Fowlpox virus thymidine kinase: nucleotide sequence and relationships to other thymidine kinases

VIROLOGY

156, 355-365

(1987)

Fowlpox Virus Thymidine Kinase: Nucleotide Sequence and Relationships to Other Thymidine Kinases DAVID B. BOYLE,“,’ BARBARA E. H. COUPAR,* ADRIAN J. GIBBS,t LINDA J. SElGMAN,+ AND GERALD W. BOTH+ *Department of Microbiology, tDepartment of Evolutionary Australia; and *Commonwealth

John Curtin School of Medical Research, Australian National University, Canberra, A.C. T. 2601, Australia; Biology, Research School of Biological Sciences, Australian National University, Canberra, A.C. T. 260 1, Scientific and Industrial Research Organization, Division of Molecular Biology, North Ryde 2113, Australia Received

July

17, 1986;

accepted

October

15, 1986

The thymidine kitiase (TK) gene of fowlpox virus (FPV) is located in a 2.2-kb Hindlll-C/al fragment derived from a 5.5-kb EcoRl fragment of the FPV genome. The TK gene was mapped to the region of a 700-bp Xbel fragment contained within this Hindlll-C/al fragment. Nucleotide sequence analysis of this region revealed an open reading frame of 183 codons. Identification of this region as the FPV TK gene was confirmed by its homology with the vaccinia virus TK at both the nucleotide and amino acid levels. The derived FPV TK polypeptide has a calculated molecular weight of 20,380 and is six amino acids larger than the vaccinia virus TK gene product. We have reported previously that the FPV TK gene operates in vaccinia virus without the requirement for a vaccinia virus promoter. The sequence homologies between the two TK promoters substantiated this observation. Northern blot analysis of RNAs from cells infected with a vaccinia virus recombinant expressing the FPV TK gene showed major (700 nucleotide) and minor (1000 nucleotide) transcripts from the FPV TK gene. The deduced amino acid sequence of the FPV TK has significant homology with the TKs from chicken, man, and three other poxviruses, but shows no homology with herpes simplex virus TK. Comparisons of the homologous sequences indicated that the “core” of the enzyme has probably evolved in poxviruses four times as quickly as in vertebrates. Characterization of the FPV TK gene may facilitate the construction of recombinant FPVs as vehicles for the delivery of vaccine antigens to poultry and other avian species. Q 1997 Academic PWSS. IIPC.

region of 60 to 100 nucleotides preceding the mRNA start site is enriched in A+T and differs significantly from eukaryotic and prokaryotic regulatory sequences. The nature of promoters from genes of other poxviruses has not been studied. Using vaccinia virus as a selectable cloning vehicle we recently identified a 5.5-kb EcoRl fragment of the fowlpox virus (FPV) genome containing a functional TK gene (Boyle and Coupar, 1986). The location of the TK gene in this fragment was further mapped to the region of a 700-bp Xbal fragment. In this paper we report the nucleotide sequence of the TK gene of FPV and the amino acid sequence derived from it. These sequences have homology with the vaccinia virus TK gene and other TK genes and proteins. Further, the nucleotide sequence immediately upstream of the FPV TK gene is very A+T rich, similar to vaccinia virus promoter regions. This region operates as a promoter in both FPV and vaccinia virus since vaccinia virus recombinants containing the 5.5-kb EcoRl FPV DNA fragments express TK activity without a requirement for a vaccinia virus promoter (Boyle and Coupar, 1986). Finally we analyze the homologies of the vertebrate and poxvirus TKs and speculate on the phylogeny of this family of enzymes.

INTRODUCTION Poxviruses are large DNA viruses that replicate within the cytoplasm of infected ‘cells. Vaccinia virus, the type species of the orthopoxvirus group, has been widely studied while members of other poxvirus groups have been largely neglected. Mapping and nucleotide sequence determination of the thymidine kinase gene (TK) of vaccinia virus (Hruby et al., 1983; Weir and Moss, 1983) led to the development of a general method for the insertion of foreign DNA into vaccinia virus (Mackett et al., 1984; Boyle et a/., 1985). A variety of foreign genes have been inserted into the genome of vaccinia virus (Smith et a/., 1983; Panicali et al., 1983; Kieny er al., 1984; Boyle et a/., 1985). Expression of these foreign genes required their positioning near unique vaccinia virus transcriptional regulatory signals (promoters) recognized by the viral RNA polymerase. Promoter regions of a number of early and late vaccinia virus genes have been identified and sequenced (Venkatesan et a/., 1981; Weir and Moss, 1983, 1984; Bertholet et al., 1985; Boyle et al., 1985; Plucienniczak et al., 1985). A

’ Author

to whom

correspondence

should

be addressed. 355

0042-6822/87

$3.00

Copyright 0 1987 by Acaaemlc Press. Inc. All rights of reproduction I” any form reserved.

