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The primary structure of the thymidine kinase gene of fish lymphocystis disease virus
VIROLOGY
182, 835-840 (1991)
The Primary Structure
of the Thymidine
Kinase Gene of Fish Lymphocystis
Disease Virus
PAUL SCHNITZLER,’ MICHAELA HANDERMANN, ORSOLYA SZ~PE, AND GHOLAMREZA DARAI’ lnstitut filr Medizinische
Virologie der UniversitAV Heidelberg, Received November
Im Neuenheimer
14. 1990; accepted
February
Feld 324, 6900 Heidelberg,
Germany
12, 199 1
The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIll (0.669 map unit; nucleotide position 1) and Accl (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man. o 1991 Academic press, I~C.
in comparison to the TK genes of eukaryotic cells, e.g., man (26, 27), mouse (28), and chicken (29), are a valuable technique for calculating the evolutionary stage of each species. The exact characterization and determination of the DNA nucleotide sequence of the TK gene of FLDV genome is the subject of this paper. In order to determine the exact position and the primary structure of the DNA sequences expressing the TK gene the following strategy was used. The particular region of the viral genome located between the genome coordinates 0.669 and 0.718 which harbors the complete TK gene locus of the FLDVf was molecularly cloned into the corresponding sites of plasmid vector pAT153 (30) and termed pFLDV-TK-C19. Furthermore a defined library harboring 14 subclones (Fig. 2C) of the DNA sequences located between the genome coordinates 0.669 (HindIll site) and 0.718 (EcoRI site) was established. This gene librarywas screened forthe ability to transform 3T3 TK negative to 3T3 TK positive cells. The 3T3 cell cultures with TK negative phenotype were grown and propagated in Eagle basal medium balanced with Earle’s salt solution supplemented with 10% fetal calf serum which contained 30 pglml bromodeoxyuridine. The transfection assay was carried out according to the calcium phosphate technique (31) and the DNA of 3T3 TK negative cells was used as carrier DNA as described previously (70). The transfected cultures were processed under HATG (0.1 mM hypoxanthin, 0.4 PMaminopterin, 16 @I thymidine, 10 puM glycine) selection pressure (32). According to the
Fish lymphocystis disease virus (FLDV) is the causative virus for lymphocystis disease, a common chronic disease of pleuronectes. FLDV has been classified as a separate genus of the lridoviridae family with the proposed name Lymphocysfivirus (1) which belongs to the icosahedral cy-toplasmic deoxyriboviruses. FLDV has many interesting properties; e.g., the genome structure of FLDV was found to be circularly permuted and terminally redundant (2-6), a common genomic feature of frog virus 3 (FV3) and insect iridescent virus type 6 (CIV) and type 9 (7-9). The thymidine kinase (TK) gene of FLDVf isolated from flounder was identified by biochemical transformation of 3T3 TK negative to 3T3 TK positive cells using specific viral DNA sequences as described previously (10). These analyses revealed that the DNA sequences located between the coordinates 0.61 1 to 0.718 (Table 1 and Fig. 1) of the viral genome harbor the TK gene of FLDVf with the ability for biochemical transformation of 3T3 TK negative cells to 3T3 TK positive cells. The primary structure of the TK genes of a variety of viruses including poxviruses (1 l-16), herpesviruses ( 17-24), and recently African swine fever virus (25) has been reported. The analyses of these particular genes Sequence data from this article have been deposited with the EMBUGen Bank Data Libraries under Accession No. M61 115. ’ Present address: Department of Microbiology and Immunology, Duke University Medical Center, Box 3020, Durham, NC 27710. ’ To whom requests for reprints should be addressed.
835
0042-6822191 $3.00 Copyright 0 199 1 by Academic Press, Inc. All rights of reproduction in any form reserved
836
SHORT COMMUNICATIONS
FIG. 1. Physical maps of the genome of fish lymphocystis disease virus isolated from flounder (A) (4) and fine mapping of the EcoRl FLDV DNA fragment C (B). The position and the DNA sequence arrangement of the TK gene are shown in (B) and (C), respectively.
results of this study shown in Fig. 1 E?it was found that the DNA sequence located between the /-/indIll (0.669 m.u.) and Accl (0.691 m.u.) sites was capable to convert 3T3 TK negative cells to 3T3 TK positive cells (Table 1). The primary structure of the FLDV TK gene was analyzed by determining the complete nucleotide sequence of the insert of the recombinant plasmid pFLDV-TK-Cl9 corresponding to the viral DNA sequences between the HindIll (0.669 m.u.) and Accl (0.691 m.u.) sites. The DNA sequencing was performed by the dideoxy chain termination procedure (33-35) using a modified T7 DNA polymerase (36). The sequence of each subclone was determined from both strands by analysis of the particular DNA fragment which was inserted into both sequencing vectors M 13mpl8 and Ml 3mpl9 (Fig. 2C) as described else-
where (5, 37, 38). The strategy of DNA sequencing is given in Fig. 2B. The DNA nucleotide sequence obtained from the HindllllAccl DNAfragment (0.669 to 0.691 m.u.) of the FLDV-f genome is shown in Fig. 3. This DNAsequence of 2032 bp has a base composition of 30.4% G + C and 69.6% A + T. The presence of all restriction sites mapped within this fragment was confirmed. A computer search for detecting open reading frames (ORFs) was carried out. One ORF was identified on the lower strand (954 bp; nucleotide position 219 to 1 172) encoding a polypeptide of 3 18 amino acid residues with a molecular weight of 35.3 kDa. A high probability to encode a protein was predicted for this ORF when the DNA nucleotide sequence of this particular region was analysed by the method of Fickett (39). The amino acid sequence of this gene is given in Fig. 3. This gene J
SHORT COMMUNICATIONS TABLE 1 OF 3T3 CELLS WITH TK NEGATIVE PHENOTYPETO 3T3 CELLS WITH TK POSITIVE PHENO~PE USING SusCLONESOF THE INSERTOF RECOMEINANTPLASMID Cl 9 (4.7 kbp; 0.669 TO 0.7 18 m.u.) HARBORINGTHE TK GENE OF FLDV-f GENOME THE RESULTS OF TRANSFORMATION
DNA fragment” kbd EcoRI-C (1 1.2) Subclones of EcoRI-C Cl9 Subclones of Cl 9 Subclones of C 19
Viral map unit 0.61 1 to 0.61 1 to 0.669 to 0.669 to 0.691 to
0.718 0.669 0.718 0.691 0.718
No. of coloniesb 28 0 33 42 0
a DNA fragments were obtained from the corresponding recombinant plasmids harboring the EcoRl DNA fragments of the viral genome. * Average of two experiments (0.1 pM DNA/i X lo7 cells). The specificity of the transformation assay was determined using Southern blot hybridization experiments In which the specific FLDV TK gene DNA sequences were detected in the biochemically transformed cells as described elsewhere (10).
