M. D. 160. ORF R. IOP. N. ElI. 00K. LE K KY. K G. H. LOIG. F. H. __. 180 pol. *. V ...... from our colleague Bob Honess, who tragically died last year. .... B. E. Griffin.
JOURNAL OF VIROLOGY, Sept. 1991, p. 4670-4680
Vol. 65, No. 9
0022-538X/91/094670-11$02.00/0 Copyright © 1991, American Society for Microbiology
Characterization of the DNA Polymerase Gene of Human Herpesvirus 6 IAN A. TEO, BEVERLY E. GRIFFIN, AND MICHAEL D. JONES* Department of Virology, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, England Received 8 March 1991/Accepted 21 May 1991
The construction of a recombinant bacteriophage A library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pot gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the a and y herpesvirus families, lending further support to the notion that HHV-6 is a member of the herpesvirus group. The recently discovered human T-cell lymphotropic herpesvirus, human herpesvirus 6 (HHV-6) was first isolated from patients suffering from AIDS-related diseases (15, 51, 57). Serological analysis indicate that more than 60% of the population have antibodies against the virus by the age of 1 year, with the infected individuals carrying the virus in a latent form for the rest of their lives (9). The virus is thought to be transmitted orally, with the reported site of latency being the parotid glands (20, 38, 47). HHV-6 has been identified as the agent of the childhood disease roseola infantum (33, 62) and is associated with fatal hemophagocytic syndrome and some lymphoadenopathies in adults (27, 44). Recently, Razzaque (50) reported that HHV-6 DNA can induce transformation and tumor formation in a model system. Josephs et al. (31) and Jarrett et al. (30) demonstrated the presence of HHV-6 DNA sequences in some lymphoma tissue, although these appeared to be relatively isolated cases. The highly cytopathic effect of HHV-6 in tissue culture, coupled with the ability of the virus to reactivate in transplant cases (4, 59, 61), may point to important clinical implications for immunosuppressed individuals in general. In vitro experiments show that HHV-6 can transactivate the human immunodeficiency virus long terminal repeat linked to a reporter gene, enhancing HIV gene expression and CD4+ cell death in cultures coinfected with both viruses (1, 18, 26, 40). It is interesting that HHV-6 is easily isolated from the peripheral blood lymphocytes of AIDS patients, whereas it can be detected in normal peripheral blood lymphocytes only by polymerase chain reaction (PCR) analysis (23). Whether HHV-6 contributes to the catastrophic decline of CD4+ cells seen in the later stages of AIDS remains to be proved. Neither the range of clinical manifestations of HHV-6 infection nor the appropriate therapeutic treatment has been fully established. Some preliminary data indicate that the virus is sensitive to the effects of acyclovir and azidothymidine in tissue culture systems (10). Since the virus may have a role in the pathogenesis of AIDS and other immune dysfunctions, the characterization of HHV-6 DNA polymerase is important in terms of investigating potential antiviral *
Corresponding author. 4670
agents and establishing the relationship between the DNA polymerase of HHV-6 and those of other members of the human herpesvirus family. Commensurate with this exercise, we have recently completed the cloning and restriction site mapping of the 161-kbp HHV-6 genome from the Ugandan isolate U1102 (42; see below). With consensus primers corresponding to conserved amino acid residues of the DNA polymerase sequences in other herpesviruses, the corresponding region from HHV-6 DNA has been amplified in a PCR. Using the product generated as a probe has allowed the DNA polymerase gene to be identified, positioned on the viral genome, and isolated from the lambda libraries. We report here the analysis of the HHV-6 DNA polymerase gene. (Part of this work was presented in preliminary form at the XVth International Herpesvirus Workshop, Washington, D.C., 1990.)
MATERIALS AND METHODS
Cell culture. HHV-6 (Ugandan isolate U1102) was propagated in phytohemagglutinin (1 ,ug/ml)-stimulated cord blood mononuclear cells (CBMC) or in the T-cell line HSB-2, as described previously (15). The Gambian HHV-6 isolate AJ was propagated in J.Jhan cells (57). Viruses were propagated via cell-cell contact or from infective media previously filtered through Millipore filters (pore size, 0.45 ,um). Construction and mapping of libraries. Total genomic DNA was prepared from CBMC infected with HHV-6 U1102 (15) and partially digested with Sau3AI, and the ends were repaired with dGTP and dATP. Fragments size selected (9 to 20 kbp) on agarose gels were ligated to the XhoI restriction enzyme site, partially filled in by using dCTP and TTP, of the A replacement vector AFIX, as specified by the manufacturer (Stratagene). The DNA was packaged into phage particles in vitro (Amersham packaging system) and then plated on selective Escherichia coli P2-392 host cells. A library was also constructed in the insertion vector XgtlO (28), by using EcoRI-digested total genomic DNA from HHV-6-infected CBMC. Growth, amplification, and screening of the libraries were carried out by standard techniques (2, 41). HHV-6 DNA containing plasmid clones pHD5 (17), pSAD1, pSAD2a, pHD12, and pHD9 (from B. Honess) and pZB70
VOL. 65, 1991
HHV-6 DNA POLYMERASE GENE
and pZH14 (32) (from R. Gallo) were used as probes to screen the libraries and confirm the alignment of a series of overlapping XFIX clones. DNA from these A clones was restricted, subcloned into the plasmid vector Bluescribe (pBS; Stratagene), and in turn used to probe for isolation of additional overlapping A clones. All cloned HHV-6 DNA was hybridized to Southern blots of HHV-6 genomic DNA digested with either Hindlll, BamHI, Sall, EcoRI, or XhoI to confirm and generate correct map alignments. PCR amplffication of HHV-6 pol gene probe. For each PCR amplification, 1 ,ug of total genomic DNA from cells infected with U1102 or AJ or control cells was used together with 1 to 3 ,ug of each primer (for the nucleotide sequence of primers, see Fig. 2), 2.5 U of Taq DNA polymerase (Amplitaq; Cetus), and 200 ,uM deoxynucleoside triphosphates in PCR buffer (10 mM Tris.HCl [pH 8.5], 50 mM KCl, 2 mM MgCl2) with or without 15% glycerol, in a final volume of 50 ,ul. All DNA samples were boiled for 5 min and then rapidly cooled on ice before the PCR. PCR amplification was carried out in two stages. In the first, the reaction mixes were subjected to denaturation at 94°C for 30 s, 37°C for 1 min, and 72°C for 2 min for five cycles, and in the second, the samples were cycled at 94°C for 30 s, 55°C for 1 min, and 72°C for 2 min for 30 cycles. PCR amplification products were analyzed by electrophoresis on 1.2% agarose-TBE gels (41). Cloning and DNA sequencing of the pol gene. A 510-bp PCR product was excised from an agarose gel and labeled with 32p by the random-primer method of Feinberg and Vogelstein (19). This probe was used to screen the AFIX and AgtlO libraries. Insert DNA from the probe-positive AgtlO clone RPMS4.4 was subcloned into pBS. The isolated 4.4-kbp DNA fragment was shotgun cloned into M13 phage vectors by using randomly generated restriction enzyme fragments, as well as DNase-digested fragments (3, 52). M13 clones were sequenced by the technique of Sanger et al. (6, 53) with Klenow DNA polymerase or Taq DNA polymerase (TAQuence; United States Bio-Chemicals). Both strands of the DNA were sequenced, with each position being determined approximately two to five times. For certain regions, specific oligonucleotide primers, based upon the sequence data, were synthesized and used in sequencing reactions to enable both strands of the DNA to be determined. The PCR fragment probe was also cloned into M13 vectors and sequenced. The final sequence was analyzed on an IBM AT computer with the Pustell-IBI Software programs (48, 49) and the Multalign program (11) and on a Microvax with the AMPS suite of programs (7, 8). Mapping of the DNA polymerase gene. [32P]pRPMS4.4 DNA was used to probe Southern blot digests (BamHI, HindIlI, EcoRI, SalI, or XhoI) of HHV-6 U1102 DNA and AFIX recombinant cloned DNA. Where necessary, appropriate double-enzyme digests were used to derive a fine map of the polymerase gene region. Nucleotide sequence accession number. The DNA sequence presented has been submitted to the EMBL and GenBank data bases under the accession number M63804. RESULTS