356

BOYLE ET AL

MATERIALS

AND

METHODS

Enzymes Restriction endonucleases and DNA-modifying enzymes were obtained from several commercial suppliers and used according to the manufacturers’ instructions or those described in detail by Maniatis et

al. (1982). Viruses and cell cultures Primary and secondary chick embryo skin cell cultures were prepared from specific pathogen-free embryonated eggs (CSIRO, SPF Poultry Unit, Maribyrnong, Victoria) as described by Silim et al. (1982). Fowlpox virus (Mild Vaccine Strain: Arthur Webster Pty. Ltd., Northmead 2152, Australia) was adapted to chick embryo skin cell cultures by passage at low multiplicity. Chick embryo skin cell cultures overlaid with Eagle’s minimum essential medium with Earle’s salts containing 1% agar and 5% fetal bovine serum were used for plaque assays. Plaques were stained with MlT tetrazolium on the 5th or 6th day after inoculation (Klebe and Harriss, 1984). Virus stocks were disaggregated by digestion for 30 min at 37” with 1 mg/ml of trypsin immediately before dilution for assays or infection of cell cultures. Human 1436 cells, a TK- variant of cell line R970-5 (Rhim et a/., 1975) were obtained from Dr. K. Huebner (Wistar Institute, Philadelphia) and grown in Eagle’s basal medium with 5% fetal bovine serum and 25 pg/ ml of 5’-bromodeoxyuridine (BUdR). Cells were passaged at least twice in the absence of BUdR prior to use in experiments. An L929 cell adapted vaccinia virus, VV-WR-L929, and a vaccinia virus recombinant, W-FPV-TK, containing a 5.5-kb EcoRl fragment of the FPV genome have been described previously (Boyle and Coupar, 1986).

Preparation

of cell lysates for TK assays

Confluent monolayers of chick embryo skin cell cultures were infected with 1O-20 plaque-forming units/ cell of virus or mock infected. At appropriate times after infection cell lysates were prepared and TK enzyme assayed as described previously (Boyle and Coupar, 1986).

Nucleotide

sequence

analysis

Nucleotide sequence analysis was carried out on the 2.2-kb HindIll-C/al fragment of FPV genome in the region of the 700-bp Xbal fragment, previously shown to contain a functional TK gene (Boyle and Coupar, 1986). Nucleotide sequences were determined from 3’ endlabeled DNA fragments by the method of Maxam and

Gilbert (1977) and by the “dideoxy” method after subcloning fragments into bacteriophage Ml 3 (Messing et a/., 1981). The majority of the sequence was determined from both DNA strands.

Mapping of the 5’ end of mRNAs by primer extension Cytoplasmic RNA was prepared from cells infected with W-WR-L929 or VV-FPV-TK or control cells (Human 143B, TK- cells) as described by Cooper and Moss (1979). Early RNA was prepared at 4 hr postinoculation from cells infected in the presence of and maintained in medium containing 100 pg/ml cycloheximide. Late RNA was prepared from infected cells at 8 hr postinoculation. A 68-bp DNA fragment from the cloned FPV TK gene (Sau961 to Dral fragment, bases 1167-l 235, Fig. 1) was used as a primer to map the 5’ end of the FPV TK mRNA expressed by the vaccinia virus recombinant, VV-FPV-TK. The primer was end labeled, annealed to total cytoplasmic RNA (early or late), extended with reverse transcriptase, and the products were analyzed, after denaturation in formamide, on 3% polyacrylamide gels containing 7 M urea (Sleigh et a/., 1981). The 68-bp (Sau961-Dral fragment) has only 63% homology with the vaccinia virus TK gene in this region and therefore is unlikely to act as a primer on the vaccinia virus TK mRNA.

Glyoxal gel electrophoresis

and Northern

transfer

RNA samples denatured by glyoxal treatment were separated by electrophoresis on 1.5% agarose gels in 10 rnM phosphate buffer, pH 7.0, at 4”, as described by McMaster and Carmichael (1977). RNA was transferred to a GeneScreen membrane according to the manufacturer’s instructions using 10 mlLl phosphate buffer, pH 7.0. Parallel tracks containing glyoxalated markers (ribosomal RNA or restriction fragments of DNA) were cut off before transfer and stained with acridine orange.

Analysis of sequence

data

Nucleotide sequences and amino acid sequences derived from them were manipulated using a VAX computer. Nucleotide sequences of the TK genes for herpes simplex, monkeypox, vaccinia, and variola viruses, man and chicken, were obtained from the Genbank nucleotide database (Revision 38; 1985). Initially the derived amino acid sequences were aligned using a diagonally enhancing variant (McLachlan, 1971, 1972) of the dot-matrix method (Gibbs and McIntyre, 1970) and by the method of Needleman and Wunsch (1970); both methods used the same matrix of amino acid substitution frequencies (McLachlan, 1972). Den-

FOWLPOX VIRUS THYMIDINE

drograms were calculated from the percentage sequence homologies of the aligned sequences using the “group average” sorting strategy (Gibbs and Fenner, 1984). The NBRF protein sequence database was searched using the NBRF Align program, and another based on amino acid nearest-neighbor comparisons (Gibbs et al., 1971). RESULTS Location, nucleotide sequence acid sequence of the FPV TK