corresponds to the DNA sequences of the viral genome located within the FLDV-f EcoRl DNA fragment C between the map coordinates 0.669 and 0.691 which possesses high TK-transforming activity (Table 1). The analysis of the DNA sequences upstream and downstream of the start and termination codons of the FLDV-f TK protein revealed the presence of classical or modified transcriptional signals, which are underlined in Fig. 4. This finding is in agreement with the previously described transcriptional start and polyadenylation signals detected upstream and downstream
0.609 Hlndlll
Cio(4.7
837
of the star-t and termination codons of a variety of predicted FLDV genes (5). For detecting the evolutionary relationships of the TK gene of FLDV to the other known TK genes a comparative analysis of their amino acid sequence was carried out according to the method of progressive sequence alignment (40). The results obtained by progressive sequence alignment were in agreement with the results obtained by the multiple alignment of the protein sequences using the method developed by Higgins and Sharp (4 1, 42). This method aligns pairwise similarities at the first step of computation (43). With exception of the amino acid motif GNMSGYK located at the amino acid position 283 to 289 of FLDV TK no other significant similarities were detected when the amino acid sequence of the FLDV TK was compared to the amino acid sequence of the known TKs of herpesviridae ( 1724). This motif is similar to the amino acid consensus sequence GXXGXGK which is common in all known TK genes including TKs of herpesviruses (44). In contrast evolutionary relationship was detected between the thymidine kinase of FLDV and those of poxvirus and cellular origin. An example of the results of these studies are given in Fig. 4. In this study the amino acid sequence of TK gene of FLDV was compared to the amino acid sequence of the TK genes of representative members of the cytoplasmic deoxyriboviruses like African swine fever virus (ASFV), fowlpox virus (FPOX), shope fibroma virus (SFV), vaccinia virus (VV), and to the amino acid sequence of the cytoplasmic TK of chicken (CHI), mouse (MOU), and human (HUM). These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene
kbp)
..
.
yw,
mu
Y.. .._
‘.._ “h
0.691
mu
FIG. 2. Diagram of the strategy of DNA sequencing (B) for the insert of recombinant plasmid Cl 9 (0.669 to 0.718 m.u.). The position of the cleavage sites of some restriction endonucleases of the particular regions are given In (A). The sequence of each DNA fragment was determined from both strands using the M 13mpl8 and -19 systems. The map positions of the individual subclones established are shown in (C).
838
SHORT COMMUNICATIONS 100
1 AATGTTTCACGAGGAGGCGATTATGTCCTTAATTCCTGGATGACGGTTCGTATACCTGCCATTAAATTGAAAGCTGATAATAGAATGAATAATAACGGCA
AccI 101
(2032)
CCATTCGATGGTGCAAAAACTTATTTCATAATCTAATTAAACAAACGTCGGTTCAGTTTAATGATTTAGTAGCACAAAAATTTGAAAGTTATTTCTTAGA -
200
219119691
201 TTATTGGGCAGCGTTCGTATGTGTGGCTCTAAAAGAGCAGGTTATAATAATATGATCGGTAATACAATTGATATGATACAACCGGTTGACCATACAGGAA TCGSKRAGYNNMIGNT IOMIQPVOHTG 1
300 27
301 TGTTACCTGAAAAAGTTTTAGTGTTACCATTACCGTATTTCTTTTCTAGGGACAGCGGTGTTGCATTGCCTAGTGCAGCACTTCCTTATAACGAAATAAG 28H L P E K VLVLPL PYFFSROSGVALP SAALPYNEIR
400 61
401 62
ATTAACGTTTCATCTTAGAGATTATACGGAATTACTTATTTTTCAGCATAAACAAGATTGTACCATTATACCAATTACAGCTGCTGATTTAGAATATGGA L T F H LROVTELLIFQHKOOC T I I P I T AAOLEYG
500 94
501 95
AAACCTGATTTAAAAGACGTTCAAGTATGGATTACTAATGCAGTGGTTACAAATGAAGAACGGCGACTTATGGGTACAACTCCTCGAGATATATTAGTGG K P 0 L K OVQVWI T N A V V T N E EARLMGTT P R
601 128
AACAAGTTCAAACTGCGCCAAAACATGTATTTCAACCGTTAACTATTCCAAGCCCTAATTTTGATATTCGGTTTTCACACGCTATTAAATTGTTATTTTT E 0 V Q TAPKHVFQPL TIPSPNFOIRFSHAIKLLFF
700
701 162
TGGTGTAAGAAATACAACTCATGCAGCTGTTCAATCTAATTATACTACAGCTTCTCCTGTTATTTTAGAAGAAGCCTATGCAAGCGATCTATCTTTAGTG GVRNTTHAAVQSNVTTASPVILEEAVASOLSLV
900
601
GCAGCAGATCCAATTGCAAATGTAACGCTTGTTTATGAAAATAGTGCACGGCTTAATGAAATGGGTAGTGAATATTATTCACTCGTTCAACCATATTATT A A 0 P I A N V T L V Y E N S A R L N E H G S E Y Y S L
900 227
195
0
I
L
v
600 127
161 194 V
Q
P
V
Y
901 TTGGTGGTTCTATTCCAATAGAAACAGGATATCATATGTATTGTTATTCATTAAATATGATGGATATGGATCCAATGGGATCCACAAATTACGGACGTTT 226FGGSIPIETGYHMYCVSLNMMOMOPMGSTNVGRL
1000 261
1001 ATCAAACGTTAGTATGAAATTAAAAACCTCAGACAAAGCAGTTGTAAATGCTGGTGGCGGTGGTGGTAATATGTCTGGTTATAAAGATGCACAAAAGTTT
1100 294
262
SNVSHKLKTSOKAVVNAGGGGGNMSGVKDAQKF
1172(91S)
1101 GAATTTCTTACTATGGCTATTAATCATAATGTAATTCGTATAAAGAACGGATCAATGGGTTTTCCGGTTTTATAATGAGTATCTTACCACAACTTAAGAG 295 E F L T H A I N Ii N V I R I K N G S H G F P V L”’
1200 319
1201
TATAATCAATTTAAAATTATCGATAATTTTAAATTTGTTACTTGTTTAAGCTTAAACGTTGTACTATATCATATCATTAAAAAATGGTTTGTACTCTAGT
1300
1301
GCAATAGATGTAATGTATAAACTCTCTTCAAATGTAATATATAATAACAAGTTTAAATGCTGGTTTAAAGAATTGGGGTTATATAAGATTATTGGTGAAA
1400
1401
TTAATAATTGTAAATTGACAACGCTTCTAATACAGCGTATTAAACACAATAATGGATTTTTAGTAATATCATATTTCACAGGCACGTTAAATCGCTGTAA
1500
1501 ATTTTCTTGTGTATAAAATGGTTTTAAAAGTTGTTTTAATGTATTTAATTCTAATTGTAATTGCCATAGATATTTAGAAACAATACCATTTAACATAATA
1600
It01
GAATTATTTTGTTTTATGATTTGAATCATATATTTAAATTTTAAAATTTTACGGAAAATAGTGTGCTGTAGAAATTGCTCATCTTGTAATGTAAGACATT
1700
1701
GAAGACATTGTTTACATGGTTTAAAATAAGTACAACTGGTACAACTAGAACTTGGGAAAAATACCATTTTTTTAAACAAATAAATAAAAATGAATTATTA
1600
1901 TCAATTGCCGATTATAATTCCGTCGAGGAAAACGTTAATTTTATTATCGGAATGTATTTCTACCACGTTAAAAGCCCAAAATTTAAACGTTATTATTTCA
1900