Mapping the HHV-6 U1102
library, in the replacement vector XFIX, was thus constructed by using total genomic DNA prepared from HHV6-infected CBMC. This library contained approximately 200,000 individual recombinant X clones. It was screened with the HHV-6 plasmid probe pHD5 (17, 36). Almost 0.5% of the clones were positive with this probe. From the size of
the inserts in the XFIX clones and the HHV-6 genome, it is was estimated that around 5% of the clones in the library should contain HHV-6 sequences, which would correspond to a minimum of 1,000 copies of the virus per infected cell. A similar-sized library was also prepared from total genomic DNA digested to completion with the restriction enzyme EcoRI and cloned into the vector AgtlO. Screening the library with the probe pHD5 resulted in the isolation and characterization of the overlapping XFIX clones HD5.14, HD5.18, and HD5.12 (Fig. 1). XFIX clones are named after the plasmid probe used in screening the library with a number for the individual phage clones isolated. The use of DNA restriction fragments from the ends of clones HD5.14 and HD5.12, and subsequent rescreening of the library, gave A clones 14W2 and 12W4 (the designation W indicates that the clones were derived from a genomic walk). Further probing of the library with DNA fragments from clone 12W4 yielded the clone 12W9. Clone 14W2 was found to overlap with clone SAD2A12, isolated by probing with plasmid pSAD2a. This procedure generated overlapping clones covering ca. 67 kbp of DNA. Probing the library with plasmid clones pSADl and pZB70 resulted in the isolation and ordering of clones SAD1.2, ZB70.7, and ZB70.6 covering ca. 34 kbp. Similarly, the use of plasmids pZH14 and pHD12 resulted in the isolation of clones Z14.3 and Z14.1 (covering ca. 20 kbp) and clones HD12.6 and HD12.9 (covering ca. 19 kbp), respectively. Further attempts to complete the library by these procedures were not successful. This we attribute to the fact that certain sequences were either unstable in the original library or underrepresented (or lost) during differential amplification of the clones. Specific screening of the XgtlO library to overlap the isolated XFIX clones yielded the clones RPMS3.8, RPMS5, RPMS4.4, and RPMS3.5 (Fig. la). There is no overlap between the two clones RPMS3.8 and RPMS5 (at position 42 kbp [Fig. la]), but restriction map analysis indicates that the two EcoRI fragments are apparently contiguous. The probe used for the isolation of RPMS4.4 was generated by PCR, and the EcoRI fragment was found to be within clone Z14.1 (see below). Clone RPMS3.5 allowed linkage between clones Z14.1 and SAD2A12. From neither library were we able to isolate clones covering coordinates 132 to 134 kbp. This region has therefore been isolated by PCR amplification (42). The DNA at this position contains an array of simple repeat sequences, which may explain the instability of this region in plasmid and A vectors. The termini of the linear genome were not found in our libraries. However, owing to the nature of the directrepeat sequences, only approximately 2 kbp of sequence was missing within the 10-kbp repeat sequences (Fig. 1), Martin et al. (42) have cloned these missing sequences in plasmid vectors.
libraries. To generate an extensive and convenient library of the HHV-6 genome, we have adopted a strategy for preparing recombinant clones in a K vector. By partial digestion of the target DNA, followed by size selection and then cloning, it was expected to create an overlapping library of fragments, from which the whole genome could be isolated. A recombinant genome in
4671
X
Many XFIX clones besides those mentioned above were isolated and located onto the physical map, confirming data from the representative clones shown in Fig. 1. The Sau3AI ends of the HHV-6 inserts in the XFIX clones are fused to a XhoI restriction enzyme sequence of the XFIX vector, and so it has not been possible to delineate the authentic ends of the inserts in some of the recombinant clones.
~I
J. VIROL.
TEO ET AL.
4672
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FIG. 1. (a) Restriction endonuclease map of the HHV-6 genome showing the XhoI, EcoRI, BamHI, HindIII, and Sall restriction sites. Below the restriction sites are the locations of the clones (double-headed arrows) and their designations, named after the plasmid probe used for screening. The locations of the terminal direct repeat sequences (DRI and DRr [shaded boxes]) and the known HHV-6 genes, pol (DNA polymerase), gpB (glycoprotein B), mcp (major capsid protein), and exo (exonuclease), are shown. (b) Restriction map of the RPMS4.4 clone sequenced showing EcoRI, XbaI, BglII, XhoI, PstI, and Sall restriction sites and the location of the DNA pol gene. The arrangement of the ORFs is shown below the map. (A)n refers to AATAAA sequences present in the sequence, and TATA refers to the putative promoter region of the pol gene. These are positioned above and below the restriction map to indicate rightward and leftward transcriptional and polyadenylation signals, respectively.
Within the limits of the methods, the map presented in Fig. la is accurate, although it contains some uncertainties, particularly in the sizing of DNA fragments and the order of some of the smaller fragments. The map is in excellent agreement with that of Martin et al. (42). PCR amplffication of the probe for the pol gene. Nunberg et al. (45) have shown that PCR amplification with degenerate oligodeoxynucleotide primers, derived from conserved amino acid sequences, allows the amplification of the homologous region from a related gene. They used the approach to
clone a probe for the thymidine kinase gene of feline herpesvirus. We have used the same strategy to generate probes to map and isolate the gene for the DNA polymerase gene of HHV-6: the DNA sequences of the pol genes from five human herpesviruses are known, and a comparison between them and other DNA polymerases, including human polymerase ax and vaccinia virus DNA polymerase, has highlighted conserved regions of the DNA polymerases involved in substrate recognition and/or catalytic activity (25, 35, 60). Figure 2 shows the amino acid sequences for
VOL. 65, 1991
HHV-6 DNA POLYMERASE GENE a
Region II HCMV EBV
vzv HSV-1 HSV-2
6POL1 6POL2 HHV-6 DNA
4673
PheAspPheAlaSerLeuTyrProSerIleIleMet ValAspPheAlaSerLeuTyrProSerIleIleGln LeuAspPheAlaSerLeuTyrProSerIleIleGln PheAspPheAlaSerLeuTyrProSerIleIleGln PheAspPheAlaSerLeuTyrProSerIleIleGln 5'-tcaaagctTAYCCNTCNATHAT-3' (96) 5'-tcaaagctTAYCCNAGYATHAT-3' (48) PheAspPheGlnSerLeuTyrProSerIleMetMet
-
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5'-TATCCGAGCATTAT-3'
341
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Region III HCMV EBV
vzv HSV-1
HSV-2 6POL4 HHV-6 DNA
CysAsnAlaPheTyrGlyPheThrGlyValValAsn CysAsnAlaValTyrGlyPheThrGlyValAlaAsn CysAsnSerValTyrGlyPheThrGlyValAlaGln CysAsnSerValTyrGlyPheThrGlyValGlnHis CysAsnSerValTyrGlyPheThrGlyValGlnHis
(128) 5'-tcaaagcttGGNTTYACNGGNGT-3' CysAsnSerValTyrGlyValThrGlyAlaAlaHis 5'-GGTGTCACGGGAGC-3'
Region I HCMV EBV
VZV HSV-1
HSV-2 6POL5 HHV-6 DNA
AlaArgValIleTyrGlyAspThrAspSerValPhe LeuArgValIleTyrGlyAspThrAspSerLeuPhe ValLysValIleTyrGlyAspThrAspSerValPhe MetArgIleIleTyrGlyAspThrAspSerIlePhe MetArgIleIleTyrGlyAspThrAspSerIlePhe 3'-TADATRCCNCTRTGcttaagctc-5' (48) ValGluValIleTyrGlyAspThrAspSerIlePhe 3'-TAAATACCACTATG-5'
FIG. 2. Conserved amino acid sequences of human herpesviruses from regions I to III (25, 35, 60). Below the aligned sequences of HCMV, EBV, VZV, HSV-1, and HSV-2 are given the nucleotide sequences of the degenerate oligodeoxynucleotide primers (the number in parentheses at the left refers to the number of individual sequences present in the nucleotide mixture) and the amino acid sequence of the corresponding region from HHV-6 pol. In the oligodeoxynucleotide primers the symbols are as follows: R, A and G; Y, C and T; H, A, C, and T; D, A, G, and T; N, A, G, C, and T; lowercase letters, sequences of the restriction enzyme site extensions. The lines designated DNA give the actual sequence of the regions in HHV-6 that corresponds to the degenerate primers.
