 (4"2 kb, 2.9 kb
and 1.5 kb, respectively) with a common promoter and
1" Presentaddress: Sonnsyterain8, CH-6048 Horw, Switzerland.
The nucleotidesequencedata for the BICP22geneof BHV-1 strain
Jura havebeen submittedto the EMBLData Libraryand assignedthe
accessionnumberX76943.
Dedicated to Professor Robert Wyleron the occasion of his 70th
birthday.
0001-2219 © 1994SGM
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1704
M. Schwyzer and others
et al., 1992), equine herpesvirus type 4 (EHV-4) (Cullinane et al., 1988), pseudorabies virus (PRV) (Zhang &
Leader, 1990), varicella-zoster virus (VZV) (Davison &
Scott, 1986) and Marek's disease virus (MDV)
(P. Brunovskis & L. F. Velicer, personal communication). The MDV ICP22 homologue gene has recently
been sequenced by another laboratory and termed US537
without noting its homology to the ICP22 gene family
(Sakaguchi et aL, 1993).
Despite this structural similarity, only the ICP22 genes
of HSV-1 and BHV-1 have been shown conclusively to
belong to the IE kinetic class. The corresponding genes
of PRV and MDV are regulated as late genes and EHV1 exhibits a late promoter as well as an alternative early
promoter located within the ICP22 coding sequence
(Holden et al., 1992). For BHV-1, we observed that the
BICP22 gene was expressed with biphasic kinetics, IE
and late, under the control of a single promoter. The
IEI. 7 promoter was found to overlap with duplicated
sequence elements bearing close similarity to herpesvirus
origins of replication (oris; oriL), which might facilitate
reactivation of IER 1.7 synthesis after the onset of DNA
replication.
Methods
Virus, cell culture and transcript mapping procedures. Virus strains
(K22 and Jura), infection of Madin-Darby bovine kidney (MDBK) cell
cultures, treatment with metabolic inhibitors, isolation of total RNA,
Northern blot analysis and primer extension analysis have been
described previously (Wirth et al., 1991).
Plasmids and sequence analysis. Plasmid pJuC, containing the BHV1 strain Jura HindlII C fragment [0-733 to 0.852 map units (m.u.)] and
the corresponding strain K22 HindlII C fragment, cloned in two parts
as p601 (0-733 to 0.816 m.u.) and p615 (0'816 to 0-852 m.u.), have been
described elsewhere (Wirth et al., 1991). Cleavage of pJuC with DraI
and HindlII provided two fragments of 3"0 kb (0.811 to 0"833 m.u.) and
1-7 kb (0"833 to 0"847 m.u.) which contained the region of interest and
these were cloned into pBluescript (pBS K S + ; Stratagene) to give
pBDJ3 and pBDJ17, respectively (Fig. 1 b). From these two plasmids,
additional overlapping subfragments were generated with the enzymes
NarI, PstI, Y(hoI, EcoRI, Sinai and DralI, and cloned into pBS KS +.
The nucleotide sequence was determined for both strands by the
dideoxynucleotide chain termination method (Sanger et al., 1977) using
double-stranded plasmids as templates for Sequenase (United States
Biochemical) with 7-deaza-dGTP or dlTP instead of dGTP. Synthetic
oligonucleotides based on already determined sequences were used as
primers where required. The sequences derived were analysed using the
University of Wisconsin Genetics Computer Group programs
(Devereux et al., 1984).
Results
Kinetic analysis of the two IE transcription units
As a guide to the experiments described below, Fig. 1
shows the map locations of the hybridization probe and
oligonucleotide primers used for kinetic analysis, the
sequenced region (c) and a summary of the two IE
transcription units (d). Previously, we have shown by
Northern blot analysis that IER4.2 and IER2.9, the
transcripts specified by IE transcription unit 1, are
present at 2 h post-infection (p.i.) but cannot be detected
later in infection unless cycloheximide is used in the IE
phase to block translation (Wirth et al., 1992). Here,
similar Northern blot analysis was used to examine IE
transcription unit 2 and to determine the kinetics of
IER1.7 synthesis (Fig. 2). Hybridization of RNA from
BHV-1 Jura-infected cells with the EcoRI-DraI probe
(Fig. 1c) confirmed that IER1.7 (1.6 kb for strain Jura as
shown here; 1"8 kb for strain K22) belonged to the IE
kinetic class, since it formed a strong band in the
presence of cycloheximide (Fig. 2, lane 2) and could also
be detected at 2 and 3 h p.i. in the absence of cycloheximide (lanes 4 and 5). Surprisingly, IER1.7 accumulated to higher levels at 5 and 8 h p.i. (lanes 6 and 7). The
amount of IER1.7 which was reached at 8 h p.i. was
strongly reduced by blocking entry into the late phase of
infection with the DNA synthesis inhibitor cytosine
arabinoside (lane 8). An additional 4 kb band observed
in all lanes, even with RNA isolated from uninfected cells
(a)
0-78 0-79 0.80 0.81"~0.82 0-83 0.84 0-85 m.u.
'
'
' i '
'
. . . . . .
(c)
"
L
EcoRI-DraI
03
- "~ .
I
04
. ~i
~NNNN. . . . . .
!J
p615
p601
pJuC
pBDJ3
pBDJ17
Northern
probe,
Primer
extension
i This repor 7t[
Sequence
• ' (Schwyzer etiaL, 1993) 1determined
J
( d ) : I E !ranscfiption unit 1 i E t . . . . . ripti . . . .
::::
:
IERI 7
it 2
::'i"~'~ ',' ]
Transcripts
....!!:::. . . . . .
:;
BICP4
:,~:~~(~,~
,, ::::
:
:: : : : :::C;:,::BICP22
: r~
:
:~''~
Fig. 1. Map of the BHV-1 IE region encoding BICP22. (a) The righthand part of the internal repeat ORs; 0.756 to 0.839 m.u.) is shown
together with a short segment of the adjacent U s region. Locations of
selected restriction sites and of consensus sequences for an origin of
viral DNA replication (oris) are indicated. (b) Plasmids used in this
study. (c) Locations of the Northern blot probe, primers and nucleotide
sequences reported in this work and of sequences published elsewhere
(Schwyzer et al., 1993). (d) Locations of the transcripts (Wirth et al.,
1991). The exons of the transcripts (IER4.2, IER2.9 and IER1.7) are
shown as boxes; non-coding parts are black, and parts encoding
proteins (BICP4, BICP22) are white.