1901 AACGATAAATTATATGACTTGTATACTACTTTAAGTCAAATACCAGCTTTAGGGCAAGATAAAACATTAATTTTTGAAAAAACCGTCGAAATTGCAATTT 2001
2000
GTAACGCTTTAAATTATATAACACCCGTCATTAAAACATATTACAAAACAACAATTATCAAGTCAGGTTTAAAATCTATAAAGCTT HlndIII
(0.669
map
2086 units)
FIG. 3. Nucleotide sequence (lower strand) of a part of the EcoRl FLDV-f DNA fragment C between the HindIll site (0.669 mu.; nucleotide position 2086) and -49 upstream from the Accl site (0.691 m.u.; nucleotide position 50) which corresponds to the nucleotide positions 1 (HindIll) and 2032 (Accl) of the upper strand. Nucleotide sequence (219 to 1172 nucleotide position) and predicted amino acid residue of the fish lymphocystis disease virus TK gene are indicated. The positions of transcriptional start and termination signals are marked. The corresponding nucleotide positions of the upper strand are given in parentheses. The Sequenase sequencing kit was purchased from United States Biochemical Corp. (Cleveland, OH) or obtained from Renner GmbH (Dannstadt, FRG). For determination of the DNA sequences [a-3*P]dATP (sp act 800 Ci/mmol) or [u-%]dATP (sp act 500 Ci/mmol) were used. Both radionucleotides were purchased from New England Nuclear (Dreieich, FRG). Nucleotide sequence, and amino acid sequences were compiled and analysed using the BSA Program at the German Cancer Research Center (Heidelberg, FRG) and the PC Gene program (University of Geneva, Switzerland).
and the amino acid composition of the other TK genes tested (Fig. 4). However, significant conservations were detected at several regions of amino acid residues of the FLDV TK gene as marked in Fig. 4. It should be underlined that the conservation at the positions (160 and 161; two phenylalanine residues marked with arrows in Fig. 4) is of considerable importance because this particular region and its surrounding belong to the amino acid domains containing the nucleotide binding motifs known for TK genes of cellular and poxvirus origin (45). Moreover, the two phenylalanine residues are conserved in all known cytoplasmic thymidine kinases (14, 15, 25, 26, 28, 29). The degree of relative homology (46) between the amino acid residues of the complete TK gene of FLDV to the amino acid residues of other cytoplasmic TK
genes was found to be 8.2% for ASFV, 10.9% for FPOX, 9.790 for SFV, 9.6% for VV, 7.6% for CHI, 9.4% for MOU, and 6% for HUM. No significant variations of the ratio of homology were found when only the central region of FLDV TK protein between amino acids 72 and 247 (20 kDa) was used for alignment. However, the homology between the amino acid residues of the TK genes of ASFV and the poxvirus family had been found to be 25.1 to 31% (25). Although according to these analyses no statistical homologies between FLDV TK and other known TKs were observed, the detected conserved amino acid motifs at different regions of the FLDV TK protein, e.g., the nucleotide binding motif, are strong evidence for the relatedness of this gene to the other cytoplasmic TK genes. These data justify to conclude that the TK
839
SHORT COMMUNICATIONS Wolff for critical comments and helpful discussions dooghi for computer data analysis.
and Neda Sa-
REFERENCES
.
l
.*
*
.*
FLD” ASF” FPOX SF” “V CHI llOU HUM
177 114 112 IO6 106 122 121 121
TASP”ILEEAYASOLSL”AAIDPIAN-Y--T~””E~SA~~~~~G~E”“S~“GP”“F---GG LAGLNASFEPKnFPPI”RIF-PYCSWYKYIGRT-CnKCNPCF~”~KNAGKT~~~AGG VAALNGOFKRELFGNVYKLLS-LAETVSS-LTAICVKCV-CGASFSKRVTENKEVMDIGG YAALDGTFPAKPFSNISELI-p~AEN”T-K~NA”C~”C”KN-GSFSK~~GGK”EI~“~GG “AALDGTFPRKPFNNILNLI-PLSEnVV-KLTI\VCnKCFKE-ASFSK~~GEETEIEIIGG YAALOGTFPRKAFGSILNL”-PLAESVV-KLNIYCnECYRS”TK~~GAE~E”E”IGG YAALDGTFPRKAFGS~LNLY-PLAESVV-KLTI\VCHECF~E-AA”TK~~G~EKE”E”*GG VAALDGTFORKPFGAILNLY-PLAESVV-KLTAVCMECF~E-AAYTK~~GTEKEVEVIGG
230 171 168 162 162 178 177 177
Fmv ASF” FPOX SF” Y” CHI “0” HUM
231 172 169 163 163 179 178 178
Y l * *,* ~*PIETGYH~YCYSLN~HG~GP~GSTNYGRLSNVSHKLKTSGKA””~AGGGGGN”SG”K~ SELYVTCCNN-CLKNTFIKQLQPIK” KOKYIAYCRK-C-FFSN SOKYKSVCRK-CYFF NOWPSYCRK-CYIDS AOKY”S”CAA-CYF(lKRPPI)L--GSENKENVPHGVK(S ADKY”SYCRL-CYFKKSSA(TA-GSDNK-NCLVLGPPGEA~””~K~FASGG”~G”NSA~ ADYYHSYCRL-CYFKKASGPPA-GPGNKENCPYPGKPGEAI~”CSPA~
290 196 183 176 177 224 233 234
FLD”
291
AQKFEFLT”AINHN”IRIKNGSHGFPVL
318
FIG. 4. Multiple alignment of eight thymidine kinase genes determined by progressive method of sequence alignment (40). The amino acid sequence of TK gene of FLDV was compared to the amino acid sequence of the TK genes of African swine fever virus (ASFV (25)), fowlpox virus (FPOX (15)), shope fibroma virus (SFV (I#)), vaccinia virus (VV (11, 12)), and to the amino acid sequence of cytoplasmic TK of chicken (CHI (29)), mouse (MOU (28)). and human (HUM (26, 27)). The locations of those amino acid residues where all (100%) or at least 75% of the screened protein sequences are identical to the FLDV TK have been denoted with an asterisk (*). The locations of those amino acid residues where at least three or more (up to 75%) of the screened protein sequences are identical to the FLDV TK gene have been marked with a plus (+). The dashes represent artificial gaps which were introduced into the amino acid sequences to achieve optimal sequence homology. The arrows indicate the position of amino acids 160 and 161 (two phenylalanine residues) corresponding to the region of TK proteins known to contain the nucleotide binding motifs.