three highly conserved regions of the five human herpesvirus DNA polymerases. It was predicted that these sequences should be present in the HHV-6 DNA polymerase gene and would therefore be ideal targets for the design of degenerate oligodeoxynucleotide primers (25, 35, 60). The primers were synthesized covering all the possible DNA sequences encoding the amino acid sequences, as follows. Primers 6POL1 and 6POL2 (containing a mixture of 96 and 48 individual sequences, respectively) covered the sixfold redundancy of the codons for serine and were used separately in PCR amplifications. 6POL4 and 6POL5 each contained a combination of 128 and 48 unique sequences, respectively. The primers also had 5' extensions containing recognition sequences for the restriction enzymes EcoRI and HindIII, to allow for subsequent cloning of PCR-amplified products. The predicted sizes of the amplification products for the DNA polymerase of human cytomegalovirus (HCMV) are 586 bp (6POL1 or 6POL2 with 6POL5) and 295 bp (6POL4 with 6POL5), and those for Epstein-Barr virus (EBV) are 520 and 220 bp, respectively, including the primer extensions (5, 34). The results of PCR analysis of total genomic DNA from cells infected with HHV-6 U1102 isolate compared with data from the EBV and HCMV pol genes, are shown in Fig. 3. The PCR amplifications all used primer 6POL5 and either 6POL1, 6POL2, or 6POL4. We were able to amplify a band
FIG. 3. Agarose gel analysis of PCR amplification products (see Materials and Methods). (a) HHV-6 DNA amplifications. Lanes: 1, primer pair 6POL1 and 6POL5; 2, 6POL2 and 6POL5; 3, 6POL4 and 6POL5. (b) EBV pol cosmid PCRs (lanes 1 to 3), HCMV pol plasmid PCRs (lanes 4 to 6), and HHV-6 total genomic DNA (lanes 7 and 8). Primer pairs used were as follows: lanes 1 and 4, 6POL1 and 6POL5; lanes 2, 5, 7, and 8, 6POL2 and 6POL5; lanes 3 and 6, 6POL4 and 6POL5. Lanes 1 to 7 contained 15% glycerol, and lane 8 contained no glycerol. The extreme left- and right-hand lanes contain Bluescript size markers, generated from an equal mixture of plasmid Bluescript KS digested with TaqI and Sau3AI.
of ca. 510 bp (closer to EBV than HCMV) with either 6POL1 or 6POL2 from HHV-6-infected cells (Fig. 3a, lanes 1 and 2), but were unable to direct amplification with primer 6POL4 (lane 3). The increased yield of PCR product with primer 6POL2 compared with 6POL1 suggested a greater hybridization efficiency of 6POL2 and indicated that the serine codon used at the primer site would be AGY, not TCN (Fig. 2). Subsequent sequence analysis showed the codon to be AGC (see below). The failure to obtain any amplification with primer 6POL4 was explained by the DNA sequence of region III; the nucleotide differences between the target template and primer would prevent, or drastically reduce, correct annealing and thus prevent amplification (Fig. 2). The mismatch at the 3' end of the primer, coupled to the degeneracy of the sequence at the other positions, would appear to inhibit amplification, despite reports that a T. G
a
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FIG. 4. Southern blot of HHV-6 DNA probed with pRPMS4.4. (a) Digests were carried out as follows: lane 1, HindlIl; lane 2, BamHI; lane 3, SalI; lane 4, EcoRI; lane 5, XhoI. Lane 3 has been exposed longer to compensate for poorer transfer of high-molecularweight DNA. The correct hybridizing bands are arrowed. (b) Comparison of strains U1102 (lane 1) and AJ (lane 2) digested with EcoRI. The positions of X HindIll size markers are indicated to the left and right of the gels, respectively.
4674
TEO ET AL.
J. VIROL.
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1202
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ATGCAAACGACTTACTCTACGTTTCGAAGGACTGGACTCAACGATGTCGCCAACAACCACATCTTCAATCTTTATATGTCACAAACGACAACACA P
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AGAGACACATTAATTCTCCGTTGCCGATCTGAAAACTCCCCCCGCTCGCCAGCGACCAAATTTATGATACCACGGCATCCTGTTACGAACTT S S L M L H R V D A R N Q F L E E R A K C L R R L 0 K H 0 G V P V G 0 K R L Q T
10808
pal
CTGTTCAGTGAAAACTCCCAGAGCATCTAACCCTTATCAAGACAACAGTCACATATTTTACCAACCTTCAAAATCGCAGAGTCTTCTACCAA 0 T M H S F E V A A T Q V E E D F F L L D V I D K V V G K V F D C S T K R V L D
12000
pol
TACCCCTAAAATTAAAGCAATATCCGTCACCATGTTCTTTTTACAATCAAATAGCGACAAAAACTTTCAACTCAGCTGATGGCGCTTAACAA V G K F 0 L L S D D Q R G 0 Y R K K C I L 0 L P C L 0 K E F E L K 0 P 0 K F L R
13202
pol
6P0L5 ATCTTGATTGTTGGCATCACGGGCAACCTTGCAAGACGATAACCTATTCTGCAGAATAAGATCTATCGTTCACATAATTACTCCCTTCAAACA D T 0 H K A 0 M P A 0 R R L 5 0 N V M N R V S M F 0 5 D T 0 G Y 0 V E V E L D G
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810 770
730
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T
AGTAAATCGATACGTAAACAAATCCTGCAAAAATTGCCGGCTGCTCTTGAATAACTAATCACGTGGACAAGCACTCTTTCAAGAACGTACAAA F D 5 S T L G F K E C F F Q E S Q M K S N V Y D V T S C L M E R G L C T V S A T 6P0L4
pol
690
A
18 AGAACGAAACCGCATACCGGCCCCCCGTACCCTAACGGTACTGTGTTCGACGGCGCTTTTCAGAGAGTTACA1680CG
0
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A
V
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15606
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CACGTTAAATGCACTTCTGCAACCAGTATCGTCTTCACCAAACTAAGTCTAAACAAGATATGGCCACATATGCCGGAACAACTTGAAATCAAC
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0
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D
V
CACGTAGGACACATATAACCGTTTAGCTCCAAAGGTACCCTTTGAACCATCCTTGCTATGTGGAAGCACATACCGGAGAACATATACGCGTTT T
P
V
A
Y
Y
G
T
K
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L
V
T
A
G
K
Y
0
G
G
H
N
R
Q
S
S
V
P
S
M
0
L
N
M
R
A
K
R
TTCCTAAGATGAGGGAATACTTTTCTCTGACCTGAACCCAACATTAGCGTGCGTGGCCATCTGCGACTCGCAACTCAAATATAATAATTGTTA
19202 570 20404 530
F
21606 4 90
GAGAGCACACTAGACGAGTCTGAGACATACTACGACACAGCCTGCACTGGTCACTATAATTTTTTGTATTCCTGTACATACTGCCCTTTCTGC R V V L V 5 D Q L C Y K G V V A R G 0 P G 0 I F K K P 0 E K Y S L 0 E K K Q Q
22808 450
GAGAAGACTTACAAAGTACCATTTGAATCTGCCCGTATCTACAGAAAGAGGGTACAATCAGATCAAACTCGGAGTAACACTTGGTTGGCCTA
24000
T
F
C
L
I
0
K
P
C
F
0
A
T
0
K
L
D
0
K
K
Y
Q
G
N
Q
K
F
A
V
V
0
T
K
C
A
R
S
Y
S
V
T
P
V
L
A
H
Y
0
M
A
R
L
Y
V
L
A
E
V
V
E
S
G
T
F
F
H
Y
V
0
N
K
T
Q
0
A
K
0
L
GAACCCTTCTGACTGTCGTAGGACAGGATTCAACTTTCATCTTCGTTTGAAAACACCAAATCTAATGTAATCTATCCTCCTATAACAATATTT
pal
D
pal pol
G
K
R
Y
Q
K
H
P
5
V
0
I
G
K
G
N
K
K
L
F
S
C
0
G
Y
D
0
Y
H
K
D
M
0
R
L
C
Y
K
L
ATCAAATTTTATATTTAACGGTATAACTCCGAGCTCATCCTAAAATATAAAAACCAAAATAATCAACTCGACGAAATCATTATAGCACCCGCA 0 0 K 5 K 0 F
N
N
K
E
C
T
N
0
G
L
N
Y
G
T
G
L
T
F
L
H
R
E
0
R
D
C
E
I
D
F
S
W
C
G
Y
L
F
R
L
F
0
F
F
G
L
L
L
E
F
E
S
A
F
K
Y
I
H
V
G
D
0
G
E
T
0
F
5
0
V
S
0
0
I
V
0
D
G
L
N
E
A
D
P
F
N
G
P
W
N
V
0
E
L
S
V
L
0
S
V
H
C
N
0
E
V
E
L
N
S
G
K
P
TCCCTATCTGAGAATAATTTTTTCATTTACCTCCGAACTAAAAACCTTATGACGAGAACGATCAAACATTACGCGATTCAAAACACGATCCCCA 0 0 K M D 0 P 0 Y K K V N Y W G F S L F G N V F F L V V
410
25202 370
26404 330 290 250
30000
pol
G
pal
CTGCGAATTTTCAATCGTTAGAACATAAAATTCAAAATCATTTAACAGTTACGACGTGTGGCTTGTCCCTACACGACACTATCACCGGTCATG 31202 0 V 0 L 0 K G I R 0 5 V Y F N G F S V K F L N V V T N A N Y G Y L S L K 0 A P 170
pal
F
pal
33606 0 GATCTCTCCCGCCTCGTTCCCCAACATCTATCATGTCCCGAGGATCACAAAGTCTTACTAAAGGAAATTTCGACGAACCGAAACTTAAGTTCT K E G 0 K T R G F H R I V T G S P 0 V F N K V Y F P L N K 0 5 D F H L
pal