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ICP22 homologue of BHV-1
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
1705
8
97 nt
28S
18S
o4
.~--1.7
56 n t
BHV-1
J
T i m e p.i. ( h ) 8
Inhibitor
Ratio
10
J
8
a
3
8
-
K
5
c
K
2
.
0.3
.
K
3
K
5
K
8
.
0-7
.
2.2
10
Fig. 3. Levels o f R N A t r a n s c r i p t s f r o m the t w o I E t r a n s c r i p t i o n u n i t s
as d e t e r m i n e d b y p r i m e r e x t e n s i o n analysis. M D B K cells w e r e m o c k BHV-1
-
J
-
J
J
J
J
J
6
c
0
.
2
3
.
5
8
8
a
-
Fig. 2. Northern blot analysis of BHV-1 RNA. MDBK cells were
mock-infected ( - ) or infected with BHV- 1 Jura (J) and treated with the
inhibitors cycloheximide (c; 100 lag/ml) or cytosine arabinoside (a;
100 lag/ml) or left untreated (-). Total RNA was isolated at the
indicated times p.i. (h), it was then separated by electrophoresis, blotted
on nylon membranes and hybridized with zzP-labelled probe (Fig. I c),
and transcripts were revealed by autoradiography. The scale on the left
indicates mobilities of RNA size markers (Boehringer Mannheim) and
of 28S and 18S rRNA.
infected ( - ) or BHV-1 Jura- (J) or K22-infected (K), treated with the
inhibitor cytosine arabinoside (a; 100 lag/ml) or cycloheximide (c;
100 lag/ml) or left untreated (-), and total RNA was isolated at the
indicated times p.i. (h). Oligonucleotides 03 and 04 (antisense to IE
transcription units 1 and 2, respectively; Fig. 1c) were 5' end-labelled,
mixed in equal amounts, and hybridized to these RNA samples.
Reverse transcriptase from avian myeloblastosis virus was used to
synthesize cDNA fragments, which were analysed on an 8%
polyacrylamide-urea sequencing gel. Sizes of produced cDNAs are
given in nucleotides (nt), and the ratio of the signal intensities
(transcription unit 2/transcription unit 1), as determined by scanning
the preflashed X-ray film, is indicated below the relevant lanes.
(lanes 1, 3 and 9), was attributed to non-specific
hybridization with bulk 28S r R N A as discussed previously (Wirth et al., 1989).
F o r a direct c o m p a r i s o n o f the two I E transcription
units by a different approach, we performed primer
extension analysis. Previously, oligonucleotides 03 and
04 had been used separately as primers to m a p the 5'
termini o f the leader R N A s o f I E transcription units 1
and 2, respectively. The distance measured f r o m o3 to
the 5' end o f I E R 4 . 2 / 2 . 9 was 97 nt, with additional
signals at 95 and 94 nt, and the distance f r o m 04 to the
5' end o f I E R 1 . 7 had been determined to be 56 nt,
surrounded by weaker signals f r o m 53 to 58 nt (Wirth et
al., 1991). Since the c D N A p r o d u c t s o f the two reactions
were easily distinguishable, primer extension was performed here with equal a m o u n t s o f 5' end-labelled 03
and 04 mixed in each tube to c o m p a r e transcript levels at
different times after infection (Fig. 3). Primers extended
on R N A isolated at 8 h p.i. f r o m BHV-1 Jura-infected
cells revealed a high level o f I E R 1 . 7 but virtually n o
detectable I E R 4 . 2 / 2 . 9 (lane 1). W h e n entry into the late
phase was blocked the level o f I E R 1 . 7 was strongly
reduced (lane 2) but it was still detectable when c o m p a r e d
with R N A extracted f r o m uninfected cells (lane 3).
F u r t h e r reactions were performed with R N A isolated
f r o m BHV-1 K22-infected cells. As expected for I E
transcripts, high levels o f I E R 4 . 2 / 2 . 9 and even higher
levels o f I E R 1 . 7 were observed at 6 h p.i. if protein
synthesis was blocked by cycloheximide (lane 4). A time
course experiment performed in the absence o f cycloheximide between 2 and 8 h p.i. revealed a continuous
increase in I E R 1 . 7 levels with a c o n c o m i t a n t decrease in
I E R 4 . 2 / 2 . 9 levels (lanes 5 to 8). Densitometric scanning
o f the lanes indicated a 30-fold rise in the ratios o f
IER4.2/2.9
to I E R 1 . 7 levels. T a k e n together,
the N o r t h e r n blot a n d primer extension analyses
demonstrated that IE transcription unit 1 exclusively
Time p.i. (h) 6
Inhibitor c
.
.
.
8
a
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M. Schwyzer and others
1706
•
•
.
<=
.
.
.
i
. i.i
GGCGCCGACCCACGTGGCCCCGTACAAcCGCGGGGCGGGCGGGcCGACGAGCCTCGCGGGGCTGCTTGGCTGGCCTCCAGCGTTCGCACAAAGCTCAATAAGTTTATATATATATTATTG
iii.
.ii
=><=
iii
.
.
ii
.
orisb
GCCCGAGTGCGAG~CCTGGGACCCGCC-CCA~CTCT~GACcGTGCCCGTGAGAACC-CTGGCAGAATGCCAGCGTTC~CACAAAACTCAATAAGTATATATATATTATTAGCCCGAGTGC
•
=>
<-.
.
.
iv
.DR83
<
GAATTCTGGGACTGGCGCCAAGCCGGCCCATGGCAACCACCAACGCCGGGTTTCGATGGGGCTGCCGGGATAGCGGGAGGGCATATGCAAATCATGTCGGGTCGGAGGCGGCGCCGGGTC
DR17
.>
v
-><=
.
iii
.
ori3c
ii
=><GGAGGCGGCGCCGGGcCGGGGGCGGCGCTCGGCCGGGGGCGGGGCCCCTTACC~zG~CC~CCTGGCATTCTGCCAGTGT~CTQ/~GTGGGGTCCCGAGAGGGCTACCGGGACGGTGGGAG
iv
. DR83
v
->
.