gene of FLDV as a real member of the lridoviridae family and the prototype of the genus Lymphocystivirus should be placed at the bottom of the evolutionary tree of cytoplasmic TK genes as reported by Blasco et a/. (25). With respect to the evolutionary stage of the thymidine kinase of FLDV exact functional and biochemical characterization of this interesting enzyme is of considerable importance and the subject of further studies. ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft, Project II B 6 Da 142/2-4. The authors thank Angela R&en-
1. WILLIS, B., In “Molecular Biology of Iridoviruses” (G. Darai, Ed.), pp. l-l 2. Kluwer Academic, Boston/Dordrecht/London, 1990. 2. DARAI, G., ANDERS, K., KOCH, H. G., DELIUS, H., GELDERBLOM,H., SAMALECOS. C., and FLUGEL, R. M., virology 126, 466-479 (1983). 3. DARAI, G., DELIUS. H., CLARKE,J., APFEL, H., SCHNITZLER,P., and FLUGEL, R. M., v;fology 146, 292-301 (1985). 4. SCHNITZLER,P., DELIUS, H., SCHOLZ, J., TOURAY, M., ORTH, E., and DARAI, G., virology 161, 570-578 (1987). 5. SCHNITZI-ER,P.. and DARAI, G., Virology 172, 32-41 (1989). 6. SCHNITZLER, P., ROSEN-WOLFF,A., and DARAI, G., In “Molecular Biology of Iridoviruses” (G. Darai, Ed.), pp. 203-234. Kluwer Academic, Boston/Dordrecht/London, 1990. 7. GOORHA, R., and MURTI, K. G., Proc. Nat/. Acad. Sci. USA 79, 248-252 (1982). 8. DELIUS, H., DARAI, G., and FLUGEL, R. M., /. viral. 49, 609-614 (1984). 9. WARD, V. K., and KALMAKOFF,J. viral. 160, 507-510 (1987). 10. SCHOLZ, J., ROSEN-WOLFF. A., TOURAY, M., SCHNITZLER, P., and DARAI, G., Virus Res. 9, 63-72 (1988). 11. HRUBY, D. E., MAKI. R. A., MILLER, D. B., and BALL, L. A., Proc. Nat/. Acad. Sci. USA 80, 3411-3415 (1983). 12. WEIR, J. P., and Moss, B., J. Viral. 46, 530-537 (1983). 13. ESPOSITO,I. J., and KNIGHT, J. C., Hfology 135, 561-567 (1984). 14. UPTON, C.. and MCFADDEN, G., J. Gen. Viral. 60, 920-927 (1986). 15. BOYLE, D. B., COUPAR, B. E. H., GIBBS, A. J., SEIGMAN, L. J., and BOTH, G. W., Hrology 156, 355-365 (1987). 16. GERSHON. P. D., and BLACK, D. N., J. Gen. Viral. 70, 525-533 (1989). 17. MCKNIGHT, S. L., Nucleic Acids Res. 8, 5949-5964 (1980). 18. KIT, S., KIT, M., QAVI, H., TRKULA, D., and OTSUKA. H., Biochim. Biophys. Acta 741, 158-l 70 (1983). 19. SWAIN, M. A., and GALLOWAY, D. A., J. Viol. 46, 10.045-10.050 (1983). 20. OTSUKA, H., and KIT, S.. L/iro/ogy 135, 316-330 (1984). 21. HONESS, R. W., CFIAXTON, M. A., WILLIAMS, L., and GOMPELS, U. A., J. Gen. Viral. 70, 3003-3013 (1989). 22. ROBERTSON,G. R., and WHALLEY, J. M., Nucleic Acids Res. 16, 11,303-11,317(1988). 23. SHEPPARD, M., and MAY, J. T., J. Gen. Viral. 70, 3067-3071 (1989). 24. Scorr, S. D., Ross, N. L. J.. and BINNS. M. M.. J. Gen. !Iiro/. 70, 3055-3065 (1989). 25. BLASCO, R., LOPEZ-OTIN, C., MUNOZ, M., BOCKAMP, E.-O., SIMONMATEO, C., and VINUELA, E., Virology 178, 301-304 (1990). 26. BRADSHAW, H. D., JR., and DEININGER, P. L., Mol. Cell. Biol. 4, 2316-2320 (1984). 27. FLEMINGTON, E., BRADSHAW,H. D., J. R., TRAINA-DORGE, V., SLAGEL. V., and DEININGER,P. L., Gene 52, 267-277 (1987). 28. LIN, P. F., LIEBERMAN. H. B., YEH, D. B., Yu, T., ZHAO, S. Y., and RUDDLE, F. H., /\/lo/. Cell. Biol. 5, 3149-3156 (1985). 29. KWOH, T. J., and ENGLER, J. A., Nucleic Acids Res. 12, 39593971 (1984).
