Download Nucleotide sequence of a cytomegalovirus single

Journal of General Virology (1990), 71, 2451-2456.
2451
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Nucleotide sequence of a cytomegalovirus single-stranded DNA-binding
protein gene: comparison with alpha- and gammaherpesvirus counterparts
reveals conserved segments
David
G.
Anders
Virology Laboratories, The Wadsworth Center for Laboratories and Research, S U N Y at Albany, School of Public Health,
New York State Department of Health, Albany, New York, 12201-0509, U.S.A.
The genomic sequence encoding a cytomegalovirus
strain Colburn homologue (DB129) of the herpes
simplex virus major DNA-binding protein (ICP8) was
determined. Multiple alignments of the deduced
DB129 amino acid sequence and three alpha- and
gammaherpesvirus homologues revealed that 56 % of
the amino acid residues identical in all four homologues are contained within 12 relatively conserved
segments, which together constitute only 11.2 % of the
shortest aligned sequence. In light of published ICP8
deletion analyses, this alignment suggests conserved
segments that may participate in forming DNA
contacts. The identified conserved regions present
interesting targets for site-directed mutagenesis in
structure-function analyses.
Previous studies have identified an early nuclear
ssDNA-binding protein, present in cytomegalovirus
(CMV)-infected cells, as a probable homologue of the
herpes simplex virus type 1 (HSV-1) major DNAbinding protein (ICP8) (Anders et al., 1986, 1987) and
have mapped its gene near the centre of the long unique
component of the viral genome (Anders & Gibson, 1988;
Kemble et al., 1987). The CMV strain Colburn protein
(DB129) has an estimated Mr of 129000, whereas its
immunologically cross-reactive human CMV (HCMV)
counterpart (DB140), encoded by the UL57 open reading
frame (ORF; M. Chee, personal communication), has an
estimated Mr of about 140000 (Anders et al., 1986). The
extensive biochemical (Anders et al., 1986; Kemble et
al., 1987) and sequence (this paper) similarities of this
CMV protein to HSV-1 ICP8 argue that it plays a
comparable role in infection, although no genetic
evidence for its function(s) is available. This laboratory
is studying the CMV ssDNA-binding protein as a model
for the structure and function of this group-common gene
product. To facilitate these studies, and to allow
comparison with herpesvirus counterparts, I determined
the nucleotide sequence encoding DB129 and its flanking regions.
Strain Colburn CMV (Gibson, 1981, 1983), from
which all recombinant clones were derived, was obtained
from W. Gibson. Plasmid pDGA8 contains the EcoRI D
fragment of CMV(Colburn) and has also been described
(Anders & Gibson, 1988). All recombinant plasmids
were propagated in Escherichia coli DH5~ (Bethesda
Research Laboratories). Hybrid-arrested in vitro translation experiments previously showed that the gene
encoding CMV(Colburn) DB129 lies within EcoRI-D;
its boundaries were predicted from limited sequence data
and transcript mapping (Anders & Gibson, 1988). To
establish the structure of the DB129 gene, the 5-6 kb
HindIII subfragment of EeoRI-D containing the DB129
gene (Fig. 1) was excised from pDGA8 and ligated into
the HindIII site of pBluescribe (Stratagene Cloning
Systems) in both a and b orientations to generate
pDGA16 and pDGA24, respectively. Nested sets of
0000--9628 © 1990 SGM
K
(a) If
B
ASUXPL1L2DVRFM
t II I "~,,,~,~,~,\,/,,
. . . . . . . . . . . . I ,/
jf
E
H
P Sp S X K K X
I I
E H INGJZQ
, , t,\/,I,
P, . . . . .
HXX
X
C OTWY
~/~/
III
E
Fig. 1. Location of DB129 gene and sequencing strategy. (a) EeoRI
physical map of the CMV(Colburn) genome (LaFemina & Hayward,
1980; Jeang & Hayward, 1983). Cleavage sites are indicated by crosshatches and the larger fragments are designated with uppercase letters.
(b) Restriction map of EcoRI-D, cloned as pDGA8, showing restriction
sites designated as follows: E, EcoRI ; H, HindIII ; K, KpnI, P, PstI ; S,
Sail; Sp, SphI; X, XbaI. The approximate position of the most
abundant transcript from this region and its orientation are indicated
above the map. The HindIII fragment subcloned from EeoRI-D in both
a and b orientations, as pDGAI6 and pDGA24 respectively, is filled.
The cross-hatched segment shows the location of sequence data given
in Fig. 2.