AGCACATAATGGAAATTCACCTATATATCAAATCTTGACCAGCTTGTCAAATCGGCAGGCATTCTTGCTAGTAATACGTCGTCTGATAAACTA H 0 H ND Y D K S R A 0 0 L S P W A 0 Y 0 N F K M RK K S L Y 0 Y F H
pal
36000 K 5 D C V D K I L G A A G 0D 0I GN P L 0 R I Y S S R S K K K PCGGTCAGATCAAAACTCCTTATAATCCGCCCACATCAGCATATACGCGGGAGTATCGAAGTACTCGTCTCTTTTTTCTTAGCGATCGCTCCAA H N A K L Y 10
pal
P
D
gpB gpB
D
T
D
Y
E
V
F
V
N
210
E
AAAAAACAGACTTTTACCTGCCGTTACATCAATGTTTTAGAGCCTCAGCTTTTCCTCGAGCATCGCGTAAAATTTGCCCTTCCAACACTTCCG 32404 S C S M K V K G T L H L N N 0 T A K L S G 0 V C K C Y F Y 0 K 0 G F V N V C
130
R
R
Q
T
T
E
L
F
F
S
5
V
0
K S
I
R V P
R A K
Y V S
S
K
0
S
1
0
V
N
L
L
90
T
R
R
L
N
S
G
D
R
0
K
Y
G
R
Y
R
0
R
0
0
L
5
P
K
S
A
N
S
K
S
S
S
P
1 230
H
K
L
A
0
K
0
S
Y
K
D
0
5
V
Q
G
S
V
D
A
H
G
K
K
K
K
190
S
P
1
K
T
1
G
1
V
S
S
V
T
T
V
P
N
T
A
Y
P
F
H
H
0
150
V
0
0
0
V
0
L
0
L
H
L
G
G
G
F
P
N
K
L
F
S
V
K
V
N
K
I
A
K
L
V
0
R
V
F
V
50
S
K
V
R
V
A
K
34808
R
K
gpB
gpB
R
V V
V V
V
A
V
110
42000 70 43202 30
gpB
G
CCACCACATATCCCAGCGCCCTCCGTACACTATACCGGCCAATCAGTGCTAAGCCCAAGCCTGTAAAAAAATTATCCATTACATCGAAGTGAT 0 V I 0 G L A G A T V 5 I V 5 G L G 1 G 0 A 0 L G 0 L F S N 0 G N V Y S P 1 N
gpB
T
GTAGAATTTAGTTCTTAGTTATGTGCCTCTATAGAGTATATCACAAGATATCTCTGATCAAACGTTGCTTACTAATTGTCGGCGATATAATGA A I K A K I 1 Y L A S K Y S N Y K R L 0 N K L D F V N A R S L K 0 P 5 Y L 0 L
gpB
ACTAATGCTTCAAGTGTTTGCATAACTCAACCAGCTTTGCTGAT K 0 K 1 5 V K F D A N K L P 0 0 K L F A R
I
D
L
V
D
I
P
N
4407 1
HHV-6 DNA POLYMERASE GENE
VOL. 65, 1991
4675
FIG. 5. DNA sequence and predicted protein sequence of the U1102 pol region. The DNA sequence is given with the orientation shown in Fig. 1, i.e., rightward 5'-to-3' strand only (numbered 1 to 4407). The rightward-encoded unknown protein sequence ORF R is shown above the DNA sequence (in the single-letter code), and the two leftward-encoded protein sequences, pol and gpB, are shown below the sequence. The name of each ORF is given on the left, and the amino acid sequences, numbered from the N terminus to the C terminus, are given on the right. The pol protein sequence is given from the first ATG (bold). The five nucleotide differences observed between the U1102 and AJ strains are indicated, as well as the two resulting amino acid changes. The six potential poly(A) addition site sequences (5'-AATAAA-3') are overlined, the putative promoter (CATAAAG) is given a double overline, and the four PCR primer sequence positions are underlined.
mismatch at the 3' end can still yield specific PCR amplification (22). Surprisingly, we were initially unable routinely to obtain amplification of the EBV and HCMV pol genes from DNA isolated from either infected cells or plasmid or cosmid clones, even though the sequences of the genes are known and were covered by the degenerancy of the primer sequences. However, addition of glycerol to 15%, as suggested by the work of Smith et al. (54), overcame this difficulty and resulted in amplification of the pol sequences from EBV cosmid and HCMV plasmid targets (Fig. 3b, lanes 1 to 6). The correctly sized bands were found with all primer pairs used, although primer 6POL1 gave much lower yields of PCR product than did 6POL2 and 6POL4. Interestingly, inclusion of 15% glycerol lowered the amount of HHV-6specific product obtained by using primer 6POL2 (Fig. 3b, compare lanes 7 and 8). The HHV-6 PCR product was used subsequently to screen the libraries of HHV-6 clones; it identified a single 4.4-kbp EcoRI fragment in the clone XgtlO RPMS4.4, which was shown to contain the complete pol gene (see below). The 4.4-kbp EcoRI fragment was subcloned into Bluescribe to generate the recombinant clone pRPMS4.4. As an independent confirmation of our initial PCR results with DNA from U1102-infected cells, PCR was also performed on DNA isolated from cells infected with the AJ (Gambian) isolate of HHV-6. The respective HHV-6-specific DNA bands were isolated, digested with the restriction enzymes EcoRI and HindIII, and cloned into similarly digested M13mp8 and M13mp9 vectors. The inserts were sequenced and found to be homologous to the sequences of the other herpesvirus DNA polymerases, although not identical to any of them. In the case of the AJ-derived product, three independent M13 clones were sequenced. Two of them were identical in sequence, but the third clone contained a run of six T residues in a region where the other two contained seven T residues (at positions 1476 to 1482; see Fig. 5). This discrepancy is assumed to be due to Taq DNA polymerase slippage during the PCR cycles, an effect also observed when sequencing through a poly(A) tail present in cDNA clones.
ORF3RC
POL
C
POL
.FFL7R (96 kbp) C
0 POL
IU5
GPB
1 (7 kbp) RF27
[
POL POL
A comparison between the 465 bp of sequence obtained from the AJ strain PCR product and the corresponding sequence of the U1102 gene (see below) revealed five nucleotide differences (approximately 1% of the sequence). Three of the changes were silent third-base changes, and the remaining two resulted in amino acid changes (see Fig. 5). The sequence data also indicated the presence of an extra EcoRI restriction site in the AJ strain. Southern blot analysis of EcoRI endonuclease-digested DNA from U1102- and AJ-infected cells probed with pRPMS4.4 is shown in Fig. 4b. The presence of DNA fragments of 2.6 kbp and a doublet of ca. 1 kbp in the AJ strain indicates at least two EcoRI fragment polymorphisms within the 4.4-kbp pol region covered by the probe. Further Southern blot analysis of genomic DNA from HHV-6 U1102-infected cells with RPMS4.4 as a probe revealed single 7-kbp HindIII and 4.4-kbp EcoRI fragments, two BamHI fragments (10 and 5 kbp), two Sall fragments (>23 and 10 kbp), and two XhoI fragments (5.5 and 4 kbp) (Fig. 4a). Together with the hybridization of RPMS4.4 to XFIX clone Z14.1, this has shown that the pol gene should be positioned between 58 and 61.5 kbp from the left-hand end of the genome (Fig. 1 and 4a). Sequence analysis. The complete nucleotide sequence of the 4.4-kbp EcoRl restriction enzyme fragment was determined on both DNA strands and is shown in Fig. 5. Computer analysis for open reading frames (ORFs) within the sequence revealed the presence of three ORFs (Fig. lb). The largest ORF (3,036 bp) encodes the DNA polymerase gene with a predicted protein of 1,012 amino acids and a molecular weight of 115,811. The ATG at 3627 to 3625 is the first initiation codon in the ORF and is presumed to be the first methionine codon of the pol gene. The ORF to the right of the polymerase gene was shown by a library search to encode the C-terminal region of a putative HHV-6 homolog of the glycoprotein B (gpB) gene. The DNA polymerase and glycoprotein B genes overlap by 1 base; the A of the ATG initiation codon of the pol gene is also used as the A of the TGA termination codon of the gpB gene (Fig. 5). To the left of the pol gene, and in a rightward translational frame, is the C-terminal region of a gene (ORF R) which is homologous to ORFs conserved in human herpesviruses, i.e., HCMV UL53 HHV-6
[GPB GPB
V(
I
EBV
GPB
_U3 (6 kbp)
CMV
HSV-1
I
GPB
VzV
1 kbp
FIG. 6. Arrangement of ORFs of HHV-6 compared with the corresponding arrangements of ORFs for homologous regions of HCMV, EBV, HSV-1, and VZV. The incomplete gpB and ORF R genes of HHV-6 are shown as open arrows.