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182, 835-840 (1991)
The Primary Structure
of the Thymidine
Kinase Gene of Fish Lymphocystis
Disease Virus
PAUL SCHNITZLER,’ MICHAELA HANDERMANN, ORSOLYA SZ~PE, AND GHOLAMREZA DARAI’ lnstitut filr Medizinische
Virologie der UniversitAV Heidelberg, Received November
Im Neuenheimer
14. 1990; accepted
February
Feld 324, 6900 Heidelberg,
Germany
12, 199 1
The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIll (0.669 map unit; nucleotide position 1) and Accl (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man. o 1991 Academic press, I~C.
in comparison to the TK genes of eukaryotic cells, e.g., man (26, 27), mouse (28), and chicken (29), are a valuable technique for calculating the evolutionary stage of each species. The exact characterization and determination of the DNA nucleotide sequence of the TK gene of FLDV genome is the subject of this paper. In order to determine the exact position and the primary structure of the DNA sequences expressing the TK gene the following strategy was used. The particular region of the viral genome located between the genome coordinates 0.669 and 0.718 which harbors the complete TK gene locus of the FLDVf was molecularly cloned into the corresponding sites of plasmid vector pAT153 (30) and termed pFLDV-TK-C19. Furthermore a defined library harboring 14 subclones (Fig. 2C) of the DNA sequences located between the genome coordinates 0.669 (HindIll site) and 0.718 (EcoRI site) was established. This gene librarywas screened forthe ability to transform 3T3 TK negative to 3T3 TK positive cells. The 3T3 cell cultures with TK negative phenotype were grown and propagated in Eagle basal medium balanced with Earle’s salt solution supplemented with 10% fetal calf serum which contained 30 pglml bromodeoxyuridine. The transfection assay was carried out according to the calcium phosphate technique (31) and the DNA of 3T3 TK negative cells was used as carrier DNA as described previously (70). The transfected cultures were processed under HATG (0.1 mM hypoxanthin, 0.4 PMaminopterin, 16 @I thymidine, 10 puM glycine) selection pressure (32). According to the
Fish lymphocystis disease virus (FLDV) is the causative virus for lymphocystis disease, a common chronic disease of pleuronectes. FLDV has been classified as a separate genus of the lridoviridae family with the proposed name Lymphocysfivirus (1) which belongs to the icosahedral cy-toplasmic deoxyriboviruses. FLDV has many interesting properties; e.g., the genome structure of FLDV was found to be circularly permuted and terminally redundant (2-6), a common genomic feature of frog virus 3 (FV3) and insect iridescent virus type 6 (CIV) and type 9 (7-9). The thymidine kinase (TK) gene of FLDVf isolated from flounder was identified by biochemical transformation of 3T3 TK negative to 3T3 TK positive cells using specific viral DNA sequences as described previously (10). These analyses revealed that the DNA sequences located between the coordinates 0.61 1 to 0.718 (Table 1 and Fig. 1) of the viral genome harbor the TK gene of FLDVf with the ability for biochemical transformation of 3T3 TK negative cells to 3T3 TK positive cells. The primary structure of the TK genes of a variety of viruses including poxviruses (1 l-16), herpesviruses ( 17-24), and recently African swine fever virus (25) has been reported. The analyses of these particular genes Sequence data from this article have been deposited with the EMBUGen Bank Data Libraries under Accession No. M61 115. ’ Present address: Department of Microbiology and Immunology, Duke University Medical Center, Box 3020, Durham, NC 27710. ’ To whom requests for reprints should be addressed.
835
0042-6822191 $3.00 Copyright 0 199 1 by Academic Press, Inc. All rights of reproduction in any form reserved
836
SHORT COMMUNICATIONS
FIG. 1. Physical maps of the genome of fish lymphocystis disease virus isolated from flounder (A) (4) and fine mapping of the EcoRl FLDV DNA fragment C (B). The position and the DNA sequence arrangement of the TK gene are shown in (B) and (C), respectively.
results of this study shown in Fig. 1 E?it was found that the DNA sequence located between the /-/indIll (0.669 m.u.) and Accl (0.691 m.u.) sites was capable to convert 3T3 TK negative cells to 3T3 TK positive cells (Table 1). The primary structure of the FLDV TK gene was analyzed by determining the complete nucleotide sequence of the insert of the recombinant plasmid pFLDV-TK-Cl9 corresponding to the viral DNA sequences between the HindIll (0.669 m.u.) and Accl (0.691 m.u.) sites. The DNA sequencing was performed by the dideoxy chain termination procedure (33-35) using a modified T7 DNA polymerase (36). The sequence of each subclone was determined from both strands by analysis of the particular DNA fragment which was inserted into both sequencing vectors M 13mpl8 and Ml 3mpl9 (Fig. 2C) as described else-
where (5, 37, 38). The strategy of DNA sequencing is given in Fig. 2B. The DNA nucleotide sequence obtained from the HindllllAccl DNAfragment (0.669 to 0.691 m.u.) of the FLDV-f genome is shown in Fig. 3. This DNAsequence of 2032 bp has a base composition of 30.4% G + C and 69.6% A + T. The presence of all restriction sites mapped within this fragment was confirmed. A computer search for detecting open reading frames (ORFs) was carried out. One ORF was identified on the lower strand (954 bp; nucleotide position 219 to 1 172) encoding a polypeptide of 3 18 amino acid residues with a molecular weight of 35.3 kDa. A high probability to encode a protein was predicted for this ORF when the DNA nucleotide sequence of this particular region was analysed by the method of Fickett (39). The amino acid sequence of this gene is given in Fig. 3. This gene J
SHORT COMMUNICATIONS TABLE 1 OF 3T3 CELLS WITH TK NEGATIVE PHENOTYPETO 3T3 CELLS WITH TK POSITIVE PHENO~PE USING SusCLONESOF THE INSERTOF RECOMEINANTPLASMID Cl 9 (4.7 kbp; 0.669 TO 0.7 18 m.u.) HARBORINGTHE TK GENE OF FLDV-f GENOME THE RESULTS OF TRANSFORMATION
DNA fragment” kbd EcoRI-C (1 1.2) Subclones of EcoRI-C Cl9 Subclones of Cl 9 Subclones of C 19
Viral map unit 0.61 1 to 0.61 1 to 0.669 to 0.669 to 0.691 to
0.718 0.669 0.718 0.691 0.718
No. of coloniesb 28 0 33 42 0
a DNA fragments were obtained from the corresponding recombinant plasmids harboring the EcoRl DNA fragments of the viral genome. * Average of two experiments (0.1 pM DNA/i X lo7 cells). The specificity of the transformation assay was determined using Southern blot hybridization experiments In which the specific FLDV TK gene DNA sequences were detected in the biochemically transformed cells as described elsewhere (10).
corresponds to the DNA sequences of the viral genome located within the FLDV-f EcoRl DNA fragment C between the map coordinates 0.669 and 0.691 which possesses high TK-transforming activity (Table 1). The analysis of the DNA sequences upstream and downstream of the start and termination codons of the FLDV-f TK protein revealed the presence of classical or modified transcriptional signals, which are underlined in Fig. 4. This finding is in agreement with the previously described transcriptional start and polyadenylation signals detected upstream and downstream
0.609 Hlndlll
Cio(4.7
837
of the star-t and termination codons of a variety of predicted FLDV genes (5). For detecting the evolutionary relationships of the TK gene of FLDV to the other known TK genes a comparative analysis of their amino acid sequence was carried out according to the method of progressive sequence alignment (40). The results obtained by progressive sequence alignment were in agreement with the results obtained by the multiple alignment of the protein sequences using the method developed by Higgins and Sharp (4 1, 42). This method aligns pairwise similarities at the first step of computation (43). With exception of the amino acid motif GNMSGYK located at the amino acid position 283 to 289 of FLDV TK no other significant similarities were detected when the amino acid sequence of the FLDV TK was compared to the amino acid sequence of the known TKs of herpesviridae ( 1724). This motif is similar to the amino acid consensus sequence GXXGXGK which is common in all known TK genes including TKs of herpesviruses (44). In contrast evolutionary relationship was detected between the thymidine kinase of FLDV and those of poxvirus and cellular origin. An example of the results of these studies are given in Fig. 4. In this study the amino acid sequence of TK gene of FLDV was compared to the amino acid sequence of the TK genes of representative members of the cytoplasmic deoxyriboviruses like African swine fever virus (ASFV), fowlpox virus (FPOX), shope fibroma virus (SFV), vaccinia virus (VV), and to the amino acid sequence of the cytoplasmic TK of chicken (CHI), mouse (MOU), and human (HUM). These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene
kbp)
..