and derived amino

The TK gene of FPV was previously mapped to a 2.2kb HindIll-C/al fragment derived from a 5.5-kb EcoRl fragment of the FPV genome (Boyle and Coupar, 1986). The TK gene was further mapped to the region of a 700-bp Xbal fragment contained within this HindIll-C/al fragment (Fig. 1). Analysis of the nucleotide sequence determined from this region revealed an open reading frame of 183 codons commencing 279 bp to the right of the central Xbal site and terminating 273 bp to the left of this Xbal site (Figs. 1 and 2). Identification of this region as the FPV TK gene was confirmed by its homology with vaccinia virus TK at both the nucleotide and amino acid levels (discussed below). The derived FPV TK polypeptide has a calculated molecular weight of 20,380 and is six amino acids larger than the vaccinia virus TK gene product. The first translation initiation codon in this open reading frame appears to be the start of the FPV TK gene for a number of reasons. It is flanked by nucleotides characteristic of preferred eukaryotic initiation sites while the next ATG, 12 codons downstream, is not (Kozak, 1984, 1986). There is a highly conserved amino acid sequence in the TKs (compared below) immediately downstream of the first ATG and surrounding the second suggesting a functionally important region of the TK enzyme. Mapping of the 5’ end of the mRNA shows that the transcript commences upstream of this first ATG, at least when expressed by the vaccinia virus recombinant, VV-FPV-TK (Figs. 3 and 5). The distribution of translation initiation and stop codons in the nucleotide sequence shown in Fig. 1 in the three reading frames of both strands is presented in Fig. 2. In addition to the TK gene, a smaller open reading frame of 89 codons, one base out of phase with the TK, commences 77 bp downstream (bases 17662032). It could code for a protein of approximately 10,000 Da. Interestingly the 75 bp upstream of this open reading frame is 78% A+T (compared with the 7 19/oA+T of the whole sequenced fragment), and this suggests that it may contain a poxvirus promoter. Further, Northern hybridization analysis suggests that this

KINASE

357

short open reading frame is transcriptionally active (Fig. 4). There are no open reading frames coding for proteins of 10,000 mol wt or larger in any of the three reading frames on the complementary strand of the sequence in Fig. 1 (Fig. 2). The codon usage in the FPV TK gene compared with that of the vaccinia virus TK gene is given in Table 1. There are definite biases in codon use of the FPV and vaccinia virus TKs. The codons used by FPV and vaccinia virus TKs are very similar with some exceptions, e.g., those for leucine, valine, and proline. The derived amino acid compositions of FPV and vaccinia virus TKs are very similar (Table 2) reflecting the high degree of homology between them. However, the FPV TK contains less glutamine and proline and more serine and valine than the vaccinia virus TK. Mapping of the 5’ end of the FPV TK mRNA expressed by the vaccinia virus recombinant W-FPV-TK The primer extension method was used to locate the 5’ end of the FPV TK mRNA expressed by W-FPV-TK. Both early and late cytoplasmic RNA from uninfected, VV-WR-L929 and VV-FPV-TK infected cells was hybridized to the 68-base Sau96I-Dral primer fragment (Fig. 1) and extended with reverse transcriptase. A major product, 114 bases in length, was present with the early RNA from W-FPV-TK infected cells (Fig. 3). This result very likely maps the 5’ end of the TK mRNA to an A residue at nucleotide position 1123 (Figs. 1 and 5). The 14-base untranslated leader of the FPV TK mRNA expressed by W-FPV-TK is therefore larger than the 6-base leader of the vaccinia virus TK mRNA (Weir and Moss, 1983). The size of the TK mRNA transcript was determined by Northern analysis (Fig. 4). Hybridization of early cytoplasmic RNA from VV-FPV-TK infected cells with 32Plabeled 700-bpXbal fragment from the TK gene region (probe B, Fig. 4) showed transcripts of 450, 700, and 1000 nucleotides. When hybridized with 32P-labeled Xbal-C/al fragment (probe A, Fig. 4) a major transcript of 700 nucleotides with a minor transcript of 1000 nucleotides was identified. These data suggest that the FPV TK gene expressed by the VV-FPV-TK virus has two transcripts, a major one of 700 nucleotides and a minor one of 1000 nucleotides. The other 450-nucleotide mRNA transcript (specific to probe B) may be derived from transcription of the short open reading frame identified downstream of the TK gene (Figs. 2 and 4). Late transcripts from the FPV TK region were heterogeneous in size. No FPV TK specific transcripts were detected in RNA from uninfected cells or from those infected with VV-WR-L929 (Fig. 4).

358

BOYLE E

I

ET

HX

x

,

‘TK 1

,

16861137

0

\I

2190

AL.

C

I

I

E I

1030.

.llOO CTCCGTTTTATGGAAATATTTTCTACTATTATGTTTATTCCT~~T~TTATATTGTACGCTGCTTATATA

AGAAAAATTAAAATGAAAAATAATTAGAATCTGAAAATGTCTTCTGG~~ATCCATGTTATTACAGGCCCTATG MSSGSIHVITGPM .1250 TTTTCCGGTAAAACATCGGAGCTAGTAGT~G~G~T~GATTTATGCTATCT~CTTT~TGTATTATTATT FSGKTSELVRRIKRFMLSNFKCIII AAACATTGTGGAGATAATAGATATAATGAGGATGAGGATGATAT-C-GTATATACT~TGATCTATTGTTTATGGAG KHCGDNRYNEDDINKVYTHDLLFME .1400 GCTACGGCATCTTCTAATCTATCTGTATTAGTACCTACGC ATASSNLSVLVPTLLNDGVQVIGID Xbal . GAGGCTCAATTCTT~~ATAGTAGTAGAATTTAGTGAC EAQFFLDIVEFSESMANLGKTVIVA .1550 GCGCTTAACGGTGATTTTAGCGAATTATTATTCGGT~CGTATAT~GTTATTATCATTAGCTG~CAGTGTCC A 1. N G D F K R E L F G N V Y K L L S LA E TV