.
vi
.
,
o r i s a
120
.
iii.
240
DR17
><
360
480
vii
GGCAC~GC4~AAATCAGGCCCCG~CAGGGGCGGCC~CTCGGCCGGGGGCGGGGGGAGGAGCGCCCTAcTCAAAGACATATAAGCGCGCGACATCGCGCCCCKAGcCCACACA~GGGGGGCTA
•
viii
ix
x
.
600
.
GAGGAGC~C~-GGGCGCACTGCAGAACCTTCACCGAGGCCcAGCCCCGC4~AAA~AAC~CaCGCATCCGCCCC~CGGGGCCCTTTTTTTTTTTTTTTTTTTTTcTcGCTCACCCCC
.
x
xi
<. DR21
-><.
->
ACCCCCAcCCGAGCTCGAGCGAAcGGCTTCCTCGAGCTcAGGTC-C-GTTGcTTcCAT~C43AACTCC-CCGACGACCCCGGAcGCCCCGCCGACGACCCCGGACGCCCCGCCGACGAcCCCGC
<v.
IR35
->
<IR35
v
->.
CAAGTGAC-CTCCGCCCACCCAGCCCCCACCCTCATCTCGGGCCcGGGGGCGGC~AGACGGGGGTGGGC~CTGGGTGGGCGGAGCTCACTCGGGACCccACCCGAGCTccTGTGCTCcGCc
xii<DR5><
><
><
CCCCCGTGcAGCGCTcCATTGCAGCCGGCTCGACGAGCACCTcCAGGAACCGCGCCTCGACTGCACCTAGGGACCTGCCGCAAC~TAGCCCAGCcCAcCCCAGCCCAGCCCAGCCCACC
><
><
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><
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><
><
><
><
><
><
><
><
><
720
840
960
><
><
><
1080
><
><
><
1200
CCAGCCCAGCCCAGCCCACCCCAC~CCAGCCCAG~CCACCCCAC-cCCAGCCCACCCCAGCCCAGCCCACCCCAC~CCAC~CCCACCCCAGCCCAGCCCACCCCAGCCCAGCCCAGCCCACC
><
><
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><
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CCAGcCCAC-CCCAGCCCAGcCCAGCCCACCCCAGcCCACCCCAGCCCAGCCCAGCCCAGCCCAC-cCCACCCCAGCCCAGCCCAGCCCACCCCAGCCCACCCCAGCCCAGCCCAGCCCAcic1 3 2 0
><
><
><
><
>
440
C C A G c c C A C C C C A G C C C A G C C C A G c C A T G C T C T T G C A G A A C C C C G G C G G C G A C C C C ~ c A G A G C T G A A A C C C A G G A C C G C G c C G G T G G A C ~ A G G C ~ G A C A T T C ~ c G A G G T A A G c C A G G T T C1 C
<DR22
-><-><-.
-><-><C C T C G c C C C C C A T C C C C A T C C C C C C G A T C C C T C C - c C C C C A T C C C C C C G A T C C C T C G C C C C C A T C C C C C C G A T C C C T C G C C C C C A T C C C C C C G A T C C C T C C - C C C C C A T C C T C C C G A T C C C T1 5 6 0
-><->
xiii
1680
CGCCCCCATCcCCCCGATCCCTCGCCCGCCCGGGGCAAGCCCGcCcTCCCCGGACGCGCCCTC.CGCGGCCACTCGCTTGCCACGCGACCGGGCGGGCCCTGCCCCGGGGcC-CCCGCTTAC
v.
xiv
xv
.
1800
CGCTCCTCCTCTTCTCTGTCCCGCCCCCTTTGTCCCGGCAGACCGC-cCCc-GCcCGCCGCcC-CC1~GCCACGGCCAGcCTTGCCCCACCTGCGACGGCTCCTGCCGCCTCTGCCC-CTcG
CCCGACCGCGTGGTCTCGGGCcCcGcCCcCGCGGACGAC~AcGCTCGcCGcC`c4~CC~GCCTTCTC-cCCCGAGGACTGGCGCCCCGAGC42GcTGCGCCTCGCcATcGACGTCAAcACc
1920
CTCTTCCGCTGCATCGCCACcGGcTcCGcGTTCGTCACGGCCGACAcGCGcGCC-CTGCGCCGcGCC-CTCGTcC-C~TTCTTCCTGCTCC4C9CTACACGGGCGCCAcGCCCACGGACGCGTC-C
2040
TGGGAGGCGCTGCTGCAGC
2160
TCTCGCCCGAGCAGGCCGGCCCGCTGCGCCGGCT
TTTGCGCGCCGCCGCCGCCGCCGGGC6CAGGGCGCGCCCGCTGTCGCCCCCGGCGCGCCTGCC~GC
CCGCTTTTCGGAGcCGAGTGCGACGTGAGcGGcAGCGACTCCAGCAGCGAAGACTACGAGGAGGACGAGGAGGCGGACGAGGAGGGC43AGGAGAACGGCGGCGAGGGG~CGCCC-CCGAA
2280
AGCCCCCCCCGGTGCCGCCCGCGAGCCGCGGTCCTCTCCTCCGCCTCGCCCC-CC-GCCTCGGCCGTCTCCTTCGTCTCGTCGCCCACCGCGTCCTCTTCCACCTCCTCGTCGTCGCTGTCC
2400
TCGTTCTCCGCCTCGGACGGCGACGACGACGTGTTCTTCCCCGGGCCcGGGGACCCGCGCCcGGCCGGCGCCGGGCCCGGCGCCGGCGGCCCCccCGCGCGCGCGGGCCGGC~c~CCC
2520
C-CGCCCTGCTGGCC-CTGGTCCTCCGGCTCCTCGCCGGGCTCCTCGCCATACCCGTcGCC~TcGCCCTCCGGTCGCC-CCCGGGCGCGGCCCGCGCCGGCcAAGCGCCGCcAGCGA
xvi
xvii
.
xviii
GTT2~3~-C~GGG~CCCGCGGCCCCCTCTCCcGCCCTCTCCcCGGCCCTTGTcATATTTTTTTAAATAAAACGCCC-CGCGcAGGCC-CGcCTTGcCTCTC~TATATCTTGTGGTTCTAG
xix
.<DR17
-><-.