.
yw,
mu
Y.. .._
‘.._ “h
0.691
mu
FIG. 2. Diagram of the strategy of DNA sequencing (B) for the insert of recombinant plasmid Cl 9 (0.669 to 0.718 m.u.). The position of the cleavage sites of some restriction endonucleases of the particular regions are given In (A). The sequence of each DNA fragment was determined from both strands using the M 13mpl8 and -19 systems. The map positions of the individual subclones established are shown in (C).
838
SHORT COMMUNICATIONS 100
1 AATGTTTCACGAGGAGGCGATTATGTCCTTAATTCCTGGATGACGGTTCGTATACCTGCCATTAAATTGAAAGCTGATAATAGAATGAATAATAACGGCA
AccI 101
(2032)
CCATTCGATGGTGCAAAAACTTATTTCATAATCTAATTAAACAAACGTCGGTTCAGTTTAATGATTTAGTAGCACAAAAATTTGAAAGTTATTTCTTAGA -
200
219119691
201 TTATTGGGCAGCGTTCGTATGTGTGGCTCTAAAAGAGCAGGTTATAATAATATGATCGGTAATACAATTGATATGATACAACCGGTTGACCATACAGGAA TCGSKRAGYNNMIGNT IOMIQPVOHTG 1
300 27
301 TGTTACCTGAAAAAGTTTTAGTGTTACCATTACCGTATTTCTTTTCTAGGGACAGCGGTGTTGCATTGCCTAGTGCAGCACTTCCTTATAACGAAATAAG 28H L P E K VLVLPL PYFFSROSGVALP SAALPYNEIR
400 61
401 62
ATTAACGTTTCATCTTAGAGATTATACGGAATTACTTATTTTTCAGCATAAACAAGATTGTACCATTATACCAATTACAGCTGCTGATTTAGAATATGGA L T F H LROVTELLIFQHKOOC T I I P I T AAOLEYG
500 94
501 95
AAACCTGATTTAAAAGACGTTCAAGTATGGATTACTAATGCAGTGGTTACAAATGAAGAACGGCGACTTATGGGTACAACTCCTCGAGATATATTAGTGG K P 0 L K OVQVWI T N A V V T N E EARLMGTT P R
601 128
AACAAGTTCAAACTGCGCCAAAACATGTATTTCAACCGTTAACTATTCCAAGCCCTAATTTTGATATTCGGTTTTCACACGCTATTAAATTGTTATTTTT E 0 V Q TAPKHVFQPL TIPSPNFOIRFSHAIKLLFF
700
701 162
TGGTGTAAGAAATACAACTCATGCAGCTGTTCAATCTAATTATACTACAGCTTCTCCTGTTATTTTAGAAGAAGCCTATGCAAGCGATCTATCTTTAGTG GVRNTTHAAVQSNVTTASPVILEEAVASOLSLV
900
601
GCAGCAGATCCAATTGCAAATGTAACGCTTGTTTATGAAAATAGTGCACGGCTTAATGAAATGGGTAGTGAATATTATTCACTCGTTCAACCATATTATT A A 0 P I A N V T L V Y E N S A R L N E H G S E Y Y S L
900 227
195
0
I
L
v
600 127
161 194 V
Q
P
V
Y
901 TTGGTGGTTCTATTCCAATAGAAACAGGATATCATATGTATTGTTATTCATTAAATATGATGGATATGGATCCAATGGGATCCACAAATTACGGACGTTT 226FGGSIPIETGYHMYCVSLNMMOMOPMGSTNVGRL
1000 261
1001 ATCAAACGTTAGTATGAAATTAAAAACCTCAGACAAAGCAGTTGTAAATGCTGGTGGCGGTGGTGGTAATATGTCTGGTTATAAAGATGCACAAAAGTTT
1100 294
262
SNVSHKLKTSOKAVVNAGGGGGNMSGVKDAQKF
1172(91S)
1101 GAATTTCTTACTATGGCTATTAATCATAATGTAATTCGTATAAAGAACGGATCAATGGGTTTTCCGGTTTTATAATGAGTATCTTACCACAACTTAAGAG 295 E F L T H A I N Ii N V I R I K N G S H G F P V L”’
1200 319
1201
TATAATCAATTTAAAATTATCGATAATTTTAAATTTGTTACTTGTTTAAGCTTAAACGTTGTACTATATCATATCATTAAAAAATGGTTTGTACTCTAGT
1300
1301
GCAATAGATGTAATGTATAAACTCTCTTCAAATGTAATATATAATAACAAGTTTAAATGCTGGTTTAAAGAATTGGGGTTATATAAGATTATTGGTGAAA
1400
1401
TTAATAATTGTAAATTGACAACGCTTCTAATACAGCGTATTAAACACAATAATGGATTTTTAGTAATATCATATTTCACAGGCACGTTAAATCGCTGTAA
1500
1501 ATTTTCTTGTGTATAAAATGGTTTTAAAAGTTGTTTTAATGTATTTAATTCTAATTGTAATTGCCATAGATATTTAGAAACAATACCATTTAACATAATA
1600
It01
GAATTATTTTGTTTTATGATTTGAATCATATATTTAAATTTTAAAATTTTACGGAAAATAGTGTGCTGTAGAAATTGCTCATCTTGTAATGTAAGACATT
1700
1701
GAAGACATTGTTTACATGGTTTAAAATAAGTACAACTGGTACAACTAGAACTTGGGAAAAATACCATTTTTTTAAACAAATAAATAAAAATGAATTATTA
1600
1901 TCAATTGCCGATTATAATTCCGTCGAGGAAAACGTTAATTTTATTATCGGAATGTATTTCTACCACGTTAAAAGCCCAAAATTTAAACGTTATTATTTCA
1900
1901 AACGATAAATTATATGACTTGTATACTACTTTAAGTCAAATACCAGCTTTAGGGCAAGATAAAACATTAATTTTTGAAAAAACCGTCGAAATTGCAATTT 2001
2000
GTAACGCTTTAAATTATATAACACCCGTCATTAAAACATATTACAAAACAACAATTATCAAGTCAGGTTTAAAATCTATAAAGCTT HlndIII
(0.669
map
2086 units)
FIG. 3. Nucleotide sequence (lower strand) of a part of the EcoRl FLDV-f DNA fragment C between the HindIll site (0.669 mu.; nucleotide position 2086) and -49 upstream from the Accl site (0.691 m.u.; nucleotide position 50) which corresponds to the nucleotide positions 1 (HindIll) and 2032 (Accl) of the upper strand. Nucleotide sequence (219 to 1172 nucleotide position) and predicted amino acid residue of the fish lymphocystis disease virus TK gene are indicated. The positions of transcriptional start and termination signals are marked. The corresponding nucleotide positions of the upper strand are given in parentheses. The Sequenase sequencing kit was purchased from United States Biochemical Corp. (Cleveland, OH) or obtained from Renner GmbH (Dannstadt, FRG). For determination of the DNA sequences [a-3*P]dATP (sp act 800 Ci/mmol) or [u-%]dATP (sp act 500 Ci/mmol) were used. Both radionucleotides were purchased from New England Nuclear (Dreieich, FRG). Nucleotide sequence, and amino acid sequences were compiled and analysed using the BSA Program at the German Cancer Research Center (Heidelberg, FRG) and the PC Gene program (University of Geneva, Switzerland).