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2452
Short communication
-240
CACCGAGCATCACGcTATTTTCGGAAGCGcCT~TGGAGGACAGTTCGCCGAGCCGGCGGCGGGTGGCGCGGGGTGTGTTTGGGTCGCAAATCGGGCGCATAACCGTCGGAGGGGGCGGCG -121
-120
CGCGTCGGGGACAGTGTGCA~TTCCTT~CTGGTGGTCTACGTATT~CGTCAACTATGACG~C~AGCC~CTCC~AGGTATAAAGTTCACTT~ATTA~CG~AGTCGTTACA~ACCACC
-i
i
~TGAGCAACGAGGA~CTC~GT~CTCTcGCTCCC~TGGGTCCGGCGGCCTATGTATACTTTACCAAAACCAACCAT~AAA#GAACGAGGTTTTAGCCACGTTATCGCTGTGCGATTC~T~T
M S N E E L S A L A P V G P A A Y V Y F T K T N H E M N E V L A T L S L C D S S
120
40
121
~CGCCCGTGGTGATCGCCCCGCTCTTGATGGG~CTC~CCGTCGATCAAGATTTCT~TACCTCGGTTCGCACCCCGGTCGTGTGTTACGACGGT~GGGTGCTC~CCAAGGTGA~GTCTTTC
S P V V I A P L L M G L T V D Q D F C T S V R T P V V C Y D G G V L T K V T S F
240
80
241
TGTC~CTTTGCTCTGTATTTCTACAACACTCAGGGGATCGT~GATTTCTCGG~GCCGCATGGTGACGTACAACGGCTGTGTGACGAAAC~CGTCAAAGATACGCCATTGAGAGCTACATG
C P F A L Y F Y N T Q G I V D F S E P H G D V Q R L C D E T R Q R Y A I E S Y M
360
120
361
CCGGAAGAAG~C~T~CCCCAC~GACCTTGC~GCCCTCTGCACGGCCGCCGGGTGCGATCCTCAAGAGGTGTTGGT~CAC~TCGTCGTG~GCAACGGCATGAAAGAGTTCATGTATGCG 480
P E E G R A P I D L A A L C T A A G C D P Q E V L V H V V V G N G M K E F M Y A
160
481
GGCC~GCTcATcC~GTGCT~CGAGGA~CGGCGCCCACTCGACTGAACGATTGCG~CGCGGTGCGCGTCCCGCTGTATCCT~CCACCCTCTTTGGTT~TTTGCAGGCCGATGTGGATTCT
G Q L I P C F E E A A P T R L N D C D A V R V P L Y P P T L F G S L Q A O V D S
600
200
601
GACGAGC~GlC~CTAGACAAGCGCAGCT~GTTCGTGGAATCTCGGGGATTGTACGTGCCTGCGGTGAGCGAAACCCTGT~CTACTATGTTTACA~TTCGTGGTGCCAGGCCCTGCGCTT
D E L S L D K R S S F V E S R G L Y V P A V S
T L F Y Y V Y T S W C Q A L R F
720
240
721
TCAGAGACcAAGGTGCTGATCGAAGCGGCCCTGAAGCAGTTCGTGAACGACAGCCAGCAGTcCGTGAAGCTGGCTCCGcACAAGAAGTACTTCGGGTACACGAGccAGAAGCTGAGCAGT 840
S E T K V L I E A A L K Q F V N D S Q Q S V K L A P H K K Y F G Y T S Q K L S S
280
841
CTGGAGAAAGACCACTTAATGCTCAGCGACGCCGTGATCTG~GAGCTGGGGTTCAGCTTcGCCTCGGTGTTTCTGGACTCGGCCTACGGGGCA~CGGATTCCATGGTTTAcTcGGAATGG
L E K D H L M L S D A V I C E L G F S F A S V F L D S A Y G A S D S M V Y S E W
960
320
96]
CcTGTCGTGGTGAACGCCAcGGA~CATCG~GATCTCATCCGAGCTCTCACCGAGCTCAAATTGcATCTCTcTACCCATATTAGTGCACTGcTGTTTAGTTGTAATT~TATTCTGTAT~AT
P V V V N A T D H R D L I R A L T E L K L H L S T H I S A L L F S C N S I L Y H
]080
360
1081
AA~CGGCTTGTG~ATCT£ACTTCCAACAAGAACGC~AGCGGTACCGGAGCCAGCCAGGAGGTGCT~CTGAAGTCTATTCACTTCGCCAACGGCCTGACGGGGCTGTG~GAAGACACGTAT 1200
N R L V Y L T S N K N A S G T G A S Q E V L L K S I H F A N G L T G L C E D T Y
400
1201 AACGACGCCAGGAAACTGATCAAGTGTTCTGGCGTGGTTG~CAAGGACGAACGTTATGCGCCGTATCA~CTGTC~CTCATCTGCGGTACGTGTCCTCAACTTTTC~CTG~TTTCATA~GG
N D A R K L I K C S G V V A K D E R Y A P Y H L S L C G T C P Q L F S A F I W
1321
1320
440
TACCT•AATCGAGTTTCTGTTTACAATACCGGGTTGACAGGG•CTTCGACTTTGAGTAATCATTTAATCGGTTGTTCG•CTA•TCTGT•TGGGGCCTGTGGTGGGACATGTTGTCATACC
1440
Y L N R V S V Y N T G L T G S S T L S N H L I G C S S S L C G A C G G T C C H T 480
]44] TGTTATAACACGGCATTCGTGCGGGTACAGACCCGTCTGCC~CAGATGCCGAGGCTCCCGAAGAAGGAGCCCTCCGTCGTGGTCATGCAA~TcGATTTCTCAACGATGTGGATGTGCTG
1560
C Y N T A F V R V Q T R L P Q M P R L P K K E P S V V V M Q S R F L N D V D V L 520
1561
GGTACGTTCGGACGCCGCTATAGCGCGGAG~CTAAAGAAGCGAGTCTA~ACGCGAAAG~CGACGAGGGTTCCGCGTCGACGTCTAATCGCACCGCGAGCTCGAGCGTGGACCGCACCCAT 1680
G T F G R R Y S A E S K E A S L D A K A D E G S A S T S N R T A S S S V D R T H
560
1681
CGT~TCAACCGCA~CTTGGACTATTGTAAAAAAA~GAGA~TCATAGACTcGGTTACGGGTGAAGACACCATGACTATCAACGGCAGGAGCGATTTTATTAATCTGGTGTCCTCGCTTAAT 1800
R / N R I L D Y C K K M R L I D S V T G E D T M T I N G R S D F I N L V S S L N
600
1801