4676
HHV6 CMV
EBV HSV1 HSV2 VZV
TEO ET AL.
1 1 1 1 1 1
J. VIROL.
MDSVSFFNPYLEANR-L---------------------KKKSRSSYIRILPRGIMD-----GAAGLIKDVCDSEPRMFYRD MFFNPYLSGGVTGGA-VAGGRRQRSQPGSAQGSGKRPPQKQFLQIVPRGVMFD----GQTGLIKHKTGRLPLMFYRE MSGGLFYNPFLRPNKGLL--------------------KK-PDKEYLRLIPKCF--QTP--GAAGVVDVRGPQPPLCFYQD
MFSGGGGPLSPGGKSAARAASGFFAPAGPRGASR-GPPPCLRQNFYNPYLSPVGTQQ------KPTGPTQRHTYYSEC----DEFRFIAPRVLDEDAPPEKRAGVHDGHLKRAPKV-YCG MFCAAGGPASPGGKSAARAASGFFAPHNPRGATQTAPPPCRRQNFYNPHLAQTGTQP------KAPGPAQRHTYYSEC----DEFRFIAPRSLDEDAPAEQRTGVHDGRLRRAPKV-YCG MAIRTGFCNPFLTQASGIKYNPRTGRGSNREFLHSYKTTM----SSFQFLAPKCLDEDVPMEERKGVHVGTLSRPPKV-YCN
CON
HHV6 CMV
EBV HSV1 HSV2 VZV
F NP L
56 73 57 109 110 78
CON
156 173 167 228 229 193
HHV6 CMV EBV HSV1 HSV2 VZV CON
250 268 263 330 331 311
HHV6 CMV EBV HSV1 HSV2 VZV CON
335 378 350 426 427
HHV6 CMV EBV HSV1 HSV2 VzV CON
HHV6 CMV EBV HSV1
HSV2 VZV CON
HHV6 CMV EBV HSV1
HSV2 VZV CON
519 499
HHV6 HSV1 HSV2
574 719 586 719 724 684
691 836 706 836 840 800
786 956 801 932 937 897
905 1076 920 1051 1056 1016
HHV6
971 1196 986 1168 1173
HSV1 HSV2
VZV CON
YFY
*E VD
R
*D
*FGW
RPPTAFGTSSDVEFNCTADNLAVEGAMCDLPAYKLMCFDIECKAGGEDELAFPVAERPEDLVIQISCLLYDLSTTALEHI -----------------------LLFSLGSCDLPES-HLS RPASRQLTLSDVEIDCMSDNLQAIPNDDSWPDYKLLCFDIECKSGGSNELAFPDATHLEDLVIQISCLLYSIPRQSLEHI -------------------.----LLFSLGSCDLPOR-YVQ *
334 377 349 425 426 406
----------- IYEFASEFELLLGFFIFLRIESPEFITGYNINNFDLKYLCIRMDKIYHYDIGCFSKLKNGKI ------GISVPHEQYRKGFLQ------------------AQTKVFT
418
RDSYA-----ELEYDCEVGDLSVRREDSSWPSYQALAFDIECL-G---EEGFPTATNEADLILQISCVLWSTGEEAG-------RYRR-------------ILLTLGTCEDIEGVE-AAPMAFGTSSDVEFNCTADNLAIEGGMSDLPAYKLMCFDIECKAGGEDELAFPVAGHPEDLVIQISCLLYDLSTTALEHV ---------------------- LLFSLGSCDLPES-HLNN *E *C
*L
PY
*
G
FDIEC
D *.*QIS-* *
FP A
L * G.C
----------DVYEFPSEYELLLGFMLFFQRYAPAFVTGYNINSFDLKYILTRLEYLYKVDSQRFCKLPTAQG------GRFFLHSPAV-GF--KRQYAAAFPSASHNNPASTAATKVYI 478 R---PHD-AGKGFL--R-----------------ANTKVRI 433 ELAARGLP-TPVLEFDSEFEMLLAFMTLVKQYGPEFVTGYNI IINFDWPFLLAKLTDIYKVP5LDGYGRMNGRGVFRVWDIGQS --------HF-QKR-------------------SKIKV 517 DLASRGLPAPWLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQS--------HF-QKR-------------------SKIKV 518 EMKDAGLPEPTVLEFDSEFELLIAFMTLVKQYAPEFATGYNIVNFDWAFI MEKLNSIYSLKLDGYGS4INRGGLFKIWDVGKS------- GF-QRR-------------------SKVKI 498 * EF SE o.L oF *
-----------VYEFPSELDMLYAFFQLIRDLSVEIVTGYNVANFDWPYILDRARHIYSINPASLGKIRAGGVCEVR
FD
.
*Y
*
oK
F
IV ----___________
538 SGVLYLDMYPVYSSKI5TANYKLDTIAKICLGQEKEQLSYKEIPKKFISGPSGRAVVGKYCLODSVLVRLFKQI3NYHFEVAEVARLAHVTARCVVFEGQQKKIFPCILTEAKRRNMILP
AGS9I8DMYPVCMAKTNSPNYKLNTMAELYLRQRKDDLSYKDIPRCFVANAEGRAQVGRYCLQDAVLVRDLFNTINFHYEAGAIARLAKIPLRRVIFDGQQIRIYTSLLDECACR6FILP 598 TGLIPIDMYAVCRDKLSLSDYKLDTVARHLLGAKKEDVHYKEIPRLFAAGPEGRRRLGMYCVQDSALVMDLLNHFVIHVEVAEIAKIAHIPCRVLDDGQQIRVFSCLLAAAQKENFILP 553 NGMVNIDMYGIITDKIKLSSYKLNAVAEAVLKDKKKDLSYRDIPAYYAAGPAQRGVIGEYCIQDSLLVGOLFFKFLPHLELSAVARLAGINITRTIYDGQQIRVFTCLLRLADQKGFILP 637 38 NGMVNIDMYGIITDKVKLSSYKLNAVAEAVLKDKKKDLSYRD6IPAYYASGPAQRGVIGEYCQDSLLVGQLFFKFLPHLELSAVARLAGINITRTIYDGQQIRVFTCLLRLAGQKGFILP6618 NGLISLDMYAIATEKLKLSSYKLDSVAREALNESKRDLPYKDIPGYYASGPNTRGIIGEYCIQDSALVGKLFFKYLPHLELSAVARLARITLTKAIYDGQQVRIYTCLLGLASSRGFILP G0- *DDMY-o K YKL---A L K *- Y**IP * * * R *G YCoQDo LV L H E * *AR-A * * * -
. -L
ILP
GIGYKGATVLEPKTGYYA VPTVVFDF SMVSSHNRQQ------------------------------------------------------------------------------------NHYSKGTTVPETNSVAVSPNAAIISTAAVPGDAGSVAAMFQMSPPLQSAPSSQDGVSPGSGSNSSSSVGVFSVGSGSSGGVGVSNDNHGAGGTAAVSYQGATVFEPEVGYYNDPVAVFDF MPSASDRD---------------------------------------------------------------------------------------GYQGATVIQ PLSGFYNSPVLVVDF DTQGRFRGAGGEAPKRPAAAREDEERPEEEGEDEDEREEG----GGEREPEGARETA----------------------------------GRHVGYQGARVLDPTSGFHVNPVVVFDF DTQGRFRGLDKEAPKRPAVPRGEGERPGDGNGDEDKDDDEDGDEDGDEREEVARETG ---------------------------------- GRHVGYQGARVLDPTSGFHVDPVVVFDF DGGYPATFEYKDVIPDVGDV------- EEEM-DEDE------------------------------ SVSP-T-GT-SS ------------GRNVGYKGARVFDPDTGFYIDPVVVLDF P V DF *Y GA V *P Go
QSLYPSIMMAHNLCYSTLVL--DERQIAGLSESDILTVKLGDE-THRFVKPCIRESVLGSLLKDWLAKRREVKAEMQNCSDPMMKLLLDKKOLALKTTCNSVYGVTGAAHGLLPCVAIAA
573 718 585 718 723 683
690
835 ASLYPSIIMAHNLCYSTLLVPGGEYPVDPAD---VYSVTLENGVTHRFVRASVRVSVLSELLNKWVSQRRAVRECMRECQDPVRRMLLDKEQMALKVTCNAFYGFTGW3NGMMPCLPIAA
ASLYPSIIQAHNLCYSTMITPGEEHRLAGLRPGEDYESFRLTGGVYHFVKKHVHESFLASLLTSWLAKRKAIKKLLAACEDPRORTILDKQOLAIKCTCNAVYGFTGVANGLFPCLSIAE
ASLYPSIIQAHNLCFSTLSLRADAVAHLEA--GKDYLEIEVGGRRLFFVKAHVRESLLSILLRDWLAMRKQIRSRIPOSS-PEEAVLLDKQQAIKVVCNSVYGFTGVOHGLLPCLHVAA ASLYPSIIQAHNLCFSTLSLRPEAVAHLEAD--RDYLEIEVGGRRLFFVKAHVRESLLSILLRDWLAMRKQIRSRIPQSP-PEEAVLLDKQQIKWCNSVYGFTGVQHGLLPCLHVAA
ASLYPSIIQAHNLCFTTLTLNFETVKRLNPS---DYATFTVGGKRLFFVRSNVRESLLGVLLKDWLAMRKAIRARIPGSS-SDEAVLLDKQQAAIKWCNSVYGFTGVAQGFLPCLYVAA SLYPSIo AHNLC'-To FV- *- S Lo LL Woo R- *- * * *LDK 0 A*K CNo YG TG G PC- *A ---------------------------- III -------- VI ------II --------------