and the amino acid composition of the other TK genes tested (Fig. 4). However, significant conservations were detected at several regions of amino acid residues of the FLDV TK gene as marked in Fig. 4. It should be underlined that the conservation at the positions (160 and 161; two phenylalanine residues marked with arrows in Fig. 4) is of considerable importance because this particular region and its surrounding belong to the amino acid domains containing the nucleotide binding motifs known for TK genes of cellular and poxvirus origin (45). Moreover, the two phenylalanine residues are conserved in all known cytoplasmic thymidine kinases (14, 15, 25, 26, 28, 29). The degree of relative homology (46) between the amino acid residues of the complete TK gene of FLDV to the amino acid residues of other cytoplasmic TK
genes was found to be 8.2% for ASFV, 10.9% for FPOX, 9.790 for SFV, 9.6% for VV, 7.6% for CHI, 9.4% for MOU, and 6% for HUM. No significant variations of the ratio of homology were found when only the central region of FLDV TK protein between amino acids 72 and 247 (20 kDa) was used for alignment. However, the homology between the amino acid residues of the TK genes of ASFV and the poxvirus family had been found to be 25.1 to 31% (25). Although according to these analyses no statistical homologies between FLDV TK and other known TKs were observed, the detected conserved amino acid motifs at different regions of the FLDV TK protein, e.g., the nucleotide binding motif, are strong evidence for the relatedness of this gene to the other cytoplasmic TK genes. These data justify to conclude that the TK
839
SHORT COMMUNICATIONS Wolff for critical comments and helpful discussions dooghi for computer data analysis.
and Neda Sa-
REFERENCES
.
l
.*
*
.*
FLD” ASF” FPOX SF” “V CHI llOU HUM
177 114 112 IO6 106 122 121 121
TASP”ILEEAYASOLSL”AAIDPIAN-Y--T~””E~SA~~~~~G~E”“S~“GP”“F---GG LAGLNASFEPKnFPPI”RIF-PYCSWYKYIGRT-CnKCNPCF~”~KNAGKT~~~AGG VAALNGOFKRELFGNVYKLLS-LAETVSS-LTAICVKCV-CGASFSKRVTENKEVMDIGG YAALDGTFPAKPFSNISELI-p~AEN”T-K~NA”C~”C”KN-GSFSK~~GGK”EI~“~GG “AALDGTFPRKPFNNILNLI-PLSEnVV-KLTI\VCnKCFKE-ASFSK~~GEETEIEIIGG YAALOGTFPRKAFGSILNL”-PLAESVV-KLNIYCnECYRS”TK~~GAE~E”E”IGG YAALDGTFPRKAFGS~LNLY-PLAESVV-KLTI\VCHECF~E-AA”TK~~G~EKE”E”*GG VAALDGTFORKPFGAILNLY-PLAESVV-KLTAVCMECF~E-AAYTK~~GTEKEVEVIGG
230 171 168 162 162 178 177 177
Fmv ASF” FPOX SF” Y” CHI “0” HUM
231 172 169 163 163 179 178 178
Y l * *,* ~*PIETGYH~YCYSLN~HG~GP~GSTNYGRLSNVSHKLKTSGKA””~AGGGGGN”SG”K~ SELYVTCCNN-CLKNTFIKQLQPIK” KOKYIAYCRK-C-FFSN SOKYKSVCRK-CYFF NOWPSYCRK-CYIDS AOKY”S”CAA-CYF(lKRPPI)L--GSENKENVPHGVK(S ADKY”SYCRL-CYFKKSSA(TA-GSDNK-NCLVLGPPGEA~””~K~FASGG”~G”NSA~ ADYYHSYCRL-CYFKKASGPPA-GPGNKENCPYPGKPGEAI~”CSPA~
290 196 183 176 177 224 233 234
FLD”
291
AQKFEFLT”AINHN”IRIKNGSHGFPVL
318
FIG. 4. Multiple alignment of eight thymidine kinase genes determined by progressive method of sequence alignment (40). The amino acid sequence of TK gene of FLDV was compared to the amino acid sequence of the TK genes of African swine fever virus (ASFV (25)), fowlpox virus (FPOX (15)), shope fibroma virus (SFV (I#)), vaccinia virus (VV (11, 12)), and to the amino acid sequence of cytoplasmic TK of chicken (CHI (29)), mouse (MOU (28)). and human (HUM (26, 27)). The locations of those amino acid residues where all (100%) or at least 75% of the screened protein sequences are identical to the FLDV TK have been denoted with an asterisk (*). The locations of those amino acid residues where at least three or more (up to 75%) of the screened protein sequences are identical to the FLDV TK gene have been marked with a plus (+). The dashes represent artificial gaps which were introduced into the amino acid sequences to achieve optimal sequence homology. The arrows indicate the position of amino acids 160 and 161 (two phenylalanine residues) corresponding to the region of TK proteins known to contain the nucleotide binding motifs.