-><
DR18
TTGTTTTATTCC-CCCGcGGGGCGGGAGGGGGAAGGGGGAGCcGGAGCTTTGGCCCGCTCGCTcGGCCGGCCCGAATcCTCC.C-CCGGCCCGAATCCCCTCCTTCCCCTCCCTCcCcTCCTT
-><-><-><-><.-><-><CCCCTCCCTCCCCTCCTTCCCCTCCCTCCCCTCCTTCCCCTCcCTCCCCTCCTTCCCCTCCCTCcCCTCCTTCCCCTCCCTCCCCTCCTTCCCCTccCTCCCCTCCTTCCCCTccCTCCC
- > < .
-><-><.
-><.
•
2640
2760
-><-
2880
-><-
3000
.
.
CTcCTTCCCCTCCCTCCCCTCCTTCCcCTCcCTCCCCTCCTTCCccTCCCTCCCCTCCTTCCCCTCCcTCCCCTCCTTcCccTCCCTAAGAAGAcA~C-CGGGCGTC42C-GAAAAAATTA
.
.
.
.
.
.
3120
XX
GAcAG~cTTT~TcGcG~cGGAcGGGGGGGAGGGGGGaAGGGGAcAGTcGGGccc~GccccAGGGccTcAGGGccGGGGGTcTcGGGG~
3210
Fig. 4. Nucleotide sequence of the BHV-1 (Jura) genome segment encoding BICP22, displayed in the sense of IER1.7 transcription.
Symbols < = = > above the sequence delimit two segments (orisa, or%b) and a truncated version (orisc) exhibiting close similarity
to previously characterized origins of D N A replication of other alphaherpesviruses; imperfect direct repeats and inverted repeats are
delimited by < - - > and marked D R , and IR n in the first repeat unit (n represents size in nt); all other features, underlined and labelled
with roman numerals above the sequence, are described in the text.
B H V - [ orisa G C G G G C 4 2 T ~ T G G
.... C C T .... ~ A C A A A G C T .
C A A T A A G .... T T T A T A T A T A T A
B H V - I or/sb T G C ~ G A A C G C T G G C A . . G A A . . T G C C A G C G T T C C ~ A C A A A A C T . C A A T A A G
..... T T A T T G G C C C G A , f i ~ d ~ Q ~ ~ C C G C G C C
..... A C a C ~ T ~ F , G A C C G
..... T A T A T A T A T A ...... T T A T T A C - C C C G A . G T C ~ , G A A T T C T G C 4 ~ C T G G C C 4 3 C A A . . G C C ~ T C ~ , ~
B H V - I orisc
CCCCTTAC.~7~'CCCCTGG..CATTCTG..CCAGTGTTCTC~GTC-C~
H S V - I oriL
~ C ~ A C G C
.......
GAA . . . . . . .
GCGTTOSCACTTTGTCCTAATAA. . . . . . .
TATATATA. . . . . . .
TTATTAGGACAAA~OGC
.......
TTC . . . . . . .
H S V - I oris
AA~GAAOGC
.......
GAA . . . . . . .
GCGTTCC-CACTTCGTCCCAATA . . . . . . . .
TATATATA. . . . . . .
TTATTA~GTGCGA~
......
TC-GCG. . . . . .
oriL
E H V - I oris
P R V oriL
MDV
ori
TAGAT~aACC~QA
EHV-1
A A ~ a ~ A C
GGTATGTGCGAATT
........ T A A ........ C G T T O G C A C T ~ G T T A C A A T A A
....... G T A ....... ~
G
C
A
A
T
A
A
..... T T ~ T T A T T A T A A
A T ~ ~ G A C G C G T C A G C G T T C C ~ A C ~ C C A A T A T A A G .
CAC'lkn-'J-'I°rC-,ATCT. . . . . . . . .
VZV orisb
CATGT~,%~CQA
G .........
ATTATATATATAAT
......... C . . . . . . . . . C G T T O C 4 2 A C T T T C T T T C T A T A T .
TCTGGGCCAATCAGGGTG~GGATTTGT
T~TCGGGAAGOCKq~TCCTA
G T T T T ~ T G T . . ~ i ' X 2 C . 2 ~ f ~ 7 ~ A C. . . . . . . . . . . . . . . . . .
ATATATAGAG~AGA&~AC~GA~
~
......... A ...... -GCGTTCGCACTT-GTT-CAATAA
GTGGCCCAATCATTTTCCTTGGATTTG
.ATATTATTGGCGCAAGGTGCGAACGCCG...
ATATATATATATATATATAT.
~
=
...... TATTATTATA ...... TTATTAGCAC-A-~CGC-G--
O~C,,C~C~GACTCCG
.GC. T C T G G G C C A A T C A A C C A G T C T A A A A C C a A
.... C T T A T T G G T G A T T ~ .
QATTOGC~CTTCCC..G'I-t~-.~-I-I%?A..CTGTAT~T...
Palindrome
AGA-~a-GAACGC
_
....... T T T C G ...... G C G T T C ~ A C . T T G T T G T A T A T A A . . . A A T A T T A T A T C C T A . T A T A T A T T A G C A A T T G G T G C G A A C G T G A C
V Z V orisa
Consensus
...... T T A T T A T A T A ...... T T A T T A G C A A T T
G C G ~ T T T
CGTT~CTFIL'T
....... T T T C T ....... T G T T C G C G ~ G T G T T
- -
--
-CTGGC-C-A-C-GCGTTC-Fa~-TC
Fig. 5. Multiple sequence alignment of alphaherpesvirus origins of D N A replication. Alignments were generated by using the Pileup
program (Devereux et al., 1984) and were adjusted further manually. The three lines at the top represent the BHV-1 sequence reported
here (Fig. 4), the lines below show the replication origins of HSV-I (McGeoch et al., 1986, 1988; Stow & McMonagle, 1983), EHV1 (Baumann et aL, 1989; Telford et al., 1992), PRV (Klupp et al., 1992), M D V (Camp et al., 1991) and, in two overlapping segments,
VZV (Davison & Scott, 1986; Stow & Davison, 1986). The consensus recognition site (CGTTCGCAC) for the HSV-I UL9 gene
product (origin-binding protein; Olivo et al., 1988) and its inverted complement are underlined. The consensus displays additional bases
if they match in at least five of the sequences. The palindromic structure discussed in the text extends over virtually the entire sequence•
It is centred at the two dots in the middle ( e e ) and consists of two long arms with complementary bases marked ( = ) . Each ann
represents a smaller palindromic structure with centres marked (e).