705 835 840 799
SVTCLGREMLCSTVDYVNSKMQSE----QFFC-EEFGL------------------TSSDFTGD--LEVEVIYGDTDSIFMSVRNMVNQSLMRIAPMIAKHITDRLFKSPIKLEFEKILC 785 SI9TRIGRDMLERTARFIKDNFSEPCFLHNFFNQEDYVVGTREGDSEESSALPEGLETSSGGSNERVEARVIYGDTDSVFVRFRGLTPQALVARGPSLAHYVTACLFVEPVXLEFEKVFV955 TVTLQGRTMLERAKAFVEALSPANLQALAPSPDAWAPLNPE
---------------------Q GLRVIYGDTDSLFIECRGFSESETLRFADALAAHTTRSLFVAPISLEAEKTFS TVTTIGREMLLATREYVHARWAAFEQLLADFPEAADMRAPGP ------------YSMRIIYGDTDSIFVLCRGLTAAGLTAVGDKMASHISRALFLPPIKLECEKTFT
TVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGP- -------------------YSMRIIYGDTDSIFVLCRGLTGEALVAMGDKMASHIShALFLPPIKLECEKTFT TVTTIGRQMLLSTRDYIHNNWAAFERFITAFPDIESSVLSQKA----------------------YEVKVIYGDTDSVFIRFKGVSVEGIAKIGEKMAHIISTALFCPPIKLECEKTFI * *-T GR ML * ** *IYGDTDSoF * * *A * LF Po LE EK __________ I
800 931 936 896
--
PLILICKKRYIGR-QDDSLLIFKGVDLVRKTSCDFVKGVVKDIVDLLFFDEEVQTAAVEFSHMTQTQLREQGVPVGIHKILRRLCEAREELFQNRADVRHLMLSSVLSKEMAAYKQPNLA
904
SLMMICKKRYIGKVEGASGLSMKGVDLVRKTACEFVKGVTRDVLSLLFEDREVSEAAVRLSRLSLDEVKKYGVPRGFWRILRRLVQARDDLYLHRVRVEDLVLSSVLSKDISLYRQSNLP 1075 CLMLITKKRYVGVLTDGKTL-MKGVELVRKTACKFVQTRCRRVLDLVLADARVKEAASLLSHRPFQESFTQGLPVGFLPVIDILNQAYTDLREGRVPMGELCFSTELSRKLSAYKSTQMP 919 KLLLIAKKKYIGVIYGGKML-IKGVDLVRKNNCAFINRTSRALVDLLFYDDTVSGAAAALAERPAEELARPLPEGLQAFGAVLVDAHRRI0TDPERDIQDFVLTAELSRHPRAYTNKRLA 1050 KLLLIAKKKYIGVICGGKML-IKGVDLVRKNNCAFINRTSRALVDLLFYDDTVSGAAAALAERPAEEWLARPLPEGLQAFGAVLVDAHRRITDPERDIQDFVLTAELSRHPRAYTNKRLA 1055 KLLLITKKKYIGVIYGGKVL-MKGVDLVRKNNCFINDYARKLVELLLYDDTVSRAAAEASCVSIAEWNRRAMPSGMAGFGRIIADAHRQITSPKLDINKFVMTAELSRPPSAYINRRLA 1015 L KGVoLVRK
CF
*
--
L
D V AA
* .*
ooP G
*-A
*
*
---V
*
LS-
Y
HLSVIRRLAQRKEEIPNVGDRIMYVLIAPSIGN -------------------------K-------------------------KQTH---NYELAEDPNYVIEHKIPIHAEKYFDQIIK
970
HIAVIKRLAARSEELPSVGDRVFYVLTAPGVRTAPQGSSDNGDSVTAGVVSRSDAIDGTDDDADGGGVEESNRRGGEPAKKRARPSAVCNYEVAEDPSYVREHGVPIHADKYFEQVLK 1195 HLAVYQKFVERNEELPQIHDRIQYVFVEPKGGV- ----------------------------------KGARKT-EMAEDPAYAERHGVPVAVDHYFDKLLQ 985 HLTWYKLMARRAQVPSIKDRIPYVIVAQTREVEETVARLAA-LRELDAAAPGDEPAPPAALPSPAKRPRETPSPADPPG-GASKPRKLLVS-ELAEDPAYAIAHGVALNTDYYFSHLLG 1167 HLTWYKLMARRAQVPSIKDRIPYVIVAOTREVEETVARLAA-LRELDAAAPGDEPAPPAALPSPAKRPRETPSHADPPG-GASKPRKLLVS-ELAEDPGYAIARGVPLNTDYYFSHLLG 1172 HLTVYYKLVMRQGQIPNVRERIPYVIVAPTDEVE-ADAKSVALLRG-D----------P-LQNTAGKRC------------GEAK-RKLIIS-DLAEDPIHVTSHGLSLNIDYYFSHLIG 1108 H-oV * R *-P * -R- YV
1109
249 267 262 329 330 310
-
KGSNL----- EVEINCHVSDLVSL-EDVNWPLYGCWSFDIECL-G--QNGNFPDAENLGDIVIQISVISFDTEGD ---------RDER--------------HLFTLGTCEKIDGVH--
L-I KKoYoG
VZV CON
CMV EBV
V*G
RASTC ----DIEVDCDVSDLVAVPDDSSWPRYRCLSFDIECMSG --EGGFPCAEKSDDIVIQISCVCYETGGNTAVDQGIPNGNDGRGCTSEGVIFGHSGLHLFTIGTCGQVGPDV--
-
CMV EBV
*V
G
TQNDATLNGDKNAFHGTSFKSASPESFRVEVIERTDVYYYDTQPCAFYRVYSP SSKFTNYLCDNFHPE- -LKKYEGRVDATTRFLMDNPGFVSFGWYQLKPGVDGERVRV
---
HHV6 CMV EBV HSV1 HSV2 VZV CON
G
*GQQ
539 599 554 638 639 619
y
LAAALRES----------------PGASFRGISADHFEAEVVERADVYYYETRPTLYYRVFVRSGRALAYLCDNFCPA--IRKYEGGVDATTRFILDNPGFVTFGWYRLKPGRGNAPAQP LAECLRSSMI
TGYNo
419 479 434 518
P
- --NLML -------------- -- ----TGEVKMSCSFVIEPADKLSLYGYNANTVVNLFKVSFGNFYVSQRIGKI LQNEGFVVYE IDVDVLTRFFVDN-GFLSFGWYNVKK--YIPQDMG --SVGEL-------------------VPEPRTPYAVSVTPATKTSIYGYGTRPVPDLQCVSISNWTMARKIGEYLLEQGFPVYEVRVDPLTRLVIDR-RITTFGWCSVNR--YDWRQQG --SALKA------------------- STFDRRTPCRVSVEKVTRRSIMGYGNHAGDYHKI TLSHPNSVCHVATWLQDKHGCRIFEANVDATRRFVLDN-DFVTFGWYSCRR--AIPRLQH MAAALRES --------------- PGASFRGISADHFEAEVVERTDVYYYETRPALFYRVYVRSGRVLSYLCDNFCPA--IKKYEGGVDATTRFILDNPGFVTFGWYRLKPGRNNTLAQP
----------------
HHV6 CMV EBV HSV1 HSV2 VZV CON
P G V-
Y
407
G*
*
RQ--YLLSKEMT--WPSL-----DIARSKD-YDHMRMKFHIYDAVET--LMFTDSIENLPFQYRHFVTPSGTVIRMFGRT-EDGEKICVNVFGQEQYFYCECVDGRSLKATIN ------ 155 IK--HLLSHDMV--WPCP ---1WRETLVGRV-VGPIR--FHTYD--QTDAVLFFDSPENVSPRYRQHLVPSGNVLRFFGAT-EHGYSICVNVFGQRSYFYCEYSDTDRLREVIA ------- 172 SLT1GGDEDGKGMWWRQRAQEGTARPEADTH-GSPLDFHVYDILETVYTHEKCAVIP-SDKQG-YVPCGIVIKLLGRRKADGASVCVNVFGQQAYFYASAPQGLDVEFAVL-------166 GDERDVLRVGSGGFWPRRSRLWGGVDHAPAGFNPTVTVFHVYDILENVEHAYGMRAAQFHARFMDAITPTGTVITLLGLT-PEGHRVAVHVYGTRQYFYMNKEEVDRHLQCRAPRDLCER 227 GDERDVLRVGPEGFWPRRLRLWGGADHAPEGFDPTVTVFHVYDILEHVEHAYSMRAAQLHERFMDAITPAGTVITLLGLT-PEGHRVAVHVYGTRQYFYMNKAEVDRHLQCRAPRDLCER 228 GKEVPILDFRCSSPWPRRVNIWGEIDFRGDKFDPRFNTFHVYDIVETTEAA----SNGDVSRFATATRPLGTVITLLGMS-RCGKR'WAVHVYGICQYFYINKAEVDTACGIRSGSELSVL 192 W FH YD
HHV6 CMV EBV HSV1 HSV2 VzV CON
.P*
*
55 72 56 108 109 77
K
AVTNAISPIFPKTD-IKKEK-LLL-YLL-PMKVYLDETFSAIA----EVM AVTNVLSPVFPGGETARKDK-FLH-MVL-PRRLHLEPAFLPYSVKAHECC GAANILQCLF-DNNS-GAALSVLQNFTARPPF
AACVTFKALF-GNNA-KITESLLKRFI--PEVWHPPDDVAARLRTAGFGAVGAG-----------ATAEE-TRRMLHRAFDTL-A AACVTFKALF-GNNA-KITESLLKRFI--PETWHPPDDVAARLRAAGFGPAGAG-----------ATAEE-TRRMLHRAFDTL-A TASVTFKALF-GNDT-KLTERLLKRFI--PETRVVNVKMLNRLQAAGFVCIHAPCWDNKMNTEAEITEEEQSHQIMRRVFCIPKAILHQS F