S

AGTTTGACAGCTATTTGCGTGAAATGCTATTGCGACGCTTCGTTTTCT~CGAGTTACAG~T~G~GTA SLTAICVKCYCDASFSKRVTENKEV .1700 ATGGATATAGGTGGTAAAGATAAATACATACATAGCCGTGTGTA~~TGTTTTTTTAGT~TT~~GGTTTAGTGT MDIGGKDKYIAVCRKCFFSN*

.1850 TATTAGTTCTTGCAGAATGATATATTCTGTTCTCGAACAATCA

.2000 TCACTTTGTAAGATACATAATTAACAAATTCAGGGGGAAACTATAGATAT

.-. ATCAAAAGGTAGACAACAAATAATCAGAACCTAATTTTTTTTATCAAAAAATTAAAATATAAATAAAATGAT .2150 AACTTGTATGAAGAAAAAA TGAACATGAGTAAGAAACAAGTAAAAACTCAAAGTAAATGTAATAATAACGCAT~ Xbal

.

.

Hindlll.2190

AGATTTACATGCTTGGATGCGGTGCAATAC~T~~T FIG. 1. Location, nucleotide sequence, and derived amino acid sequence of the FPV TK gene and flanking regions. The nucleotide sequence was determined as described under Materials and Methods. Restriction enzyme cleavage sites are indicated as follows: E, EcoRI; H, HindIll; X, X&l; C, C/al. Not all HindIll, Xbal, and C/e1 sites present in the 5.5-kb EcoRl fragment are shown. The position of the FPV TK gene was confirmed by its homology to the vaccinia virus TK gene. The derived amino acid sequence is presented using the single-letter amino acid code. The Sau96I-Dral fragment used as primer for 5’ end mapping of mRNA is indicated by the line above the sequence. The initiation and termination codons of the second open reading frame identified in this fragment are similarly indicated. *Termination codon.

359

FOWLPOX VIRUS THYMIDINE KINASE

l

II II

III. I

l

*

I ,I1111 II II II II

III.. I IW, I,,,

b

580 ’ START

1;o

Illll Ill,,

I.

IIU II II III 1 IIII

l&O

.I,

260

I, II If

IIII I, ,I

II

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1I., I II

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III IIII

II

!1111/ II 300

1

IIII II

I

I

l

I

I

4,II 1 3’4- 5’ 350

2 3 4ho

t STOP

FIG. 2. Distribution of translation initiation and stop codons in the nucleotide sequence of Fig. 1. The sequence was translated in all three reading frames from both strands. Initiation codons are indicated by the diamonds and stop codons by the vertical lines. The scale units are codons. Open reading frames coding for potential proteins of greater than 10,000 mol wt are indicated by the arrows above the reading frames.

Comparison of FPV and vaccinia virus TK gene promoters The FPV TK gene is expressed in vaccinia virus without requiring a vaccinia virus promoter. This suggests that the vaccinia virus RNA polymerase is able to recognize the FPV TK gene promoter sequences. A comparison of the first 100 bp upstream of FPV and vaccinia virus TK genes shows that both regions are A+T rich, containing more than 75% A+T in the first 50 to 60 bp (Fig. 5). There is significant nucleotide sequence homology (43%) between their first 60 bp, but thereafter it declines to less than 30%. Weir and Moss (1983) identified two A+T rich consensus sequences located near each other and close to the transcription start points for the vaccinia virus

TK and three other early mRNA species. These sequences are conserved in the proposed FPV TK promoter region. From a comparison of 11 vaccinia virus gene promoters, Plucienniczak et a/. (1985) proposed a more general consensus sequence TATAIAATAA spaced by 20 to 24 bp which is also conserved in the FPVTK promoter region (Fig. 5). The distances between these consensus sequences and the 5’ ends of the two TK mRNAs differ, perhaps due to inaccurate initiation in this heterologous system. Nevertheless, the proposed FPV TK promoter region is recognized by vaccinia virus RNA polymerase in expressing FPV TK activity at a level comparable to that observed with wild type vaccinia virus. The FPV region appears to operate as an early promoter since its activity is not affected if DNA synthesis is inhibited by cytosine arabinoside (Boyle and Coupar, 1986). Comparison of the FPV TK gene with orthopoxvirus, herpes simplex virus, and cellular TK genes

PRIMER 68 b FIG. 3. Mapping of the 5’ end of the FPV TK mRNA expressed by the vaccinia virus recombinant, W-FPV-TK. Early (E) and late (L) cytoplasmic RNA from uninfected (U), W-WR-L929, and W-FPV-TK infected cells was hybridized with 68-base Sau96I-Dral primer (Fig. 1) which was extended with reverse transcriptase. Hinfl-f’vull fragments isolated from plasmid pJC119 were used as size markers (M) when the products were analyzed on polyacrylamide gels.