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ICP22 homologue of BHV-1
kb
0
0.2
0-4
0-6
i
Otis
Direct repeats::
~
0-8
iili
F---I i
i
, ~3
cP
i.~i~
i ~ i i i
NF1
:~-,~-~-~i
~
Spl
! i
Oct
AP4 i
Egr-2 i
Ets-1 i
ELP !
Myc-1 ,~
NF-IL6
NFI.tE3i
TEF2 :
UBP-I::
:
!
i
'.
:
i
:
i
:
i
,
:
i~:
~i !
i-~-:~
!
:
: : i
i ~::
i
:
"
i
:
4i
1-2
i
IIHIHIHfl IHlfll
1-4
1-6
1.8
ili
['f:f'i'fT]:
:
!
:
:
:
:-I~- i
:
!
i g ~ !
:
i
i
!
i
i
i
i
:
i
IERI.~, i ::
',
:
1-0
i.
: i
i i i
i i i , I., -ID,,i ...~.i
i'~: i
i :: i
i
i
i
i
4- ~ - i - ~ t4i i !
:: TAT/~' ~,,
i i4-i
',
Fig. 6. Schematic representation of the oris, promoter and untranslated
regions upstream of the BICP22 coding sequence. The scale at the top
marks the first 1.7 kb of the sequence reported here; the boxes below
summarize the locations of ori s and direct repeat elements (Fig. 4).
Arrows on the lines below indicate map locations and orientations of
consensus signals for the following transcription factors (recognition
sequences are shown in parentheses using the IUPAC ambiguity code
for nucleotides): CP, CCAAT box binding factor (RRCCAATS); NF 1
(YGGMns_rGCCAA); Spl ( K R G G C G K R R or G G G G A G G G G ) ;
Oct, octamer binding factor (ATGCAAAT); AP4 (YCAGCTGYGG);
Egr-2 (CCGCCCCCGC); Ets-I (SMGGAWGY); ELP (CAAGGTCA); Myc-1 (CACGTG); NF-IL6 (TKNNGNAAK); NF,uE3
(GCCACRTGACC); UBP-1 (CTCTCTGG). Sequences were identified using the Find program (Devereux et al., 1984), allowing no
mismatch for Spl, Ets-1, Myc-1 and NF-IL6, and one mismatch for all
other factors (for references see Faisst & Meyer, 1992; Vlcek et al.,
1990). The two non-coding exons, the two introns, and the start codon
for BICP22 are indicated at the bottom.
displays IE kinetics, whereas IE transcription unit 2 is
regulated with dual kinetics involving the IE and late
phases of infection.
Nucleotide sequence of transcription unit 2 and of an
adjacent duplicated ori s region
To investigate possible reasons for these dual kinetics
and to characterize the gene specifying IER1.7, we
sequenced the relevant portion of the HindlII C fragment
of BHV-1 Jura, which is displayed in Fig. 4 in the sense
of IER1.7 transcription. Features requiring comment
are underlined and marked with roman numerals above
the lines. The sequence begins at a NarI site (i) located in
the IR s at 0"814 m.u. and continues for 3210 nt to the
IRs/U s junction (0.839 m.u.) which was identified by
comparison with sequences at the TRs/U s junction
(M. Schwyzer & B. Vogt, unpublished results); the last
nucleotide common to IR s and TR s is underlined (xx).
The transcript boundaries of IER1.7, which had been
determined previously by S1 nuclease and primer
1707
extension analysis relative to restriction endonuclease
sites and by partial sequence analysis of cDNA clones
(Wirth et al., 1991), could now be aligned precisely with
specific consensus signals based on the sequence. Thus,
transcription of IER1.7 is initiated 30 nt downstream
from a TATA box (vi), the main start point being located
at nt 587, and additional start points are located at nt 585
to 590 (vii), as previously established by primer extension
with o4 (viii). The experimentally determined splice site
boundaries of IER1.7 are within a few nucleotides of
consensus sequences which define a 122 nt intron 1
comprising nt 657 (ix) to nt 778 (xi) and a 677 nt intron
2 comprising nt 1045 (xii) to nt 1721 (xiv). The 3'
boundary of IER1.7 was located in a cDNA clone after
nt 2743 (xviii); a polyadenylation signal is present about
30 nt upstream (xvii). Together, IER1.7 consists of exon
1 (67 to 72 nt) spliced to exon 2 (266 nt) and exon 3
(1022 nt) with a total length of 1355 to 1360 nt which
seems compatible with the size of 1.6 kb determined for
IER1.7 of BHV-I Jura, including the poly(A) sequence.
The coding region for BICP22 was found to be
confined to exon 3 of IER1.7, because the entire spliced
transcript contained only a single ATG codon (xv)
located 22 nt after the 5' splice site of exon 3; it initiated
an open reading frame (ORF) consisting of 300 codons
and ending in a TAG stop codon (xvi) about 100 nt
before the 3' terminus of IER1.7 (xviii). This ORF was
favoured by the high similarity of the predicted BICP22
sequence to other ICP22-related sequences (see below),
by the absence of any other ATG codons and also by
codon usage analysis, 96 % of the third base positions in
the BICP22 codons being occupied by G or C, which is
typical for herpesviruses with high G + C content.
Although translation in another frame might occur, in
theory, by initiation at one of the GTG, CTG or ACG
codons present in exons 1 and 2, this was considered
unlikely because both of the other frames had only 70 %
G or C in the third base positions and their putative
translation products exhibited no discernible amino acid
sequence homology with other known herpesviral proteins.
Upstream of IER1.7, the most conspicuous feature of
the nucleotide sequence was a duplicated segment
extending from nt 56 to 163 (orisa) and from nt 164 to
276 (orisb) which exhibited similarity to previously
characterized origins of DNA replication of other
alphaherpesviruses. An additional truncated segment of
homology was observed from nt 405 to 448 (orisc).