AAGTTTG~AGA~GA~GAAGCCATGAGC~CGTG~CCGAGG~CCGTA~GAAAAGTAATCGCGACGAGGTTTTAGG~GCTA~GCAGGCCTT~AACCTCGATCTCAACCC~TCGccGTTTCG 1920
K F V D D E A M S F V S E V R M K S N R D E V L G A T Q A F N L D L N P F A V S
640
]92]
TTCAGTCCCATTCTTGCGTATGAGTACTATCGGGTGATTTTCGCCATCATTCAGAACGTCGCCCTGATCACGGC~ACGTCCTACATTGTAGAcAA~cCCCTCACCACGAGTTTGGTT~CC 2040
F S P I L A Y E Y Y R V I F A I I Q N V A L I T A T S Y I V D N P L T T S L V S
680
2041
CGGTGGGTGACTCAA~ACTTC~AGT~TATCCACGGGGCT~7TTCCACCAcTTCCTcCCGAAAGGGTTTTCTCTTTATTAGGAATGTGAAATCCTCAAAAAACGCGGATCATGACCGCCTC 2160
R W V T Q H F Q S I H G A F S T T S S R K G F L F I R N V K S S K N A D H D R L
720
2161
CccGACTTTAAA~TcTATGCCCGCGGCACGTACT~GGTCAT~TCCATGGAGATCAAGCTCTCccGcCTCTCTGTCC~TAGTCTGCTCATGTTCAGGGTcAAAAACCGCCCCATCTcTAAG
P D F K L Y A R G T Y S V I S M E I K L S R L S V P S L L M F R V K N R P I S K
2281
GCTAGCAAGGGTACGACGGCTCACGTGTTTTTTCGCCGCGAGCACGTACCTAAGAAAAATCCAGTcAAGGG~TGTTTGGGCTTTCTGCTCTACAAGTATCATGATAAGT~ATTTCCCGAT 2400
A S K G T T A H V F F R R E H V P K K N P V K G C L G F L L Y K Y H D K L F P D
800
2280
760
2401 TGCGGGTTCTCATGTTTACAGTTCTGG~AAAAAGTGTGTGCCAACGCACTG~CCAAAAACGTGAATATCGGGGA~ATGGGGGAGTTCAA~AATTTTG~CAAGTTCGTCATCTCGGTCA~C
2520
C G F S C L Q F W Q K V C A N A L P K N V N I G D M G E F N N F V K F V I S V T 840
2521 GCCGATTATAACGAGCATGACC~GATTGACGTGCCGCCCGATTGCATGCTTAACTATCTGGAGAACCGA~TCACAA~AAGTTCC~TTG~TTTTACGGGTTTAAAGATTACATAGGCACG
2640
A D Y N E H D L I D V P P D C M L N Y L E N R F H N K F L C F Y G F K D Y I G T 880
2641 TTG~ACGGCCTGACAA~GAGGCTAACGTATCAGAATCACGCCCAGTTTCcCTACCTCTTGGG~GAGAGTCCCAATTTTG~GTCAGCTGCCGATTTTGCCETGCGCTTAAAGGATCTCAAA
2760
L H G L T T R L T Y Q N H A Q F P Y L L G E S P N F A S A A D F A L R L K D L K 920
2761 GCGACCGG~GTTA~GGCGCCG~T~CGTCTACGGTTACGCGAGAGTCCTTGA~GCGCACCATTTTTGAGCAACGCTCCCTGGT~ACTGTGAGTTTTTCCATTGAGAAGTAC~CGGGGGTG
2880
A T G V T A P L A S I V T R E S L M R T I F E Q R S L V T V S F S I E K Y A G V960
2881 AACAACAACAAGGAAATT~A~CAGTTTGG~CAGATTGGGTACTTTTCG~GCAACGGGGTGGAGcGCAGCCTGAATACCAAT~CATAGGG~GTCAGGAT~ATAAATTCATGCGTCAGCGC
3000
N N N K E I Y Q F G Q I G Y F S G N G V E R S L N T N S I G G Q D Y K F M R Q RIO00
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Short communication
2453
3001
TGTATCCTGGC~ACCAAACTCTCAGAcGTTCTCATCAAGCGTTCACGGCGCGATAACGTGCTGTTTGACGAGGACATTATCAAGAACAGGGTCATGGCGGCCCTGGATTCGGAGAACCTG
3120
3121
GATGTTGA~CC~GAGCTCATGGCTATGTACGAGATA~TGAGCACTCGGGAGGAGATTCCCGAGCGGGACGA~GTTTTGTT~TTTGTAGATGGATGTCAGGCCGTGGCCGATTCCCTGATG
3240
C I L A T K L S D V L I K R S R R D N V L F D E D I I K N R V M A A L D S E N 1040
L
D V D P E L M A M Y E I L S T R E E I P E R D D V L F F V D G C Q A V A D S L M
1080
3241 GAGAAGTTTTCGCGCTTGCAGGAGAT~GGAGTGGACGAC$TTTCCCTGGTGAAT~TGCAGCAGGTGCTGGA~A~CCGGCCGGAGTGCGGCGG~GGCGGGGGCGAGGTTCACGACCTGTCG
3360
E K F S R L Q E M G V D D F S L V N L Q Q V L D S R P E C G G G G G E V H D L S1120
3361 G~GCTG~TTACCGCCGCCTCCGGGGAGGCG~TGGGCAACTCTGTGGGCCG
i AACGCGCGEGGGGGGGAGEACGCCTJTGL~GAGGATTGG
i GTCTGTTGECGGCCAAGAGAGGCC~CCTG
3480
A L F T A A S G E A V G N S V G L N A R G G E H A F D E D C G L L P A K R G R L1160
3481 TAATAAACGCCGTGCACGCCGTTATATATTAACGTCGGTGTGCACGGCAEACTGCAGAGC
3540
Fig. 2. Nucleotidesequence of the DB129-codingsegment of ~oR]-D and flanking regions, and deduced amino acid sequence. The
first nucleotideof the probable initiation codon is taken as + 1. The TATA consensus, the polyadenylationsignal and other potential
regulato~ or signal sequencesdiscussed in the text are underlined. The deduced amino acid sequenceis given beneath the nucleotide
sequence.