The deduced amino acid sequence of FPV TK was compared with the reported sequences of the TKs of man (Bradshaw and Deininger, 1984) chicken (Merrill et al,, 1984) herpes simplex virus 1 (McKnight, 1980) and three other poxviruses: vaccinia (Weir and Moss, 1985) variola, and monkeypox (Esposito and Knight, 1984). The TK of herpes simplex virus showed no statistically significant sequence homology with the other TKs and was not studied further. In contrast, the TKs of the four poxviruses and two vertebrates showed unequivocal sequence homology with one another. This is an extension of the observations reported by Kwoh and Engler (1984) that the chicken and vaccinia TKs have significant sequence homology. The enhanced dot-

360

BOYLE ET AL. U El

Ll

E2 L2

E2 L2

mologous. Similarly, the additional C-terminal regions of the vertebrate enzymes are related and insertion of a single space in the chicken enzyme renders a 31. residue portion 42% homologous, though the human enzyme is 10 residues longer. The splicing sites of the six introns in the genomic sequence of chicken TK (Merrill et al,, 1984) show no relationship either to the regions of greatest homology in the six amino acid sequences, or to the places where spaces were inserted in order to maximize homology.

28s p

18s~

Time course of TK enzyme expression

in FPV-infected 5%

probe

(6)

(A)

HX-(8)-XI

(A)

-

C 1

Yir700

RNA TRANSCRIPTS

&Q

.

l

1000

FIG.4. Size of FPV TK mRNA transcripts expressed by VV-FPV-TK and proposed partial transcription map of the HindIll-C/al fragment, Total cytoplasmic RNA from uninfected, W-WR-L929, and W-FPVTK infected cells was analyzed on agarose gels as described under Materials and Methods. The RNA was hybridized with 32P-labeled Xbal-Xbal fragment (probe B) or Xbal-C/al fragment (probe A). Restriction enzyme cleavage sites are indicated as follows: H, HindIll; X, Xbal; C, C/al. The length of the RNA transcripts is indicated in nucleotides. RNA preparations are indicated as follows: U, uninfected; E, early, L, late; 1, W-WR-L929; 2, W-FPV-TK.

diagram method was then used to align all six TKs by inserting the minimum number of spaces to maximize their homology. The result (Fig. 6) shows that most of the central (“core”) region of each enzyme is homologous to the same region of each of the other five enzymes; the fowlpox enzyme is longest in this region, and the others were aligned by inserting five or six spaces. The “core” regions of the enzymes vary in homology from 97.2% for the variola-vaccinia comparison to 48.6% for the fowlpox-variola and fowlpoxmonkeypox comparison (disregarding all residue comparisons that involved an inserted space) (Table 3). The two vertebrate enzymes differ most noticeably from the virus enzymes in that they possess additional N-terminal and C-terminal sequences of 15 and 31 (chicken) or 42 (man) residues, respectively. The additional N-terminal sequences of the vertebrate enzymes are both 15 residues in length and 47.1% ho-

cell cultures

To show that the FPV TK gene is active during FPV infection of chick embryo skin cells, cytoplasmic extracts were assayed for TK enzyme at various times after virus infection. Uninfected chick embryo cell cultures had very low thymidine kinase activity in cytoplasmic extracts. Following FPV infection, elevated levels of TK enzyme activity were first detected at 4-8 hr and increased to maximum levels by 24 hr. Enzyme levels remained constant over the next 24 hr, then declined slowly (Fig. 7).

DISCUSSION We have determined the nucleotide sequence of a FPV genome fragment, previously shown to contain a functional TK gene. This sequence has an open reading frame of 183 codons, which is homologous at the nucleotide and deduced amino acid levels with the vaccinia virus TK gene. This confirms that the fragment contains the FPV TK gene. The calculated molecular weight of the FPV TK is slightly larger than that of the vaccinia virus TK. Native vaccinia virus TK has a molecular weight of about 80,000 Da and thus it probably exists as a tetramer (Kit et al., 1977). The functional FPV TK enzyme has not been characterized. We have shown previously that the FPVTK promoter appears to function when cloned into vaccinia virus (Boyle and Coupar, 1986). The sequence homologies between the two TK gene promoters substantiate this observation. The ability of the vaccinia virus RNA polymerase to recognize the FPV TK promoter extends beyond its ability to initiate synthesis of a mRNA transcript. Temporal regulation of the FPV TK gene in W-FPV-TK appears also to be maintained. We previously reported that the FPV TK promoter operates as an early promoter in the recombinant VV-FPV-TK (not being inhibited by cytosine arabinoside). Here we showed that FPV TK enzyme activity in FPV-infected chick embryo skin cells is turned on between 4 and 8 hr after infection. FPV DNA synthesis in these same cells commences between 12 and 16 hr after infection (Prideaux, Coupar,

FOWLPOX

VIRUS

THYMIDINE

TABLE

361

KINASE

1

CODON USAGE OF FPV AND VACCINIA VIRUS TKse Amino acid Phe Leu

Ile

Met Val

Codon

FPV

WC

m -IX TTA TG Cl7 CTC CTA CTG AT ATC ATA ATG G-I-T GTC GTA GTG

IO 2 7 2 1 0 6 0 6 1 7 6 4 0 8 4

7 3 2 5 1 1 3 0 6 3 9 6 3 1 2 5

a Vaccinia virus data from * ***STOP codons. c W-Vaccinia virus.