Alignment of these segments (Fig. 5) revealed their
inherent palindromic structure. The centres of the
palindromes (marked by two dots) were located in the
characteristic A + T-rich regions which were flanked by
the nonanucleotide CGTTCGCAC (ii) and its complement GTGCGAACG (iii; underlined in Fig. 4 and 5).
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1708
M. Sehwyzer and others
I
MSHGQPCPTC
DGSCRLC ..........................
RSPDRVV
MPHGQPCGAC
DGSCRMAQRG
TPSTSPLIPS
L.TPSPPAGD PSPRSSQRID
........... GSCRMSQRG APSTSPIIPS
L.SPS.SGGN
PSPRSSQRID
MDRVW.ADWY
EPVPSPPFSP
VDPPGPRPTT
PVPGSSPPSP
MFCTSPATR GDSSESKPGA
SAESTTGTET
DASVSDDPDD
TSDWSYDDIP
PRPKRARVNL
RLTSSPDRRD
MSRDRD RARPDTRLSS
............
S ...........
S ...................
SS-R--
SGPAPADE ............
AVRVPARLPG
..GSD...HP
SVRVPARLPG
.. G S D . . . HP
ASTPTPPKRG
RYVVE...HP
SVDVNGKM ............
GVIFPKMGRV
RSTRETQpRA
SDNESDDEDY
QLPHS...HP
sv--P .............
HP
32
64
63
56
27
160
33
BHV-I
EHV-I
EHV-4
PRV
VZV
HSV-I
MDV
BHV-1
EHV-1
2
.............
HARRGPG
EYGMPLSPRA
LRPYLARGPG
EYGLPLSPRS
LRPYLSRGPG
EYGPPPDPEE
VRVHGARGPG
EYGSAPGPLN
GRD.TSRGPG
PTPSAPSPNA
MLRRSVRQAQ
EYGSDSSDQD
...FELNNVG
EYG---SP--R .... RGPG
AFCPEDWRPE
AFCAPPWRPD
AFCAPPWRPD
AFCAAPWRPD
AFCTPGWEIH
RRSSARWTPD
KFCPLPWKPD
AFC--PWRPD
ALRLAIDVNT
LFRCIATGSA
VNRLAGDVNR
LFRGISTSSI
VNRLAGDVNR
LFRGISTSSI
TRRLGADVNR
LFRGIAVSAA
PARLVEDINRVFLCIAQSSG
LGYMRQCINQ
LFRVLRVARD
VARLCADTNK
LFRCFIRCRL
--RL--DVNR
LFR-I--SS-
FVTADTRALR
HVTEDSRTLR
HVTEDSRVLR
DVTGDTRALR
RVTRDSRRLR
PHGSANR.LR
NSGPFHDALR
-VT-D-R-LR
RALVGFFLLG
RALLDFYAMG
RVLLDFYAMG
RALFDFYAMG
RICLDFYLMG
HLIRDCYLMG
RALFDIHMIG
RALLDFYLMG
89
134
133
126
96
229
i00
EHV-4
PRV
VZV
HSV-I
MDV
YTGATPTDAC
YTHTRPTLEC
YTHARPTLEC
YTRQRPSAPC
RTRQRPTLAC
YCRARLAPRT
RMGYRLKQAE
YT--RPT--C
Q.AGPLRRLL
Q.SFPLRATL
Q.SLPLRATL
Q.SAPLRSAL
Q.TQCLRATL
TWGMHLRNTI
Q.SLHLRRTL
Q-SLPLR-TL
...... GP.RARPL
RALNSE ...... DRYEQRFL
RAINSE ...... DKYEQRFL
RELNER ...... DVYDPRVL
MEVSHR ...... PPRGEDGF
REVEARFDAT
AEPVCKLPCL
RDADSRSAHP
ISDIYASDSI
RE---R ...... D-Y--R-L
SPPARLPGPL
EPPSDPPNTL
DPPSKPPKTL
SPPVIEG.PL
IEAPNVPLHR
E T R ...... R
FHPIAASSGT
-PP---P--L
FGAECDVSG
FGEECDVSG
FGEECEVSG
FGEECDVDE
SALECDVSD
YGPECDLSN
ISSDCDV..
FG-ECDVS-
150
196
195
187
158
292
166
BHV-I
EHV-I
EHV-4
PRV
VZV
HSV-I
MDV
EEADEEGEEN
EEDEASGESS
...EASGNST
EEGDEDGETD
SDDDGSTPSD
ISDATDLEAA
-E-D--GE--
GGEGAAAESP
V S E ..... FS
I S E ..... FS
VYEEDDEAED
VIEFRD...S
GSDHTLASQS
V-E ...... S
181
219
215
257
181
324
BHV-I
EHV-I
WEALLQLSPE
WQSLLQLLPE
WQALLQLMPE
WQALLQLSPE
WEELLQLQPT
WCRLLQVSGG
WETIMNLTPR
W--LLQL-PE
3
SDSSS .......................................
EDYEED
DESPS ..........................................
EEE
DESPS ..........................................
EEE
DDAGSDTTVA
SEFSFRGSVC
EDDGEDEDEE
EDGEEEDEDE
EGEEEEDEEE
DGG ............................................
EDD
LE ......................................
IHLSATSDDE
DE--S ..........................................
EEE
PRCRPRAAVL
PEEETASSEY
PEEESASSDF
EEDEEDGDDF
DAESSDGEDF
DTEDAPSPVT
--EE .... DF
SSASPAASAV
SFVSSPTASS
DSFSDVGED ...... DSSCT
ESFSD.EED ...... DSCCT
DGASVGDDDV
FEPPEDGSDG
IVEEESEEST DSCEPDGVPG
LETPEPRGSL
AVRLEDEFGE
---S---E ....... D ---i
STSSSSLSSF SASDGDDDVF FPGPGDPRPA
GAGPG.AGGP
GKWSSSES ...............
ESDSESD APTNNHHPTT
GKWSS..S ...............
ESDSEAD VPTN..PPTT
EGSGSDDGGD
GEDEDEDEDE
DEDEDDGEDE
EDEEGEDGGE
DCYRDGDGCN
TPSPKRPQRA
IERYAGAETA
EYTAAKALTA
FDWTPQEGS ........ QPW LSAVVADTSS
VERPGPSDSG
--w-s ....................