progressive unidirectional deletions, in both directions
with respect to the DB129 gene, were prepared using the
exonuclease III method of Henikoff (1984). The sequence across the deletion junction of selected clones was
determined using the dideoxynucleotide chain termination method (Sanger et al., 1977). When it was necessary
to fill in gaps, reactions were primed with an oligonucleotide corresponding to a known sequence within the
insert. Primers were extended with modified T7 D N A
polymerase (Sequenase; U.S. Biochemical) according
to the supplier's instructions, in the presence of
[~-35S]dATP (Amersham). Nucleotide sequence data
were assembled and analysed on a Digital Equipment
Corporation VAX/VMS computer using the Genetics
Computer Group Sequence Analysis Software Package,
version 5.3 ( G C G ; Devereaux et al., 1984). Both strands
were essentially completely sequenced. A preliminary
report of this work was presented at the 13th International Herpesvirus Workshop, 7 to 13 August, 1988,
Irvine, California, U.S.A.
The data revealed a 3480 nucleotide (nt) contiguous
O R F in the region to which the DB129 gene was
previously mapped, transcribed from right to left with
respect to the standard orientation of the Colburn
genome (LaFemina & Hayward, 1980; Jeang &
Hayward, 1983). The nucleotide sequence and deduced
amino acid sequence are shown in Fig. 2. Three lines of
evidence indicate that this ORF encodes the complete
DB129 polypeptide. First, the O R F predicts a protein of
Mr 129005, in good agreement with the estimated Mr of
DB129 (Anders et al., 1986) and similar to the Mr of the
HSV major DNA-binding protein (Quinn & McGeoch,
1985). Second, as described below, sequence comparisons show similarity to the full length of herpesvirus
homologues. Third, the ORF is flanked by sets of
elements that probably define the structure of encoding
transcripts. Upstream there is a T A T A box homology
(TATAAA), at nt - 43 to - 38 relative to the predicted
translational start site, and several candidate regulatory
elements (discussed below). Immediately downstream of
the ORF, forming a portion of the termination codon, is
the polyadenylation signal A A T A A A (Proudfoot &
Brownlee, 1976); further downstream are several blocks
of GT-rich sequence (e.g. nt 3518 to 3522; additional
blocks are not shown) and another short consensus,
C A C T G (nt 3529 to 3533), present distal to the
polyadenylation signals of many genes (McLauchlan et
al., 1985; Berget, 1984). The most abundant transcript
detected by coding-region probes on Northern transfers
was estimated to be 3.9 kb in length (Anders & Gibson,
1988), consistent with the utilization of these flanking
signals. Although the cap site has not been determined,
upstream probes do not detect the 3.9 kb transcript, also
consistent with the use of the T A T A box at - 43 to - 38
(Anders & Gibson, 1988; D. G. Anders, unpublished
results). These data predict an unspliced m R N A with a
short 5' untranslated region, similar to the organization
of the HSV-1 ICP8 transcription unit (Rafield & Knipe,
1984; Su & Knipe, 1987). The first A T G of the ORF, at
nt + 1 to + 3, is likely to be the translational start codon
because (i) it is the first A T G after the putative T A T A
box at position - 4 3 to - 3 8 and, because transcription
usually initiates 19 to 27 nt downstream from the T A T A
sequence, this A T G is probably 3' to the cap site, (ii) it is
in a favourable sequence context (5' A C C A C C A T G A 33
to initiate translation (Kozak, 1987) and (iii) the aminoterminal amino acid sequence deduced from this site
shows significant similarity to that predicted for ICP8
and other herpesvirus homologues, as shown below.
Alternatively, or in addition, the next in-frame ATG, at
nt + 79 to + 81, also in a favourable context, may be
utilized to begin translation. More detailed transcript
mapping and peptide sequence analysis will be required
to establish the organization of the transcription unit and
confirm the predicted DB129 protein sequence.
The region from nt - 2 4 0 to - 6 functioned as an
orientation-specific promoter, albeit a weak one, in
transient assays using reporter constructs (D. G. Anders
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Short communication
& S. Punturieri, unpublished results). Inspection of this
sequence revealed several motifs which have been
implicated in regulating transcription in various systems.