Weir

Amino acid Ser

Pro

Thr

Ala

and Moss

Codon

FPV

VV

TCT TCC TCA TCG CCT ccc CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG

7 3 1 2 2 0 0 0 1 0 6 2 6 2 1 1

3 2 3 1 0 1 2 1 3 1 3 2 3 1 4 0

DERIVED AMINO

2

ACID COMPOSITIONS VACCINIA VIRUS TKs Number

Amino

acid

FPV

Ala Au Asn Asp CYS Gln Glu GIY His Ile Leu LYS Met Phe Pro Ser Thr Tw Tyr Val

’ Vaccinia

OF FPV AND

in TK of Vaccinia

IO 7 11 11 7 2 10 11 3 14 16 14 6 12 2 77 9 0 5 16

Total data from

Weir

virusa 8 9 10 9 7 6 14 13 2 18 12 12 6 10 4 10 9 1 6 11

183 virus

177 and Moss

Tyr l ***

His Gln Asn Lys Asp Glu

Codon

FPV

VV

TAT TAC

4 1 1 0 3 0 1 1 7 4 13 1 8 3 6 4

4 2 1 0 2 0 4 2 7 3 9 3 7 2 IO 4

TM

TAG CAT CAC CAA CAG AAT PAC AAA AAG GAT GAC GAA GAG

Amino acid CYS *** Trp Arg

Ser Arg GIY

Codon

FPV

VV

TGT TGC TGA TGG CGT CGC CGA CGG AGT AGC AGA AGG GGT GGC GGA GGG

4 3 0 0 0 1 1 0 3 1 4 1 7 1 3 0

5 2 0 1 3 0 2 0 1 0 4 0 4 2 6 1

(1984).

and Boyle, unpublished observations). These results suggest that the FPV TK promoter operates as an early promoter in both FPV and the VV-FPV-TK recombinant. TABLE

Amino acid

(I 984).

Further, the production of early mRNA transcripts of discrete size suggests that the 3’ termination signals of the FPV TK gene are also recognized by vaccinia virus RNA polymerase. Since we have not yet mapped the 5’ end of FPV TK mRNA expressed by FPV, we do not know if the vaccinia virus and FPV RNA polymerases use the same 5’ start site on the FPV TK promoter. It will be interesting to determine if other FPV early and late promoters operate in vaccinia virus and vice versa. The apparent transcriptional activity of an 89-codon reading frame in the sequenced fragment suggests that this may be so. The possibility exists that promoters from other poxviruses will also operate in vaccinia virus. The apparent ability of the vaccinia virus RNA polymerase to faithfully recognize promoters of other poxviruses may be one of the mechanisms that enable nongenetic reactivation of poxviruses from different genera to occur (Hanafusa et al., 1959; Joklik et al., 1960). Cells infected with FPV show a marked increase in TK enzyme activity in the first 24 hr following infection. At least part of this increase can be attributed to viruscoded TK enzyme, since we have shown that the FPV genome contains a TK enzyme gene. Some of the increased TK activity might also be due to the induction of host TK enzyme. Since there are no TK- chicken cell lines available, it is not possible to determine the contribution of host TK activity to the overall level or to generate TK- FPV mutants by BUdR mutagenesis. The relatedness of the poxvirus and vertebrate TKs is much greater than that reported to exist between

362

BOYLE

FPV vv

ET

AL

1040 1050 1060 1070 1080 1090 TATGGAAkTATTTTCTACTATTATGTTTATTCCTGGAfiTAkTTPlTATTGTA~GCTG~TTATATAAGAAAkATTkk~~TGA :: :::: : :: : : : : : : : : : :: :: TGT-TkGkTA-CATAGA-TCCT-CGTCGCAATATCGCkT-~GTG~ 400 410 420 430 440 11 v 1130 A a+ A STTAG~ATCTGAAA------ATG ~~~~~~~-IITcTT~~T~~*T~ . . . . 480 . ..a :

:::

:

FIG. 5. Comparison of proposed FPV and vaccinia Consensus sequences proposed by Weir and Moss line above the sequence. The 5’ ends of the mRNAs

r-.

1110

. . :

:::

450

::

: : : :

460

: : : 470

ATG virus TK gene promoter regions. The first 100 bp upstream of the TK genes (1983) are underlined while those of Plucienniczak et al. (1985) are indicated are indicated by the arrowheads.

certain alphaherpesvirus proteins and members of the protein kinase family from oncogenic retroviruses, yeast, and cows (McGeoch and Davison, 1986). The relationships, and hence the possible phylogeny, of the

1

fowlpox variola vaccinia monkeypox human chicken

w&s”

10

is presented, by a broken

four poxvirus and two vertebrate enzymes was assessed from their aligned sequences (Fig. 6). It is a common practice with such aligned sequences either to disregard those parts of the sequences that are not

20

40

30

MS---------------SGSIHVITGPMFSGKTSELVRRIKRFML MN---------------GGHIQLIIGPMFSGKSTELIRRVRRYQI MN---------------GGHIQLIIGPMFSGKSTELIRRVRRYQI MN---------------GGHIQLIIGPMFSGKSTELIRRVRRYQI MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQI MNCLTVPGVHPGSPGRPRGQIQVIFGPMFSGKSTELMRRVRRFQL + + tt t ttttttttttt ttttt t