gene of FLDV as a real member of the lridoviridae family and the prototype of the genus Lymphocystivirus should be placed at the bottom of the evolutionary tree of cytoplasmic TK genes as reported by Blasco et a/. (25). With respect to the evolutionary stage of the thymidine kinase of FLDV exact functional and biochemical characterization of this interesting enzyme is of considerable importance and the subject of further studies. ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft, Project II B 6 Da 142/2-4. The authors thank Angela R&en-
1. WILLIS, B., In “Molecular Biology of Iridoviruses” (G. Darai, Ed.), pp. l-l 2. Kluwer Academic, Boston/Dordrecht/London, 1990. 2. DARAI, G., ANDERS, K., KOCH, H. G., DELIUS, H., GELDERBLOM,H., SAMALECOS. C., and FLUGEL, R. M., virology 126, 466-479 (1983). 3. DARAI, G., DELIUS. H., CLARKE,J., APFEL, H., SCHNITZLER,P., and FLUGEL, R. M., v;fology 146, 292-301 (1985). 4. SCHNITZLER,P., DELIUS, H., SCHOLZ, J., TOURAY, M., ORTH, E., and DARAI, G., virology 161, 570-578 (1987). 5. SCHNITZI-ER,P.. and DARAI, G., Virology 172, 32-41 (1989). 6. SCHNITZLER, P., ROSEN-WOLFF,A., and DARAI, G., In “Molecular Biology of Iridoviruses” (G. Darai, Ed.), pp. 203-234. Kluwer Academic, Boston/Dordrecht/London, 1990. 7. GOORHA, R., and MURTI, K. G., Proc. Nat/. Acad. Sci. USA 79, 248-252 (1982). 8. DELIUS, H., DARAI, G., and FLUGEL, R. M., /. viral. 49, 609-614 (1984). 9. WARD, V. K., and KALMAKOFF,J. viral. 160, 507-510 (1987). 10. SCHOLZ, J., ROSEN-WOLFF. A., TOURAY, M., SCHNITZLER, P., and DARAI, G., Virus Res. 9, 63-72 (1988). 11. HRUBY, D. E., MAKI. R. A., MILLER, D. B., and BALL, L. A., Proc. Nat/. Acad. Sci. USA 80, 3411-3415 (1983). 12. WEIR, J. P., and Moss, B., J. Viral. 46, 530-537 (1983). 13. ESPOSITO,I. J., and KNIGHT, J. C., Hfology 135, 561-567 (1984). 14. UPTON, C.. and MCFADDEN, G., J. Gen. Viral. 60, 920-927 (1986). 15. BOYLE, D. B., COUPAR, B. E. H., GIBBS, A. J., SEIGMAN, L. J., and BOTH, G. W., Hrology 156, 355-365 (1987). 16. GERSHON. P. D., and BLACK, D. N., J. Gen. Viral. 70, 525-533 (1989). 17. MCKNIGHT, S. L., Nucleic Acids Res. 8, 5949-5964 (1980). 18. KIT, S., KIT, M., QAVI, H., TRKULA, D., and OTSUKA. H., Biochim. Biophys. Acta 741, 158-l 70 (1983). 19. SWAIN, M. A., and GALLOWAY, D. A., J. Viol. 46, 10.045-10.050 (1983). 20. OTSUKA, H., and KIT, S.. L/iro/ogy 135, 316-330 (1984). 21. HONESS, R. W., CFIAXTON, M. A., WILLIAMS, L., and GOMPELS, U. A., J. Gen. Viral. 70, 3003-3013 (1989). 22. ROBERTSON,G. R., and WHALLEY, J. M., Nucleic Acids Res. 16, 11,303-11,317(1988). 23. SHEPPARD, M., and MAY, J. T., J. Gen. Viral. 70, 3067-3071 (1989). 24. Scorr, S. D., Ross, N. L. J.. and BINNS. M. M.. J. Gen. !Iiro/. 70, 3055-3065 (1989). 25. BLASCO, R., LOPEZ-OTIN, C., MUNOZ, M., BOCKAMP, E.-O., SIMONMATEO, C., and VINUELA, E., Virology 178, 301-304 (1990). 26. BRADSHAW, H. D., JR., and DEININGER, P. L., Mol. Cell. Biol. 4, 2316-2320 (1984). 27. FLEMINGTON, E., BRADSHAW,H. D., J. R., TRAINA-DORGE, V., SLAGEL. V., and DEININGER,P. L., Gene 52, 267-277 (1987). 28. LIN, P. F., LIEBERMAN. H. B., YEH, D. B., Yu, T., ZHAO, S. Y., and RUDDLE, F. H., /\/lo/. Cell. Biol. 5, 3149-3156 (1985). 29. KWOH, T. J., and ENGLER, J. A., Nucleic Acids Res. 12, 39593971 (1984).
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30. TWIGG, A., and SHEFIAT, D., Nature (London) 283, 216-218 (1980). 31. STOW, N. D., and WILKIE, N. M., J. Gen. Viral. 33, 447-458 (1976). 32. KIT, S., QAVI, H., HAZEN, M., TRKULA, D., and OTSUKA, H.. Wro/ogy 113,452-464 (1981). 33. SANGER, F., and COULSON, A. R., FEBS Lett. 87, 107-l 10 (1978). 34. SANGER, F., NICKLEN S., and COULSON, A. R., Proc. Nat/. Acad. Sci. USA 74, 5463-5467 (1977). 35. VIEIRAJ., and MESSING, J., Gene 19, 259-268 (1982). 36. TABOR, S., and RICHARDSON, C. C., Proc. Nat/. Acad. SC;. USA 74, 4767-4771 (1987). 37. FISCHER, M., SCHNITZLER,P., DELIUS, H., and DAR,C+I, G., Virology 167, 485-496 (1988).
38. FISCHER, M., SCHNITZLER, P., SCHOLZ, J.. ROSEN-WOLFF,A., DELIUS, H., and DARAI, G., virology 167, 497-506 (1988). 39. FICKET~,J. W., Nucleic Acids Res. 10, 5303-5318 (1982). 40. FENG, D. F., and DOOLIITLE, R. F., J. Mol. ho/. 25, 351-360 (1987). 4 1. HIGGINS, D. G., and SHARP, P. M., Gene 73, 237-244 (1988). 42. HIGGINS, D. G., and SHARP, P. M., CAB/OS 5, 151-l 53 (1989). 43. WILBUR, W. J., and LIPMAN, D. J., Proc. Nat/. Acad. SC;. USA 80, 726-730 (1983). 44. MITTAL, S. K., and FIELD, H. J., J. Gen. Viral. 70, 901-918 (1989). 45. GORBALENYA,A. E., and KOONIN, E. V., Nucleic Acids Res. 17, 8413-8440 (1989). 46. MYERS, E. W., and MILLER, W., CAB/OS 4, 11-17 (1988).