At nt - 66 to - 59, immediately upstream of the TATA
box, is a close match (ATGACGTCT) to the cyclic AMP
response element (CRE) consensus (Montminy et al.,
1986) and an adjacent partial CRE (CGTCA). This
motif, recognized by the factor A T F / C R E B (Montminy
& Bilezikjian, 1987; Lee et al., 1987), is present in the
adenovirus E2 promoter, upstream of the HCMV 2.2 kb
early promoter described by Staprans et al. (1988), and in
multiple copies upstream of the CMV(Colburn) and
HCMV major immediate early genes (Hunninghake et
al., 1989; Chang et al., 1990). Situated further upstream
are two GC-rich regions at n t - 1 3 0 to -111 and at nt
- 189 to - 168, each of which contains the 7 bp sequence
GGCGGCG. A CCAAT box consensus was not found
within the 240 nt 5' to the ORF. However, between the
two GC-rich blocks, at nt - 157 to - 148, is the sequence
TCGCAAATCG, intriguingly similar to the octamer
binding site (Pruijn et al., 1987). Also present in this
region is a direct repeat of the 7 nt sequence GGACAGT
at positions - 112 to - 106 and -203 to - 197.
The deduced DB129 amino acid sequence shares
about 72% identity with its HCMV counterpart UL57
and the similarity is roughly collinear from the amino to
the carboxyl terminus (M. Chee, personal communication), consistent with their shared biochemical properties and immunological cross-reactivity (Anders et al.,
1986, 1987). The observed higher M r of the HCMV
protein may be accounted for by the presence in UL57 of
a glycine-rich 40 amino acid segment between residues
545 and 585 and two other short glycine-rich stretches
near the carboxyl terminus, which are absent in DB 129,
if the encoding sequences are not spliced out of the
transcript. Similarity matrices comparing DB129 with
alphaherpesvirus [HSV-1 ICP8; Quinn & McGeoch,
1985; varicella-zoster virus (VZV) gene 29; Davidson &
Scott, 1986] and gammaherpesvirus [Epstein-Barr
virus (EBV) BALF2; Baer et al., 1984) homologues also
revealed collinear nucleotide and amino acid sequence
similarity (not shown). Pairwise alignments made using
BESTFIT and GAP, which maximize the quality
statistic, substantiated the impressions conveyed by
homology matrices. BESTFIT alignment of DB 129 with
ICP8 yielded a quality of 502-5, alignment of DB 129 with
VZV gene 29 a quality of 500.1 and alignment of DB 129
with BALF2 a quality of 581.7, whereas alignment of the
two alphaherpesvirus proteins, ICP8 and VZV gene 29,
gave a quality of 1101.3. For comparison, alignment of
DB129 with HCMV UL57 yielded a quality of 1396.7,
and self-alignment of DB129 (i.e. a perfect match) gave a
quality of 1740. Similar results were obtained using
GAP, which does not truncate paired sequences. This
hierarchy of similarities is consistent with previous
studies comparing herpesvirus homologues (e.g. Chee et
al., 1989).
To resolve those regions most conserved during the
evolutionary divergence of alpha-, beta- and gammaherpesvirus major DNA-binding protein homologues,
their deduced amino acid sequences were compared in
multiple alignments generated using two different
methods. First, an iterative approach using BESTFIT
and GAP to make rounds of pairwise comparisons was
applied (not shown). Second, the CLUSTAL programs
(Higgins & Sharp, 1988) were used to produce a multiple
alignment (Fig. 3). Results obtained using either approach were similar. Inspection of the alignments
revealed clusters of residues conserved in all four
compared sequences. These segments, numbered I to
XII, are ungapped and were further defined on the basis
of arbitrary criteria (given in the legend to Fig. 3). Two
larger conserved regions, a and b, which contain a gap or
fell below the arbitrary standards, but which are clearly
more conserved than the mean, are enclosed by broken
lines. Adding HCMV UL57 to this alignment caused no
significant changes in the conserved regions defined by
these criteria (data not shown). Together, the identified
segments contain 51 of a total of 91 amino acids
conserved in all four aligned proteins, or 56%, within a
combined 130 residues (11.2% of the shortest aligned
sequence). If regions a and b and the carboxyl terminus
proximal KR sequence (boxed, discussed below) are
included, the corresponding numbers become 69-2% of
conserved residues in 16.4% of the shortest aligned
sequence. The density of identical residues (fraction
identical/fraction total) is about fivefold higher in the
conserved segments than in the whole sequence, and
about 10-fold higher than that of the excluded (i.e.
unboxed) sequence.
Several laboratories have investigated the functional
organization of the prototype homologue HSV-1 ICP8
and it is of interest to consider their results as regards this
alignment. ICP8 mutants missing as few as 36 residues of
the carboxyl terminus fail to accumulate in the nucleus
although they still bind to ssDNA in vitro (Gao & Knipe,
1989); the conserved sequence Lys-Arg (Fig. 3), along
with a nearby Pro, may form all or part of a nuclear
localization signal. Leinbach & Heath (1988, 1989)
showed that fragments of ICP8 made using in vitro
transcription and translation, containing either residues
571 to 1196 or 332 to 564, can bind ssDNA in vitro.