D-E ............
250
268
259
327
251
386
EHV-4
PRV
VZV
HSV-I
BHV-I
EHV-I
EHV-4
PRV
VZV
HSV-I
4
PARAGRRRPA
PCWRWSSGSS
PGSSPYPSPG
KASAAKKRR ........ KRQ PPKGERPTKS
RARAAQKRR ........ GRP VPKGGRPAKS
DGEDGEEDE ........ DED GEGEEGGKDA
LGEGGVDWK ...............
RRRHEA
AGRAAEDRKC
L ....... D G C R K M R F S T A C
.....................
KGMNDLSVD
---A---R .......................
GSPSGRARAR
PAPAKRRQRV*
ARR*
ARR*
ARRGTRAPTR
PAAAP*
PRRHDIPPPH
GV*
PYPCSDTFLR
P*
SKLH*
RR .................
300
293
284
364
278
420
179
BHV-I
EHV-I
EHV-4
PRV
VZV
HSV-I
MDV
Fig. 7. Multiple sequence alignment of ICP22-related proteins. Alignments were generated by using the Pileup program (Devereux et
al., 1984) with the following amino acid sequences: BICP22 of BHV-1 ; IR4 gene product of EHV-1 (Holden et al., 1992; Telford et
al., 1992); protein 4 of EHV-4 (Cullinane et al., 1988); Rsp40 of PRV (Zhang & Leader, 1990); protein 63 of VZV (Davison & Scott,
1986); ICP22 (Vmw68) of HSV-1 (McGeoch et at., 1985); US1 gene product of MDV (P. Brunovskis & L. F. Velicer, personal
communication). Some N-terminal sequences are omitted from the HSV-1 and EHV-4 proteins. The four distinct regions, of which
region 2 exhibits high similarity and region 3 and 4 exhibit similar composition, are described in the text.
These elements have been reported to be essential for
origin activity (Martin e t al., 1991). Additional incomplete copies o f the nonanucleotide motif were found
near the ends o f the displayed segments. Since these
motifs were again inverted relative to their neighbours,
they formed a pair o f flanking lower-order palindromes
(centres marked by one dot), which together made up the
higher-order palindrome. The overall h o m o l o g y observed with established origin sequences o f other herpesviruses suggested that the corresponding BHV-I
sequences may also possess origin activity; this is
supported by preliminary experiments using D p n I resistance assays (Yi Geng, unpublished observations).
The close proximity of this duplicated viral origin o f
replication to the IE1.7 promoter may facilitate reactivation of I E R 1 . 7 synthesis after the onset o f viral
D N A replication.
N u m e r o u s consensus signals for the regulation of
transcription were observed not only upstream from the
I E R 1 . 7 transcription start site, overlapping with the o r i s
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ICP22 homologue of BHV-1
sequences, but also in the introns and non-coding exons.
Among the more prominent consensus signals, underlined in Fig. 4, are octamer boxes (iv) and putative
binding sites for Spl (v), Ets-1 (x) and NF/zE3 (xiii). To
illustrate how these signals are spread over the 1.75 kb
region preceding exon 3, Fig. 6 indicates their location
and orientation with arrows and displays additional
signals not marked in Fig. 4. No such cumulation of
regulatory motifs was observed in the BICP22 coding
region except for a few possible Spl binding sites. It
should be noted that the identification of these sites is
based on sequence analysis alone. Their functions have
not been tested except by the transient expression assays
described previously (Wirth et al., 1992; Schwyzer et al.,
1993).
Another striking feature of the sequence presented in
Fig. 4 is the presence of numerous direct repeats. Most
are imperfectly repeated and occur in the untranscribed
regions, resulting in the amplification of some consensus
signals. One repeat unit (DR21) was located near the
beginning of exon 2 and provided an explanation for the
strain difference in transcript size (1"6 kb for Jura; 1"8 kb
for K22); the Jura sequence (Fig. 4) contained only two
DR21 repeats, whereas cDNA clones derived from K22
mRNA were found to harbour a variable number (nine
to 15) of these repeats (M. Schwyzer & B. Vogt,
unpublished results).
A polyadenylation signal was observed at nt 2770 to
2765 (xix) on the strand opposite to that encoding
BICP22, suggesting that depending on its location in IR s
and TR s, it may serve for two different, as yet
unidentified transcripts starting at either end of U s.
Amino acid sequence homology of BICP22 with ICP22related proteins
The predicted amino acid sequence of BICP22 is
displayed as the top line of an alignment with five other
ICP22-related proteins (Fig. 7). The alignment was
found to consist of four distinct parts. The N-terminal
part (region 1) contained sequences varying in length
from 32 (BICP22) to 176 residues (ICP22 of HSV-1) and
exhibited only scattered clusters of homology. Region 2,
represented by BICP22 residues 33 to 150, exhibited
close similarity in pairwise comparisons between the
bovine, equine and porcine herpesviral ICP22 sequences
(60% identical residues), and to a lesser extent also
between the other sequences (30 % to 50 % identity). The
other half of the alignment presented a striking similarity
in amino acid composition with a preponderance of
hydrophilic residues but little detailed sequence homology. It could be divided into region 3, rich in acidic
(Asp and Glu) and hydroxyl (Ser and Thr) residues, and
a shorter C-terminal region 4 rich in basic (Arg and Lys)
1709
and proline residues. The small amino acids glycine and
alanine were also quite frequent in both regions. In
BICP22, these nine amino acids together accounted for
131 of the 150 residues. Among the different proteins,
regions 3 and 4 varied considerably in length from just 13
residues for the MDV homologue, from which region 3
was missing entirely, to 177 residues for the PRV
homologue, in which region 3 was particularly long and
acidic.
Discussion
Among the four IE genes of BHV-1, only the BICP4 gene
follows strict IE kinetics in the sense that its transcription
does not require prior protein synthesis and that it is
turned off at the end of the IE phase by autoregulation.