60 70 80 100 50 90 110 SNFKCIIIKHCGDNRMEDDINKWTHDLLFMEATASSNLSVLVPTLLNDGVQVIGIDEAQFFLD AQYKCVTIKYSNDNRY----GTGLWTHDKNNFEALEATKLCDVL-EAI-TDFSVIGIDEGQFFPD AQYKCVTIKYSNDNRY----GTGLWTHDKNNFEALEATKLCDVL-ESI-TDFSVIGIDEGQFFPD AQYKCVTIKYSNDNRY----GTGLWTHDKNNFAALEVTKLCDVL-EAI-TDFSVIGIDEGQFFPD AQYKCLVIKYAKDTRY---- SSSFCTHDRNTMEALPACLLRDVAQEAL--GVAVIGIDEGQFFPD AQYRCLLVKYAKDTRY---CTTGVSTHDRNTMEARPACALQDVYQEAL--GSAVIGIDEGQFFPD ttttt ttt t tt ttt t tt ttt t tttttttttttt

120 130 140 160 170 150 IVEFSESMANLGKTVIVAALNGDFKRLFGNVYKLLSLAETVSSLTAICVKCYCDASFSKRVTEN WEFCERMANEGKIVIVAALDGTFQRKPFNNILDLIPLSEMWKLTAVCMKCFKEASFSKRLGTE IVEFCERMANEGKIVIVAALDGTFQRKPFNNILNLIPLSEMVVKLTAVCMKCFKEASFSKRLGEE WEFCERMANEGKIVIVAALDGTFQRRPFNNILNLIPLSEMCFKEASFSKRLGTE IMEFCEAMANAGKTVIVAALDGTFQRKPFGAILNLVPLAESWKLTAVCMECFREAAYTKRLGTE IVEFCEKMANTGKTVIVAALDGTFQRKAFGSILNLVPLAESWKLNAVCMECYREASYTKRLGAE ttttt ttt tt tttttttttttt t tt t tt t ttttttttt t ttt tt+t t 180 190 200 210 KEVMDIGGKDKYIAVCRKCFFSN-------------

220

230

--____-_---------------------

240

TKIEIIwNDMYQSVCRKCYIDS--------------------------------------------TEIEIIwNDMYQSVCRKCYIDS---------------------------------------------

________-_-----___-----------------------TEIEIIGGNDMYQSVCRKCYIDS KEVEVIGGADKYHSVCRLCYFKKASGQPAGPDNKENCPVPGKPGEAVAARKLFAPQOILQCSPAN REVEVIGGADKYHSVCRACYFQK-RPPQLGSENKENVPMGIFAS---------t t ttt t t tttt t-t FIG. 6. Amino their homology;

acid sequences of the TKs of four poxviruses and two vertebrates aligned using the minimum number of spaces (-) to maXimiZe a plus (+) has been placed under each column where five or more of the six residues in that position are the same.

FOWLPOX

VIRUS

THYMIDINE

TABLE HOMOLOGY Enzyme

Homology

from:

of the “core”

Fowlpox Variola Vaccinia Monkeypox Chicken Homology Fowlpox Variola Vaccinia Monkeypox Chicken B Residues * Inserted

Variola (%)

(%I

Chicken (%I

Man (%)

aligned

50.3 97.2

48.6 97.2 96.6

53.1 68.2 68.7 67.6

52.2 66.5 67.6 65.9 81.2

60.8 97.9

59.6 97.9 97.5

39.2 52.1 52.5 51.7

43.3 55.0 55.8 54.6 72.5

sequences*

of Fig. 6, disregarding any comparisons as though a 2ist amino acid.

involving

ii

. .

, 72 POST

I&CTION

FIG. 7. Time course of TK enzyme expression in FPV-infected chick embryo skin cells. TK activity is expressed as picomoles of dT phosphorylated in 30 min at 37’ per 10’ cells. l , FPV infected; l , mockinfected.

space.

/iI b)

ij 70-

12 24 HOURS

an inserted

ocally, that the sequences of the two vertebrate enzymes are more closely related to those of the mammalian poxvirus enzymes than to that of FPV; the two branch points involved are at dissimilarities of 67.3 and 50.89/o with standard errors of 0.43 and 0.92%, respectively. This branching pattern implies that the “ancestral” enzyme was poxviruslike, and that after separating from the mammalian poxvirus progenitor, the mammalian TK ancestor acquired additional N- and Cterminal portions. These portions might have been acquired by the insertion of a poxviruslike TK into another

jso

4

KINASES

regions”

represented in all the sequences being compared, or to consider that a series of contiguously inserted spaces has arisen from a single evolutionary “event.” We started by adopting the first strategy, and assessed the percentage homology of each pair of enzymes using only sequences from the common “core” region (residues 18 to 198) disregarding the comparisons that involved an inserted space. These homology estimates (Table 3) were used to calculate, by “group average” sorting strategy (Gibbs and Fenner, 1984) the dendrogram shown in Fig. 8a. This dendrogram confirms the close sequence similarity between the three vertebrate poxvirus enzymes (ca. 97% homologous), and between the two vertebrate enzymes (81.2% homologous). However, it also shows surprisingly, but quite unequiv-

L..

VERTEBRATE THYMIDINE

Monkeypox

Vaccinia (%I

59.6

18 to 198 inclusive spaces were treated

363

3

OF THE AMINO ACID SEQUENCES OF FOUR POXVIRUS AND Two

48.6

of the entire

KINASE

60-

FIG. 8. Dendograms illustrating the relatedness, and hence possible phylogeny, of the TKs of four poxviruses and two vertebrates. (a) Was calculated from the homologies of the “core” regions, disregarding any comparisons involving an inserted space; (b) was calculated from the homologies obtained when inserted spaces (-) in the aligned sequences were treated as though they were a 21st amino acid (Table 3).

