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J. gen. Virol. (1989), 70, 3067-3071. Printed in Great Britain 3067 Key words: BHV-2/thymidine kinase/nucleotide sequence Location and Characterization of the Bovine Herpesvirus Type 2 Thymidine Kinase Gene By M I C H A E L S H E P P A R D ~* AND J O H N T. M A Y 2 ~CSIRO, Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, P.O. Parkville, Victoria 3052 and 2Department of Microbiology, La Trobe University, Bundoora, Victoria 3083, Australia (Accepted 4 August 1989) SUMMARY The precise genomic location and the nucleotide sequence of the bovine herpesvirus type 2 (bovine herpes mammillitis virus) thymidine kinase (TK) gene have been determined. The genomic location of the TK gene was found to be in a similar position to that of herpes simplex virus. The coding region consists of 918 bases, which is slightly smaller in length than other reported herpesvirus TK genes. However with an Mr of 38108 the individual protein is similar in size to other herpesvirus TK enzymes. Despite there being only limited overall sequence homology with the T K genes of other herpesviruses, there are several regions of extensive homology at the amino acid level. Bovine herpesvirus type 2 (BHV-2) is one of several herpesviruses known to infect cattle. Although BHV-2 is known to cause swelling, inflammation and ulceration of the teats and udder in domestic cattle (Ludwig, 1983; Martin et al., 1964, 1966) it is not a major problem in cattle. BHV-2 can be considered as a potential bovine-specific vector virus as some strains produce no disease and none are transmitted to people or other animals in field situations (Maragos et al., 1986; Maragos & May, 1987). If the virus has a thymidine kinase (TK) gene it can be used for D N A manipulative purposes to produce vaccines with less pathogenicity (Kit, 1985). BHV-2 virus is an alphaherpesvirus with a linear dsDNA genome approximately 135 kb in size and a genomic structure similar to that of herpes simplex virus (HSV; Buchman & Roizman 1978b; Roizman et al., 1982). The overall base sequence homology between HSV-1 and BHV-2, as defined by D N A hybridization studies, is 14~ (Roizman et al., 1982; Sterz et al., 1973/74). In this report we describe experiments which have determined the precise location of the T K gene on the physical map of BHV-2 strain BHM-1 (Maragos et al., 1986; Maragos & May, 1987) and the complete nucleotide sequence of this gene. We have also compared this sequence to those of known TK genes from other herpesviruses. Recently it has been demonstrated that several BHV-2 genes map to a collinear position with equivalent genes in HSV-1 (Maragos & May, 1987; Hammerschmidt et al., 1988a, b). This knowledge was used to determine the approximate location of a BHV-2 TK gene. Based on the known location of the HSV- 1 TK gene at map units (m.u.) 30-0 to 31.3 (McGeoch et al., 1988; Reyes et al., 1982) the equivalent region in the BHV-2 genome was determined and found to be contained within the 11-5 kb HindlII I fragment of m.u. 27.1 to 35.8 (Buchman & Roizman, 1978 a). Previously we have reported that the HindlII I ti'agment and a smaller 2.69 kb SalI internal fragment