The BICP0 and circ genes initially share IE kinetics with
the BICP4 gene because they are expressed from the
same promoter by alternative splicing. At the end of the
IE phase however, the BICP0 gene exhibits an additional
early promoter that is located just upstream of the splice
site and provides high levels of an alternative unspliced
transcript with identical BICP0 coding potential. Similarly, the circ gene is expressed from an alternative late
promoter. We report here that the BICP22 gene is
expressed with IE and late kinetics under the control of
a single promoter. Thus, BICP22 is synthesized from
identical spliced transcripts (IER1.7) throughout infection.
Sequence analysis revealed that these biphasic kinetics
could be due to the close proximity of a putative origin
of DNA replication, or to the presence of numerous
signals for transcription regulation, or to both. As in
other viruses, the expression of BHV- 1 late genes depends
on prior viral DNA synthesis (Misra et al., 1981 ; Wirth
et al., 1989). Conversely, in many viruses, origins of
replication are affected by adjacent transcription units or
by regulatory factors acting on both transcription and
DNA replication (Depamphilis, 1993; McCarty et al.,
1992; Sugden & Warren, 1989; Wong & Schaffer, 1991).
Such effects have not yet been shown for BHV-1, but
sequence elements exhibiting close similarity to origins
of DNA replication (otis; oriL) of other alphaherpesviruses were identified just 300 nt upstream from the
IER1.7 start site. Surprisingly, these elements occurred
in two imperfectly repeated copies and an additional
truncated version. Similar duplications have been noted
in the otis and oriL regions of HSV isolates (Gray &
Kaerner, 1984; Whitton & Clements, 1984).
The non-coding region between the two IE transcription units of BHV-1 is exceptionally large (3.1 kb);
in addition to the 0"6 kb upstream of the BICP22 gene
reported here, 2-5 kb of upstream sequences have been
reported for the BICP4 gene (Schwyzer et al., 1993). The
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1710
M. Schwyzer and others
entire intergenic region, including oris, and even the
introns and non-coding exons of the BICP22 gene
contain numerous potential recognition sites for transcription factors. Transient expression assays indicate a
strong constitutive activity of the IE 1.7 promoter which
is presumably mediated by some of these recognition
sites. The IE4.2/2.9 promoter contains a TAATGARAT element responsive to the BHV-1 homologue of the
alpha-trans-inducing factor (Carpenter & Misra, 1992;
Schwyzer et al., 1993), but the IE1.7 promoter does not.
Comparison of the seven ICP22-related proteins (Fig.
7) showed that each could be divided into four distinct
regions. The proteins were highly similar in a domain of
120 to 130 amino acid residues (region 2). The adjacent
C-terminal sequences exhibited little homology but were
remarkably similar in amino acid composition, acidic
and hydroxyl amino acid residues predominating in
region 3 and basic and proline residues in region 4. These
regions may constitute a PEST sequence implicated in
short protein half-life (Rogers et al., 1986) or, alternatively, an acidic domain involved in transcriptional
regulation. The size of these regions varies considerably;
in the PRV homologue, region 3 is longer and contains
a larger number of acidic residues, whereas in the MDV
homologue, region 3 is entirely missing and only a
truncated version of region 4 is present. Similarly, region
1 is much longer in ICP22 of HSV-1 than in all the other
proteins. These non-conserved regions may not be
essential or they may serve different functions in the
different homologues.
The nuclei of BHV-l-infected cells have previously
been shown to contain multiple species of an abundant
phosphoprotein (IEP-55) with apparent Mr values
ranging from 52000 to 57000 (Hayes & Rock, 1990).
Several properties of IEP-55 make it appear likely to
represent the BICP22 gene product. It can be labelled
with [32P]orthophosphate but not [3SS]methionine, reflecting the absence of internal methionine residues in the
predicted sequence of BICP22. It is synthesized under IE
conditions using a cycloheximide-actinomycin D block
but also incorporates abundant label from 9 to 11 h p.i.,
consistent with the IE and late kinetics observed here.
Using the PROSITE (Bairoch, 1992) database, we
identified six putative phosphorylation sites for casein
kinase II and two sites for protein kinase C which may
account for the observed multiple species of IEP-55
differing in apparent M r and isoelectric points. Although
the predicted M r of BICP22 is only 30 855, phosphorylation, additional post-translational modifications and
unusual amino acid composition may all contribute to
increased apparent M r . Similar observations have been
made for ICP22 of HSV-1, which consists of five
electrophoretically distinct species and is phosphorylated, guanylylated and adenylylated (Ackermann et al.,
1985; Blaho et al., 1993; Purves et al., 1993), and exhibits
a similar discrepancy between predicted and reported
MrS (46 524 and 68 000, respectively).
Little is known about the function of ICP22-related
proteins. Deletion mutants of HSV-1 in which the ICP22
gene alone (Poffenberger et al., 1993) or together with
additional genes (Sears et al., 1985) was disabled,
demonstrated that ICP22 strongly affects the host range
of the virus. Furthermore, the absence of ICP22 caused
prolonged expression of early and delayed expression of
late genes, presumably due to a delay in viral DNA
synthesis (Poffenberger et al., 1993). Transient expression
assays were used to show that the corresponding VZV
protein 63 acted as a feedback inhibitor of gene 62
(encoding the ICP4 homologue), whereas it did not affect
the glycoprotein I and II promoters and even stimulated
the thymidine kinase promoter (Jackers et al., 1992). We
employed similar transient expression assays (C. Fraefel,
B. Vogt & M. Schwyzer, unpublished results), which
seemed to indicate that BICP22 was a general transcriptional repressor. However, this assignment of
BICP22 function, listed in a recent compilation of BHV1 genes (Schwyzer, 1993), must be considered tentative
because we have not yet been able to demonstrate that
the effect is due to BICP22 protein rather than to
competition by flanking promoter sequences. Work is in
progress to express BICP22 in a baculovirus system in
order to characterize the protein and elucidate its
function. We have also produced antipeptide antibodies
which should help to determine whether BICP22 produced during the IE phase differs in structure and
function from the protein that is made late in infection.
We thank Cestmir Vlcek for help with some of the sequences, Yves
Choffat for primer synthesis, Anita Hug for photography, Peter
Brunovskis for a personal communication and Mathias Ackermann for
critical reading of the manuscript. This work was supported by grant
31-263346.89 from the Swiss National Science Foundation.
References
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(Received 5 November 1993; Accepted 26 January 1994)
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