364

BOYLE

gene: however, a search of the NBRF protein data base for amino acid sequences related to the concatenated N- and C-terminal sequences of the vertebrate TKs found only short homologies with other sequences and these did not span the possible point of insertion of the poxviruslike TK. Thus the possible phylogeny of the “core” of these enzymes, and hence of the organisms that encode them, is unexpected because, except for the orthopox viruses, the major groupings of most poxviruses reflect the relationships of their natural hosts (Matthews, 1982), and it is thus likely that most poxviruses coevolved with their hosts, rather than before them! An alternative and more intuitively acceptable interpretation of the unexpected relationships shown by the aligned TKs is that the “core” sequence of the enzyme has evolved in vertebrates more slowly than in poxviruses, and therefore a single dendrogram cannot directly reflect the phylogeny of these enzymes. It is noteworthy that different parts of the vertebrate TKs appear to have evolved at different rates: the N- and C-terminal portions are only 47 and 42% homologous, whereas their “cores” are 8 1% homologous. One way to “correct” for the possible diminished rate of evolution of the “core” is to include all residues when estimating homologies of the TKs, individual inserted spaces being treated as though they are a 2 1st amino acid (Table 3). A dendrogram (Fig. 8b), calculated from data obtained in this way, shows a branching pattern more like that predicted from the taxonomy of the poxviruses and their natural hosts, i.e., all the poxvirus enzymes in one primary division, and the two vertebrate enzymes in the other. If this alternative interpretation is correct, and if the correlation between the taxonomy of poxviruses and their natural hosts resulted from their coevolution (i.e., the fowlpox/mammalian pox TK division was at about the same time as the man/chicken TK division), then one can deduce by simple arithmetic, after correcting for reversions (Dayhoff et al., 1972), that the rate of evolutionary change of the poxvirus TK “core” sequences is about four times that of the homologous vertebrate sequences. A similar difference between virus and host proteins was reported by Soeda et al. (1980), who concluded from sequence comparisons that the proteins of three papovaviruses had changed five times more quickly than the globins of their coevolutionary hosts, and, more generally, Britten (1986) has shown that the single copy DNA of several different taxonomic groups evolve at rates that differ over a fivefold range. These conclusions leave unresolved whether a fulllength vertebratelike enzyme was the ancestral protein and lost portions of its N and C termini when it became

ET

AL

part of the genome of the progenitor of poxviruses, or whether a poxviruslike enzyme was the ancestor. To distinguish which of these conclusions is correct will require data on TKs from more “primitive” organisms. It would be of particular interest to know the structures and sequences of the TKs of an insect and an entomopoxvirus. If, by analogy with the vaccinia virus TK, the FPV TK is a nonessential gene for virus growth in tissue culture, then identification and sequencing of this gene may allow strategies to be developed for the insertion of foreign DNA into FPV. Recombinant FPVs may be useful vehicles for the delivery of vaccine antigens to poultry and other avian species.

ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mr. C. Rolls and Mrs. V. Corrigan. This work was carried out as a joint CSIROANU project. Drs. Boyle and Coupar are on secondment from CSIRO, Australian Animal Health Laboratory, Geelong, to the Department of Microbiology, John Curtin School of Medical Research.

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MCLACHLAN, A. D. (1972). Repeating sequences and gene duplication in proteins. J. Mol. Biol. 64, 417-437. MCMASTER, G. K., and CARMICHAEL, G. G. (1977). Analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Nat/. Acad. Sci. USA 74,4835-4838. MERRILL, G. F., HARLAND, R. M., GROUDINE, M., and MCKNIGHT, S. L. (1984). Genetic and physical analysis of the chicken TK gene. Mol. Cell. Ho/. 4, 1769-1776. MESSING, J., CREA, R., and SEEBURG, P. H. (1981). Asystem for shotgun DNA sequencing. Nucleic Acids Res. 9, 309-321. NEEDLEMAN. S. B., and WUNSCH, C. D. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. I. Mol. Biol. 48, 443-453. PANICALI, D., DAVIS, S. W., WEINBERG, R. L., and PAOLE~I, E. (1983). Construction of live vaccines by using genetically engineered poxviruses: Biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin. Proc. Nat/. Acad. Sci. USA 80, 53645368. PLUCIENNICZAK, A., SCHROEDER, E., ZET~LMEISSL, G., and STREECK, R. E. (1985). Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome. Nucleic Acids Res. 13, 985-998. RHIM, J. S., CHO, H. Y., and HUEBNER, R. J. (1975). Non-producer human cells induced by murine sarcoma virus. Int. J. Cancer 15, 23-29. SILIM, A., AZHARY EL, M. A. S. Y., and ROY, R. S. (1982). A simple technique for preparation of chicken-embryo-skin cell cultures. Avian Dis. 26, 182-l 85. SLEIGH, M. I., BOTH, G. W., UNDERWOOD, P. A., and BENDER, V. J. (1981). Antigenic drift in the hemagglutinin of the Hong Kong influenza subtype: Correlation of amino acid changes with alterations in viral antigenicity. J. Viral. 37, 845-853. SMITH, G. L., MACKETT, M., and Moss, B. (1983). Infectious virus recombinants that express hepatitis B virus surface Nature (London) 302, 490-495. SOEDA, E., MARUYAMA, T., ARRAND, Host-dependent evolution of three 285,165-167.

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