transformed T K - cells to a T K ÷ phenotype (May et al,, 1989). In this case we have mapped this 2.69 kb SalI fragment extensively, and by the use of the calcium phosphate transfection method (Kit et al., 1980) we have determined the precise map position of the BHV-2 TK gene to a 1.2 kb SalI/SphI fragment (Fig. 1). The SalI/SphI fragment was found to transform T K - cells to a T K ÷ phenotype with an efficiency equal to the HSV-1 1.9 kb PvulI fragment containing the TK gene; the larger fragments were much less efficient. When the SalI/SphI fragment was cleaved with XbaI (Fig. 1) before transfection no T K + ceils were obtained.This 0000-9069 O 1989 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 11:38:58 3068 Short communication M a p units 40 1 2O I 50 I 9O i 7O I 60 I Hindlll 13-8 19.2 3'4 I I I / / D" TK + TK* 6.2 | / / t I 10.8 I ? I 18-5 I 14.15 10"0 I I ~-~ ~" [ HindlII F r a g m e n t 1 H J (approx. 30 to 3 2 m . u . ) ~ ~ spsp 1 S ~c s I Nc ,I Sp TK- 3-~ 7-4 I / TK- TK + I I I.. I Sp TK + 8.2 S I ..1 TK TK + ~ I S i B / / i / s ? / J H TK + ./ 11.7 J S | S [, N | X ! I-2 kb (30-0 to 3 1 0 m u . ) Sp ~ I-2 kb Sp t 113 kb Sp ~ 0.86 kb Sp Fig. 1. HindlII restriction enzyme map of BHV-2 (Buchman & Roizman 1978a). The HindlII 11.7 kb fragment I is expanded and the various subfragments are indicated as TK ÷ or TK-. Restriction sites: H, HindlII; B, BamHI: S, Sail; N, NaeI; X, XbaI; Sp, SphI; Nc, NcoI. suggests that the XbaI cleavage site is within the coding sequence or the promoter region of the BHV-2 TK gene. The positive results in transforming TK- cells to TK + with the 1-2 kb SalI/SphI fragment led to the complete sequencing of both D N A strands of this region, using the chain termination method of Sanger et al. (1977). The results of this sequence analysis are presented in Fig. 2. With a coding sequence of 918 bp the BHV-2 TK gene is quite clearly smaller than that of other herpesvirus TK genes reported (Kit et al., 1983; Otsuka & Kit, 1984; Sawyer et al., 1988; Swain & Galloway, 1983; Wagner et al., 1981). Although the translation initiation sequence is not ideal as defined by Kozak (1987) there is an A at position - 3 which results in 60 to 90~o of the translation efficiency obtained by a perfect Kozak sequence (Kozak, 1987). Apart from the disease caused by BHV-2 this virus is also of interest as it is one of the few nonhuman herpesviruses whose D N A bears nucleotide sequence homology with the HSV-1 genome (Sterz et al., 1973/74). A direct comparison made between the BHV-2 TK gene nucleotide sequence and that of the HSV-1 TK gene showed a relatively high level of homology (57"4~o; data not shown). A comparison of the TK genes at the amino acid level between these viruses reveals a striking level of homology within two distinct regions. These two conserved regions have been identified previously in other herpesvirus TK genes (Kit, 1985). The amino acid sequences of these two conserved regions in the BHV-2 TK gene as well as the corresponding sequences for other known herpesviruses TK genes are presented in Fig. 3. These two regions in the BHV-2 TK gene share the same relative spacing and orientation as that found in other herpesvirus TK genes. Previously, Kit (1985) suggested that these conserved regions are nucleotide (Fig. 3a) and nucleoside (Fig. 3b) binding sites; this suggestion has been further supported by work by Darby et al. (1986) and Liu & Summers (1988). It is of interest to note that these two conserved regions in the BHV-2 TK gene are more closely related to three of the four human herpesviruses, HSV-1, -2 and varicella-zoster virus (VZV) than it is to BHV-1 (Fig. 3). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 11:38:58 Short communicat~n 3069 GCATGCCAGC~--~'~AGAATGCTCCGGCGGTTGACCGAGCGCATGCGCTGGAGGCCATAG 60 CCGCGCCCACTCACACGGG~'~CGGCACGGGCGAGGGCACGTCGCGCCACCTGGC 120 M S R L b ~ V ¥ V D G P H G L G TCGCGACGAACAATGAGCAGGCTACTGCGGGTGTACGTGGATGGTCCACACGGACTGGGT 17 180 R T T A A S R L A S E R G D A I ¥ L P E AAGACCACGGCGGCGTCCCGGCTGGCGTCCGAGCGTGGCGATGCGATATACCTGCCGGAG 37 240 P M S Y W S G A G E D D L V A R V ¥ T A CCTATGTCGTACTGGAGTGGTGCGGGCGAAGACGACCTGGTCGCTCGGGTGTATACCGCG 57 300 O g ~ M D R G K I D A R Z A A G V V L G CAGCACCGTATGGACCGAGGTGAGATAGACGCACGAGAAGCGGCGGGAGTAG?CCTGGGC 77 360 A O L T M S T P Y Y A L N G L I A P H I GCTCAATTGACAATGAGCACACCGTATGTGGCGCTGAACGGCCTCATCGCACCTCATATC 97 420 G E E P S P G N A T P P D L I L I F D R GGCGAGGAGCCCAGCCCCGGAAACGCCACGCCTCCGGATTTAATCCTTATCTTCGATCGC 117 480 137 H P T A S L L C Y P L A R Y L T R C L P CACCCTACTGCCTCGCTCCTCTGTTACCCGCTGGCTAGGTACCTTACTAGGTGCCTACCC I E S V L S L I A L I P P T P P G T N L 540 ATCGAATCCGTGCTGTCCCTCATCGCTCTGATACCGCCTACCCCACCCGGAACCAACTTA 157 600 I L G T A P A E D H L S R L V A R G P P ATCCTGGGAACGGCCCCGGCAGAAGATCACCTATCACGTCTGGTAGCGCGCGGACCGCCC 177 660 G E L P D A R M L R A I R Y V ¥ A L L A GGGGAACTCCCGGACGCACGAATGTTGCGTGCCATCCGGTACGTATACGCCCTGCTCGCC 197 720 N T V R Y L 0 S O G S W R A D L G S E P AACACCGTGAAGTACCTGCAGTCCGGTGGATCTTGGCGCGCAGACTTGGGGAGCGAGCCG 217 780 P R L P L A P P E I G D P N N P G G H N CCACGGCTTCCGCTGGCACCACCCGAGATCGGAGACCCCAATAACCCCGGAGGGCACAAC 237 840 T L L A L I H G A G A T R G C A A M T S ACCCTGCTGGCGCTGATCCACGGCGCTGGGGCCACCCGCGGTTGCGCCGCGATGACGTCG 257 900 W T L D L L A D R L R S M N M F T W T T TGGACTCTAGACCTGCTGGCGGACCGACTOAGGTCAATGAACATGTTTACGTGGACTACA 277 960 A R P R S L C G S D E E L A P L D A V H GCGCGCCCCCGAAGCCTGTGTGGCTCAGATGAGGAACTTGCTCCCCTCGATGCAGTGCAC 297 1020 R D P K T R I S S R * CGTGACCCCAAAACCCGAATCTCATCGCGGTAGCAGATGCCGCGCTCGCCTTTATGCGAG 1080 AAATGTCAAATTGCAAATAI~CTCCTCCCGAAACCGCTAACGAGAAGCACATTGTATATT 1140 GCGTTTGTTTATATAAAATAACAAGCGCCGGC Fig. 2. DNA nucleotidesequenceoftheentireBHV-2TK geneanditstranslatedproduct. Thepoly(A) sequenceisunderlined andthe putative CAAT and TATA sequences areenclosed. The latter exhibits a relatively low level of homology to BHV-2 within these two areas of the T K gene. Recently, the entire sequence of the BHV-1 T K gene has been published (Mittal & Field, 1989). On comparing the BHV-1 and BHV-2 T K coding regions using the L K B D N A S I S 2020 program, 5 0 . 0 ~ homology was obtained, lower than the sequence homology found between HSV-1 and BHV-2 T K genes. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 11:38:58 3070 Short communication (a) BHV-2(4-21) HSV-I(49-66) HSV-2(49-66) VZV ( 1 2 - 2 9 ) PRV (3-20) Marm. (10-27) BHV-1(IO-27) EBV (284-301) BHV-2(II5-131) HSV-I(162-178) HSV-2(162-178) VZV (129-146) Marm.