*
L
P
*-AEDP
YF
1012 1242 1015 1235 1240
1194
VOL. 65,
HHV-6 DNA POLYMERASE GENE
1991
4677
TABLE 1. HHV-6 DNA polymerase codon usage and amino acid composition Amino acid Aioai
Codon usage
composition
Codon
a.a.a
No.
Codon
a.a.
No.
Codon
a.a.
No.
Codon
a.a.
No.
a.a.
No. (%)
TTT TTC TTA TTG
F F L L
43 14 24 28
TCT TCC TCA TCG
S S S S
21 7 5 14
TAT
Y Y * *
34 11 0 0
TGT
C C * W
19 8 1 5
Ala Val Leu Pro
48 (4.74) 80 (7.91) 97 (9.58) 76 (7.51) 35 (3.46)
TAC TAA TAG
TGC TGA TGG
Ile
CTT CTC CTA CTG
L L L L
17 4 5 19
CCT CCC CCA CCG
P P P P
13 5 3 14
CAT CAC CAA CAG
H H Q Q
15 8 11 27
CGT
CGC CGA CGG
R R R R
12 3 7 4
Met Phe Trp Gly Ser
31 (3.06) 57 (5.63) 5 (0.49) 56 (5.53) 67 (6.62)
ATT ATC ATA ATG
I I I M
42 14 20 31
ACT ACC ACA ACG
T T T T
13 8 8 13
AAT AAC AAA AAG
N N K K
33 7 30 41
AGT AGC AGA AGG
S S R R
14 6 17 7
Thr
42 (4.15) 27 (2.67) 45 (4.45) 40 (3.95)
GTT GTC GTA GTG
V V V V
17 14 11 38
GCT GCC GCA GCG
A A A A
19 6 7 16
GAT GAC GAA GAG
D D E E
42 16 32 34
GGT GGC GGA GGG
G G G G
22 4 18 12
Cys Tyr Asn Gln Asp Glu Lys
Arg His
a a
38 (3.75) 58 (5.73) 66 (6.52) 71 (7.02) 50 (4.94) 23 (2.27)
ma amino acid.
(34), EBV BFLF2 (5), herpes simplex virus type 1 (HSV-1) UL31 (43), and varicella-zoster virus (VZV) RF27 (12). Nothing is known about the function of this ORF, other than that the gene is conserved in all human herpesviruses analyzed to date. The arrangement of these three genes in HHV-6 is identical to that seen in HCMV, but they are rearranged relative to the other human herpesviruses (Fig. 6). The DNA sequence of RPMS4.4 is only 41% G+C rich, and as such it has proved difficult to predict the positions of transcriptional control sequences. However, there are six potential poly(A) addition sequences, AATAAA (Fig. 5). The sequence at 3508 to 3503 is the most likely poly(A) site for the gpB gene, and the one at 554 to 549 is the most likely site for the pol gene, on the basis of their locations relative to the respective ORFs. A potential TATA transcriptional initiation sequence for the pol gene, CATAAAG, is present at positions 3753 to 3747. The codon usage of the pol gene is shown in Table 1. In most cases, owing to the low G+C content of the region, codons ending in T and A predominate as expected; e.g., only 13 of the 50 codons for arginine end in C or G. However, there are exceptions; for example, of the 80 codons for valine, 38 are encoded by GTG, and, similarly, for the amino acids leucine, lysine, and glutamic acid there is a high usage of codons ending in G (47 of 97, 41 of 71, and 34 of 66, respectively). The CG dinucleotide frequency is not underrepresented in HHV-6 and is thus similar to that found
for HCMV, whereas the lymphotropic -y herpesvirus, EBV, is deficient in CG sequences. DNA polymerase. The amino acid composition of the putative HHV-6 DNA polymerase is shown in Table 1 and is similar to that seen in other herpesvirus DNA polymerases. A multiple alignment of human herpesvirus DNA polymerase sequences is presented in Fig. 7, and the FASTA scores of a Lipman and Pearson (39) search are shown in Table 2. Surprisingly, the FASTA score of the comparison between HHV-6 and HCMV is the lowest, despite the observation they share the largest number of identical and conserved amino acid residues. FASTA is a measure of protein sequence relatedness and is used in global data base searches, but as can be seen here, the results are sometimes misleading. Our alignment is based on both pairwise comparisons of the human herpesvirus pol sequences (11) and the results from the Multalign (11) and AMPS (7, 8) programs. The data were then adjusted manually to yield maximum overall homology between the amino acid sequences from all six herpesviruses, with the minimum number of introduced gaps. As such, the alignment may not be optimal for any given pairwise comparison. Overall, among all six human herpesvirus polymerases, 215 amino acid residues are identical and another 158 residues are conserved. Relative to the HHV-6 pol sequence, this means that 21% of residues are identical and a total of 37% are conserved among all six
herpesviruses.
FIG. 7. Alignment of the predicted amino acid sequences of the HHV-6, HCMV, EBV, HSV-1, HSV-2, and VZV DNA polymerases. Gaps introduced, shown as dashes, allow for the different lengths of the proteins and generate maximum alignment of conserved residues. The bottom line (CON) shows the amino acid residues which are either identical (letters) or have conserved changes (black dots) between all six sequences. The position of the conserved regions I to VI are indicated below the consensus sequence line (25, 35, 60). The following amino acids were considered as conserved in the alignment: ILVCM, GASTP, DEQN, KRH, and YFW.