Consistent with those results, truncated ICP8 proteins,
produced by the amino-terminal region deletion mutants
dl01 and d102, efficiently bind ssDNA in vitro but fail to
support DNA replication (Gao & Knipe, 1989). Temperature-sensitive mutants tsHA1 and tsl3, reported to
show thermolabile DNA-binding, mapped to ICP8
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Short communication
__i
l
CMV DB129
EBV BALF2
HSVI DBP
VZV GENE 29
M. . . . . SNEELSALAPVGPAAYVYF---TKTNHEMNEVLATLSLCDSSSPVVIAPLLMGL
MQGAQTSEDNLGSQSQ~PcGYYIV~yY~--YPLATyPLREV-ATLGTGYAGHRCLTV~
METKPKTATTIKV--P GPLGYVY RACPSEGIEL'--LALLSARSGDSDVAVAPLVVGL
CMV DB129
EBV BALF2
HSV] DBP
VZV GENE 2g
TVE~IEANVAVVVGSRTTGLGGTAV$LKLTPSHY$$$VYVFHGGRHLDPSTQAPN--LT
CMV 08129
EBV BALF2
HSV] °BP
VZV GENE 29
TVDQ~CTSV. . . . . .
RTPVVCYDGGVLTKVTSFCPFALYFYNTQGIVD--FSEPHGDVQ
TVEPG SINVKALH-RRPDPNC-- -GLLRATSYHR-DIYVFHNAHMVPPIFEGP--GLE
**TVEKTITSSLAVVSGARTTGLAGAGITLKLTTS
*HFYP
.S
. *VFVFHGG
. KH
. VL.PSS
. AA.PN-'.LI. .
CMV DB129
EBV BALF2
HSVI DBP
VZV GENE 29
LIPCFEEAAPTRLNDCDAV~GSLQADVDSDELSLDKRS-LVAIPSLKQEVAVGQSASVIRVPLYDKEVFIPEGVPQL ...............
CMV DBI29
EBV BALF2
HSVI DBP
VZV GENE 29
PAVSETLFYYVYTSWC~-~FSETKVLIEAALKQFVNDSQQSVKLAPHKKYFGY
CMV DBI29
EBV BALF2
HSVI DBP
VZV GENE 29
.............
CMV DBI29
EBV BALF2
HSVI DBP
VZV GENE 29
CMV DB]29
EBV BALF2
HSV] DBP
VZV GENE 29
. *
.
Ill
RLCDETRQRYAIESY--MPEEGRAPTDLAALCTAAGCDPQEVLVHVVVGNGMKEFMYAGQ
ALCGETREVFGYDAYSALPRE$SKPGDF . . . . FPEGLDPSAYLGAVAITEAFK[RLYSGN
RLCERARRHfGFSDYTPRPGDLKHE33GEALCERLGLDPDRALLYLVVIEGFKEAVCINN
RACNAARERFGFSRCQGPPVDGAVETTGAEICTRLGL~PENTILYLVVIALFK[AVFMCN
SFVESRGLYV
RQFYN
......
SDLSRCMHEALYIGLA~ALR~RRVGKLVELLEKQSLQDQAKVAKVAPLK---EF . . . . . .
RPLNRLLFEAVVGPAAVIAL~RNVDAVARAAAHLAFDENHEGAALPADITFTAFEASQGTGLCHLIHDCVIAPMA
. . . .
,~,L,RRNVTAVARGAAHLAFDENHEGAVLPPDITYTYFQSSSSG
.
.
.
.
.
.
.
.
.
.
.
.
CMV 0 B 1 2 9
EBV B A L F 2
HSV1 DBP
VZV GEN£ 29
HOLSAL FTAASGEAVGNSVGLNARGGEHAFDED ........
CG . . . . . . .
L L P/~GRLQDNF | $ V A E P V S T A S Q A S A G L L L G G G G Q G S G G R - - - RIk~RLAT . . . . . . .
VLPGLE °°V"
GEVFNFGDFGCEDDNATP . . . . .
FGGPGAPGPAFAGI~KI~AFHGDOPFG- EG PPDKKGDLT
NLAFNFD--SCEPSHDTTSNVLNISGSNISGSTVPG~PPEDDELFDLSGIPIKHGNIT
CRY D B I Z 9
EBV BALFZ
HSV] DBP
VZV GENE 29
....
....
LDML
MEHI
Fig. 3. Multiplealignment of DB129, EBV BALF2, HSV-1 ICP8 and
VZV gene 29 performed using the CLUSTAL package (Higgins &
Sharp, 1988)with default parameters. Residuesthat are identical in all
aligned sequences are indicated with an asterisk below and where only
conserved substitutions have occurred a period appears. Conserved
segments, boxed and numbered I to XII, contained no gaps and met
one of the following criteria: two or more adjacent conserved residues
(i.e. asterisks), three conserved residues in 10, four conserved residues
in 15, or five conserved residues in 20.
TSQKLSSLEKDHLMLSDAVICELGFSFASVFLDSAYGASDSMVYsEVw~
.............
PASTISHPDSGALMIVOSAACELAVSYAPAMLEASHETPASLNYDSSW
-KTPRGGRDGGGKGAA . . . . . GGFEQRLASVMAGDAALALESIVSMAVFDEPPI OISA]W
VII
VVVNATDHRDLIRALTELKLHLSTHISAL~HNRLVYLTSNKNASGTGASQf
LFADCEGPEARVAALHRYNASLAPHVSTQI]fATNSVLIYVSGV . . . . . . . . SKSTGQGKE
LFEGQDTAAARANAVGAYLARAAGLVGAM~FSTNSAL~LTEV---DDAGPADPKDHSKMFIGMEGTLPRLNALGSY
. TARV
. AGV.IGAM~
. !P~A~
.I!LTEV.---EDS.GMTE
.AKDG
. GPG
.,
.