(130-146) PRV (108-124) BHV-I(132-148) EBV (392-408) LLRVYVDGPHGLGKTTAA LLRVYIDGPHGMGKTTTT LLRVYIDGPHGVGKTTTS VLRIYLDGAYGIGKTTAA ILRIYLDGAYGTGKSTTA ILRVYLDGPHGVGKSTTA VVRIYLDGAHGLGKTTTG ACSLFLEGAPGVGKTTML DRHPTASLLCYPLARYL DRHPIAALLCYPAARYL DRHPIASLLCYPAARYL DRHPIASTICFPLSRYL DRHAVASMVCYPLARFM DRHPVAATVCFPLARFI DRHPVAACLCYPFARYC DRHLLSASVVFPLMLLR Fig. 3. Comparison of the conserved regions within herpesvirus TK genes (a) suggested nucleotide binding site; (b) suggested nucleoside binding site (Kit, 1985). Marmoset herpesvirus, Mama; pseudorabies virus, PRV; Epstein-Barr virus, EBV; numbers represent position of nucleotides in the genome. Herpesvirus sequence taken from Kit et al., 1983; Kit, 1985; Mittal & Field, 1989; Otsuka & Kit, 1984; Sawyer et al., 1988; Swain & Galloway, 1983; Wagner et al., 1981. I n c o n c l u s i o n , w e h a v e l o c a t e d t h e p r e c i s e p o s i t i o n o f a B H V - 2 T K g e n e to 30-0 to 31.0 m . u . , b a s e d o n t h e r e s t r i c t i o n m a p s o f B u c h m a n & R o i z m a n (I 978 a) o f t h e B H V - 2 g e n o m e . W e h a v e also d e t e r m i n e d t h e e n t i r e s e q u e n c e o f t h i s g e n e a n d h a v e l o c a t e d a r e a s o f c o n s e r v e d s e q u e n c e w i t h i n t h i s gene. T h e l o c a t i o n a n d c h a r a c t e r i z a t i o n o f t h i s T K g e n e r e p r e s e n t s a f u r t h e r step t o w a r d s t h e full c h a r a c t e r i z a t i o n o f t h e B H V - 2 g e n o m e a n d f a c i l i t a t e s t h e i n v e s t i g a t i o n o f B H V 2 as a p o t e n t i a l b o v i n e v e c t o r virus. This research was supported in part by the Australian Research Committee REFERENCES BUCHMAN,T. G. & ROIZMAN,B. (1978a). Anatomy of bovine mammillitis DNA. I. 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Minimal size of the active fragments from HSV-I and HSV-2. Journal of General Virology62, 191-206. ROIZMAN,B., CARMICHAEL,L. E., DEINHARDT,F., DE THE, G , NAHMIAS,A. J., PLOWRIGHT,W., RAPP, F., SHELDRICK,P., TAKAHASHI, M. & WOLF, K. (1982). Herpesviridae: definition, provisional nomenclature and taxonomy. Intervirology 16, 201-217. SANGER, F., NICKLEN, S. & COULSON,A. R. (1977). D N A sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. SAWYER, M. H., INCHAUSPE,G., BIRON, K. K., WATERS, D. I., STRAUS,S. E. & OSTROVE,J. M. (1988). Molecular analysis of the pyrimidine deoxyribonucleoside kinase gene of wild-type and acyclovir-resistant strains of varicellazoster virus. Journal of General Virology 69, 2585-2593. STERZ, H., LUDWIG, H. & ROTT, R. (1973/74). Immunologic and genetic relationship between herpes simplex virus and bovine mammillitis virus. Intervirology 2, 1 13. SWAIN, M. A. & GALLOWAY,D. A. (1983). Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene. Journal of Virology 46, 1045-1050. WAGNER, i . J., SHARP, J. A. & SUMMERS,W. C. (1981). Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proceedings of the National Academy of Sciences, U.S.A. 78, 1441-1445. (Received 16 M a y 1989) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 11:38:58