J. VIROL.
TEO ET AL.
4678
TABLE 2. Percent amino acid similarity between the human herpesvirus pol sequences' % Amino acid similarity (identity)
Virus HHV-6
HHV-6 HCMV EBV VZV HSV-2 HSV-1 FASTA score
100 66.5 55.2 54.2 53.5 53.5
(45.8) (36.1) (33.7) (31.7) (31.9)
5,207
HCMV
100 52.8 44.9 45.3 44.7
(36.9) (28.6) (28.7) (28.7)
540
EBV
100 55.1 (36.7) 57.3 (38.4) 57.4 (38.2)
2,021
VZV
HSV-2
HSV-1
100 68.7 (54.8) 69.0 (54.9)
100 95.0 (90.6)
100
882
887
890
a The bottom line of the table shows the FASTA scores of the HHV-6 sequence against all six polymerase sequences, and the rest of the table shows the percentage of identical and conserved residues (identical value in parentheses) between the human herpesvirus DNA polymerases based on the alignment in Fig. 7.
DISCUSSION The isolation and restriction endonuclease mapping of overlapping K clones covering at least 155 kbp of the estimated 161 kbp of HHV-6 U1102 have been carried out. The resulting library now forms an organized resource for future detailed characterization of the HHV-6 genome. Apart from Lawrence et al. (36), who sequenced a 21.8kbp region of the HHV-6 genome which encodes the major capsid gene and putative alkaline exonuclease and phosphotransferase genes (coordinates 86 to 108 on the viral genome map [Fig. la]), no genes have been assigned to the viral genome. Using the technique of PCR amplification with consensus oligonucleotide primers corresponding to conserved amino acid sequences of the DNA polymerases of other herpesviruses, we have successfully amplified the corresponding region from HHV-6 DNA. The whole pol gene was cloned and sequenced and has been localized between positions 58 and 61.5 on the HHV-6 genome (Fig. la). Our sequence data substantiate the findings of others (36) that HHV-6 is more closely related to HCMV than to the ot or -y herpesviruses and is thus a member of the p herpesvirus group. As shown in Fig. 6, the organization of the DNA polymerase gene region is identical to that of HCMV and different from that of the other human herpesviruses. It can be seen clearly that each class of herpesvirus has a distinct organization of genes in the pol region. Relative to the p herpesvirus genome arrangement, both the cx and -y herpesviruses have rearranged their genes by an inversion event. The possibility exists that the ancestral herpesvirus was p-like, since the rearrangements necessary to generate the a and y classes are least when starting from a p-like arrangement of genes; this will become clearer when further sequence data from HHV-6 and HHV-7 (21) become available. From the available data presented here and studies carried out elsewhere (15, 25a, 33), it appears that the genome of HHV-6 is colinear with that of the long unique region of HCMV. Like HCMV, the CpG dinucleotide ratio of observed to expected frequency for HHV-6 pol does not diverge substantially from unity, implying that there is little DNA methylation activity at the site of viral latency and/or persistence. The G+C content of the amplified regions reported here (Fig. 3) are 59.8% for HCMV, 63.4% for EBV, and only 45.6% for HHV-6. This has important implications for the use of degenerate primers to amplify DNA from herpesviruses in clinical samples. Although the set of degenerate primers we have used will amplify different-sized products for each of the human herpesviruses, except HSV-1 and
HSV-2, and potentially could form the basis of a PCR diagnostic test for characterization of herpesviruses, a variety of PCR experimental conditions may be required together with addition of organic solvents to obtain specific amplification. The high G+C content of DNA in HSV-1, HSV-2, EBV, and HCMV may substantially reduce the amount of amplification product when using standard PCR amplification conditions compared with viral genomes with a lower G+C content. In agreement with Smith et al. (54), addition of glycerol might be of special relevance in cases when the PCR target sequence has a high G+C content, although the role that inclusion of glycerol in the reaction buffer plays in overcoming this problem is unclear. Restriction enzyme polymorphisms among various isolates of HHV-6 have been previously noted (29, 37), and from the limited sequence data on the AJ strain determined here, variation between isolates of HHV-6 at the nucleotide level may be as much as 1%. In this context, Pellet et al. (46) noted a 1.7% variation in sequence between the gpB genes of HSV-1 F and KOS and Spaete et al. (55) noted a 6% variation in the gpB genes of HCMV Towne and AD169. The HHV-6 enzyme appears to be the smallest of the human herpesvirus DNA polymerases, with 1,012 residues; HCMV has the largest, with 1,242 residues. The HHV-6 enzyme is therefore similar in size to the EBV-encoded enzyme (1,015 residues), and these two polymerases are essentially colinear in amino acid organisation. HCMV DNA polymerase departs from colinearity with these two polymerases by having several large insertions of unique sequence at essentially similar positions in the HHV-6 and EBV sequences. The DNA polymerases of the ot herpesviruses also have regions with insertions relative to the HHV-6 sequence. It is unclear why the FASTA score between HHV-6 and HCMV should be low compared with the other scores. It cannot just be because the HCMV polymerase has insertions relative to the HHV-6 sequence, since the ox herpesvirus polymerases which have higher scores also contain insertions. Recently, Tsai et al. (58) have isolated a monoclonal antibody that neutralizes the DNA polymerases of EBV, HCMV, HHV-6, and bacteriophage T4, but not the polymerase activity of HSV-1, HSV-2, or Escherichia coli DNA polymerase I Klenow fragment. This would imply that the DNA polymerases of HHV-6, HCMV, and EBV possess a common epitope not present in the polymerases of HSV-1 and HSV-2. By using the data in Fig. 7, it should be possible to investigate the epitopes recognized by this monoclonal antibody, by expression of the parts of the pol gene common
HHV-6 DNA POLYMERASE GENE
VOL. 65, 1991
to HHV-6, HCMV, and EBV. Discriminating monoclonal antibodies targeted against other sequences could be used in diagnostic assays. The HHV-6 DNA polymerase possesses the conserved regions seen in other herpesvirus DNA polymerases and human DNA polymerase a (see, e.g., references 25, 35, and 60). It should be noted from Fig. 7, however, that as more data are added to the multiple alignment of amino acid sequences, certain residues previously assumed to be identical are not wholly conserved. This places greater importance in terms of structure and function on the remaining common sequences. For example, in region III (Fig. 2 and 7), the previously conserved sequence Tyr-Gly-Phe-ThrGly-Val must now become the consensus sequence Tyr-GlyXxx-Thr-Gly-Xxx (where Xxx is a hydrophobic amino acid). Likewise, in conserved region I, Ile-Tyr-Gly-Asp-Thr-AspSer-Xxx-Phe-(three residues)-ArglLys-Gly, the final glycine residue is not conserved among the human herpesvirus polymerases when the HHV-6 pol sequence is included, suggesting that the Gly at position 896 in the HSV-1 enzyme may not be essential for enzyme activity, although sitespecific mutagenesis studies on HSV-1 DNA polymerase suggests the opposite (14). The inactive HSV-1 DNA polymerase resulting when glycine is changed to valine could be a function of that particular substitution. (It should be noted here that this glycine residue is not conserved in the DNA polymerase of vaccinia virus [16] and human a-DNA polymerase [60], in which the residue is serine, nor in T4 DNA polymerase [56], in which it is lysine). Interestingly, the conserved seine amino acid in region I, present in all DNA polymerase a-like sequences examined to date (14, 60), can be changed to an alanine without loss of enzyme activity. The data contributing to the alignment shown in Fig. 7 thus allow a finer mapping of residues conserved, probably for optimum DNA polymerase activity. Previous investigators have shown that expression of the HSV-1 pol gene in rabbit reticulolysates, E. coli (13), and Saccharomyces cerevisiae (24) have provided valuable target materials for use in the generation of antiviral agents. HHV-6 and EBV enzymes, as the smallest among the herpesvirus DNA polymerases, may provide simpler alternatives for the development of chemotherapeutic agents based on viral polymerases. ACKNOWLEDGMENTS
We are extremely grateful for the gifts of clones and helpful advice from our colleague Bob Honess, who tragically died last year. We are also grateful to Robert Gallo for the gift of several plasmid clones. We thank Michelle Ginsburg and Glenda Lawrence for assistance with the computer analyses. This work was supported by the MRC and the Wellcome Trust. M. D. Jones is a Wellcome Trust lecturer. REFERENCES
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