.
.
.
I
VLLKSIHFANGLTGLCEOTYNDARKLIKC . . . . . . $GVVAKDER--YAPYHLSLICGTCP
$LFNSFYMTHGLGTLQEGTWDPCRR--PCFSGWGGPDVTGTNGpGNYAVEHLVYAASFSP
PSFY'RffLVPGTHVAANPQVDREGHV~PGFEGRPTARLVG:-GTQEFAGEHUAMLCGFSP
PSFNRFYQFAGPHLAANPQTDRDGHVLSS . . . . . . QSTGS~-SNTEFSVDYLALICGFGA
IX
" "
NRVSVYNTGLTGSSTLSNHLIGCSSS-L~ICGACGGTCCHTCYNTAFVRVQ
QFCQGQKSSLTPVPETGSYVAGAAASPM~CSLCEGRAPAVCLNTLFFRLR
ERCDGAVIVGRQEMDVFRYVAOSNQTDV~CNLCTFDTRHACVHTTLMRLR
CMV DBI2g
EBV BALF2
HSVI DBP
VZV GENE 29
CMV DB129
EBV 8ALF2
HSVI DBP
VZV GENE 29
TR-R~'P~MPRLPKKEPSVVVMQSgFLNO~FGRRYSAESKEASLDAKADEGSASTSN9
DgF~PVMSTQRROPYVISGASGSYNE~DFLG~F . . . . . . . .
LNFIDKEDDGQRPDDEP
ARHP FASAARGAIGVFGTMNSMYSDC
YAA-FSA. . . . . LKRA-DGSETARTIM
~ ! ~ F G Q A ' RQPI GVF~TMNSQYSD~,DDVp~YAg-YLI ......
LRKPGDQTEAAKATM
CMV DB]29
EBV BALF2
H$V] DBP
VZV GENE 29
TA$SSVDRTHRLNRILDYCKKMR[IOSVTGEDTMTINGRSDFINLVSSLNKFVDDEAMSF
RYTYWQLNONLLERL . . . . . . $RLGIOA[GKIEKEPHGPRDFVKMFKDVDAAVDAEVVQF
QETYRAATERVMAELETLQYVDQAVPTAMGRLETIITNREALHTVVNNVRQVVDREVEQL
QDTYRATLERLFIDLEOERLLDRGAPCSSEGLSSVIVDHPTFRRILDTLRARIEQTTTQF
CMV D9129
EBV BALF~
HSVI DBP
VZV GENE 29
MNSM-AKNNITYKDLVKSCYHVMQYSCN~FAQPACPIFTQLFYRSLLTILQDISL~ICMC
MRNLVEGRNFKFRDGLGEANHAMSLTLD~YACGPCPLLQLLGRRSNLAVYQDLAL~QCHG
MKVLVETRDYKIREGLSEATHSMALTFD~YSGAFCPITNFLVKRTHLAVVQDLAL~QCHC
CMV DB]29
EBV BALF2
HSVI DBP
VZV GENE 29
YIVDNPLTTSLVSRWVTQHFQSIHGAFSTTSSRKGFLFIRNVKSSKNADHDRLPDFKLYA
YENONPGLGQSPPEWLKGHYQTLCTNFRSLAIDKGVLTAKEAKVVHGEPTCDLPDLDAAL
VFAGQSVEGRN. . . . FRNQFQPVLRRRVMDMFNNGFLSAKTLTVALSEGAAIEAPSLTAG
VFYGQQVEGRN. . . . FRNQFQPVLRRRFVDLFNGGFISTRSITVTLSEGP-VSAPNRTLG
CMV DBIZ9
EBV BALF2
HSVI DBP
VZV GENE 29
RGTYSVISMEIKLSRLSVPSLLMFRVKNRPISKASKG . . . . . . . . .
TTAHVFFRREHV
QGRVYGRRLPVRMSKVLMLCPRNIKIKNRVVFTGENA . . . . . . . . .
ALQNSFIKSTTR
QTAPAESSFEGDVARVTLGFPKELRVKSRVLFAGASANASEAAKARVASLQSAYOKPDKR
QDAPAGRTFDGDLARVSVEVIRDIRVKNRVVFSGNCTNLSEAARARLVGLASAYQRQEKR
CMV DB]29
EBV BALF2
HSV] DBP
VZV GENE 29
PKKNPV~GCLGFLLYKYHDKLF~DCGFSCLQ . . . . . . FWQKVCANALP-KNVNIGDMGEF
RENYII GPYMKFLNTYHKTLF DTKISSLY. . . . . . LWHNFSRRRSV-PVPSGASAEEY
VD--IL~GPLGFLLKQFHAAIFINGKPPGSNQPNPQWFWTALQRNQLPARLLSREOIETI
VD--ML~GALGFLLKQFHGLLF~RGMPPNSKSPNPQWFWILLQRN.Q
* MPADKLT.HEE
.ITTI..,.. . . .
CMV DB129
EBV BALF2
HSVI DBP
VZV GENE 29
b
NNFVKFVI SVTADYNEHDL~DV~CC MLNYL~ RFHNKF~F~GFKD£Y~GTLHGLTTRL
SDLALFVDGGSRAHEESNVdDVVPGNLVTYAKQRLNNAILKACGQTQFYIISLI'QGLVPRT
AFIKKF . . . . SLDYGAINFqNLAPNNVSELAMYYMANQILRYCDHSTYFIINTLTAIIAGS
AAVKRF. . . . TEEYAAIN~INLPPTCIGELAQFYMANLILKYCDHSQYLI~NTLTSIITGA
CMV 0B129
EBV BALF2
HSV] DBP
VZV GENE 29
TYQNHAQFPYLLGESPNFASAADfALRLKOLKATGVTAPL--ASTVTRESLMRTIFE~
QSVPARDYPHVLG-TRAVESAAAYAEATSSLTATTVVCAA--TDCLSQ
VCK~RP
RRPPSVQAAAAW. . . . $AQGGAGLEAGARALMOAVOAHPGAWTSMFASCNLLRPVMA~RP
RRPRDPSSVLHW-IRKOVTSAAOIETQ
.A
.*
KA.LLE.KTE
. NL.PELW
* TT
.AFTSTH
.LVRAAM
. NQR.~P
..
.
.
*.
VSEV-9MKSN--RDEVLGATQAFNLOEN~FAVSFS~ILAY~Y~]~
. . . . . . . . . . . . . . . . . . . .
.
•
CMV DB129
EBV BALF2
HSV] DBP
VZV GENE 29
2455
.
.
*
.
.
ki
. . . . . .
.
.
.
C,_,_*:
.
*"
_._._
~.-
.
_
.
.
_ :_*_~_
-
-
-~
.
-.:._*~._.3
.
~
~QNVROITATS
.
~__3
.
.
.
.
.
.
.
.
.
XII
LVIVSFSIEKYAGVNNNKEIYQFGQIGYFS~NGVERSLflTNSIGG ~
QDYKFMRQRC]
VVTLPVTINKYTGVNGNNQIFQAGNLGYFM~RGVDRNLLQAPGAGLRKQAGGSSMRKKFV
MVVLGLSISKYYGMAGNDRVFQAGNWASLM~GKNACPLLIFDRI . . . . . . . .
RKFV
M~!o,!~!~.~!~Ng!~!~!~!.~----~'o~T. . . . . . . . . . . .
!!~!
CMV DBI29
EBV BALF2
HSVI DBP
VZV GENE 29
LATKLSDVLIKRSRRDNVLFDEDIIKNRVMAALDSENLDVDPELMAMYEILSTREEIPEFATPTLGLTVKRRTQAATTYEIENIRA-GLEAIISQKQEEDCVFDVVCNLVDAMGEACAS
LACPRAGFVCAASSLGGGAHESSLCEQLRGIISEGGAAVASSVFVATVKSLGPRTQQ--IACPRGGFICPVTGPSSGNRETTLSDQVRGIIVSGGAMVQLAIYATVVRAVGARAQH---
CMV DBI29
EBV BALF2
HSVI DBP
VZV GENE 29
LTRCDAEYLLGRFSVLAOSVLETLATIASSGIE-WTAEAARO--~FLEGVW---GGPGAA
LQIEDWLALLED-EYLSEEMMELTARALERGNGEWSTDAALE---VAHEAEALVSQLGNA
MAFDOWLSLTDD-EFLARDLEELHDQIIQTLETPWTV[GALEAVKILDEKTTAGDGETPT
--RODVLFFVOGCQAVAOSLM[KFSRLQEMGVDDFSLVNLQQ--*VLDSRPECGGGGGEV
positions 348 and 450, respectively (Gao et al., 1988). I
note that these substitutions occurred proximal to, but
not at, conserved positions. Mutant n2, in which the
carboxyl terminus of ICP8 is truncated to residue 1029,
deleting 163 residues but retaining conserved segment
XII, still binds to s s D N A avidly (i.e. in 0.3 M-NaCI),
though less efficiently than the wild-type protein (Gao &
Knipe, 1989). Site-specific alteration of two cysteine
residues in the zinc-finger-like sequence within conserved region IX also greatly reduced in vitro s s D N A binding (Gao et al., 1988; Gao & Knipe, 1989). Finally, a
56K fragment of ICP8 produced by limited proteolysis,
the amino terminus of which is position 300, bound
s s D N A in vitro (Wang & Hall, 1990). The latter authors
also suggested a possible ssDNA-binding motif, which
includes the segment identified here as conserved
segment XI. Together, the above results indicate that the
ssDNA-binding domain, or domains, resides within
residues 348 to 1029, spanning the conserved segments
VI to XII. As conserved residues of homologous proteins
often form inter- and intramolecular contacts (e.g. Pabo
et al., 1990), the results further imply that some subset of
residues within one or more of these conserved segments
form the ssDNA-binding site(s) and contact D N A .
A full understanding of the interaction between this
ssDNA-binding protein and its nucleic acid substrate
awaits determination of its three-dimensional structure
in co-crystals. In the meantime, the identified conserved
segments and other conserved residues suggest themselves as targets for site-specific mutagenesis in structure-function analyses of this multifunctional herpesvirus group-common protein. Carefully chosen
substitutions might be expected to inactivate selected
functions and thus have the potential to reveal important
details of the protein's interaction with D N A and with
other elements of the herpesvirus D N A replication
apparatus.
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2456
Short communication
I thank Wade Gibson for making available virus strains and cells as
well as for advice and encouragement, Louise Belensz and Suzanne
Punturieri for technical assistance, Mark Chee for sharing sequence
data before publication, Ivan Auger for help with sequence alignments
and using GCG, and Paul Masters for helpful criticisms of the
manuscript.
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(Received 17 April 1990; Accepted 25 June 1990)
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