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Journal of General Virology (1991), 72, 1435-1439. Printedin Great Britain

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Loss of pseudorabies virus thymidine kinase activity due to a single base mutation and amino acid substitution J. Prieto, A. M. Martin Hernhndez and E. Tabards* Departamento de Microbiologia, Facultad de Medicina, Universidad Aut6noma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain

The nucleotide sequences of the coding region of the thymidine kinase (TK) gene of pseudorabies virus strain NIA3 and a TK- mutant (ATK5) were determined. The coding region of the TK gene consists of 320 codons capable of producing a polypeptide with an Mr of 34979. The mutant expressed an inactive TK

polypeptide when translated in vitro; a unique base substitution was detected in the mutant TK modifying amino acid position 13, at which aspartic acid replaces glycine. This modification affects the nucleotidebinding site, thus explaining the expression of an inactive TK polypeptide.

Pseudorabies virus (PRV) is a herpesvirus that causes an acute and often fatal infection of the nervous system (Aujeszky's disease), as well as a latent infection in swine; it causes acute infections in domestic and wild animals (Baskerville et al., 1973), but does not infect humans. However, the natural infection of swine is similar to that of herpes simplex virus type 1 (HSV-1) in man; latency is a central feature of both infections, the trigeminal ganglia being the most frequent site of PRV and HSV-I latency. PRV and HSV-1 DNAs show limited homology throughout their genomes, a fact compatible with the accepted view that, although there has been considerable evolutionary divergence, these viruses probably evolved from a common ancestor. Despite this divergence, a certain degree of collinearity between the genomes of PRV and HSV has been retained (see Ben Porat & Kaplan, 1985). Both viruses have been amenable to genetic manipulation, providing excellent models for basic virology as well as for investigation of new modes of disease intervention and the design of novel vaccines (Roizman & Jenkins, 1985; Thomsen et al., 1987). In general, these methods rely on the use of the thymidine kinase (TK) gene as the selective agent. The TK activities of both HSV and PRV are not essential for growth in culture, but are essential for virulence and latency (Efstathiou et al., 1989; Kit, 1985 a; Kit et al., 1985; McGregor et al., 1985; Tenser et al., 1983). It is known that PRV is attenuated readily by removal of the TK function (Kit, 1985a, Kit et al., 1985;

McGregor et al., 1985) and therefore, it seems that PRV might be a suitable live vaccine vector for carrying antigens of other pathogens. We report the nucleotide sequence of the PRV TK gene and of a TK- mutant lacking enzymic activity due to a single nucleotide mutation. Marker rescue experiments have demonstrated that the PRV TK gene is localized within BamHI fragment 11 of the PRV genome (Ben-Porat et al., 1983). A DNA region of 1686 bp has been sequenced (Kit, 1985a), which includes an open reading frame (ORF) of 1107 bp encoding a protein of 369 amino acids with an Mr of 40612, corresponding to the TK gene. This has shown that the TK gene is located between BamHI fragments 11 and 9 (Nunberg et al., 1989; Moormann et al., 1990). On the basis of these results, the TK gene was cloned by partial BamHI digestion of PRV strain NIA3 DNA and the insertion of fragments into a pUC 18 plasmid vector. Methods for the extraction of DNA from PRV, its digestion with restriction enzymes and purification of recombinant plasmids have been described previously (Tabar6s et al., 1987). Standard DNA manipulation techniques were used to construct recombinant plasmids (Maniatis et al., 1982); one of these, pRA3, contained BamHI fragments 9, 11 and 15, and a small fragment of about 350 bp (Fig. 1) not identified in the PRV strain NIA3 genome (Gielkens et al., 1985). The SphI-KpnI 1.8 kbp and the BamHI-KpnI 1-4 kbp TK gene fragments from pRA3a (Fig. 1) were sequenced and the ORFs on both strands determined. Nucleotide sequencing was carried out using the dideoxynucleotide chain-termination method (Sanger et al., 1977) with recombinant pUC18 or pGEM 3Z as template. The

The DNA sequencedatain thispaperhavebeenassignedthe EMBL accessionnumbersX55001 (the TK- mutantDNA) and X55002 (the TK DNA sequence). 0001-0060 O 1991 SGM

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K

8

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C

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Fig. 1. Physical map of plasmids containing the TK gene. Plasmid pRA3 consists of the PRV BamHI fragments 9, 11 and 15, and a small fragment of about 350 bp. Plasmid pRA3a was obtained by deletion of a KpnI fragment of pRA3. The SphI-KpnI 1.8 kbp TK gene fragment from pRA3a was inserted into pGEM3Z to give pGEMTK3a. The BamHI-KpnI 1.4 kbp TK gene fragment from pRA3a was inserted in pGEM3Z to give pGEMTK. Restriction enzyme sites: B, BamHI; K, KpnI; X, XhoI; S, Sphl.

DNA sequence obtained, together with the deduced amino acid sequence of the TK protein, are shown in Fig. 2. The consensus Hogness box was not found, however the two distal transcription signals CCGCCC and G G G C G G of the herpesvirus TK gene (McKnight et al., 1984) were located 341 and 157 nucleotides, respectively, upstream of the initiation codon. A typical AATAAA polyadenylation signal sequence (Proudfoot & Brownlee, 1976) is present 111 nucleotides downstream of a termination codon. Evidence that the BamHI-KpnI fragment contains the TK gene was obtained through the synthesis of active TK protein in a reticulocyte cell-free system programmed with R N A synthesized in vitro from plasmids pGEMTK and pGEMTK3a (Fig. l) using SP6 R N A polymerase, pGEMTK and pGEMTK3a were linearized at an internal KpnI site close to the T7 promoter, extracted with phenol-chloroform and ethanol-precipitated. In vitro transcription with SP6 R N A polymerase was performed as described by the supplier (Promega Biotec) in the presence of 0.5 mM-m7G(5')ppp(5')G. The resulting R N A was translated in a rabbit reticulocyte lysate system or in wheatgerm extract (Promega Biotec) in the presence of [35S]methionine. Labelled proteins were analysed by 12~ SDS-PAGE (Tabar6s et al., 1980a). Samples from the translation mixture yielded

T666CCAC6A ACACCABCA6 666CAC6A6C 6T6ATCTCCT CBCCGCCC66 666CACG6C6 6C66CGAGBA 66C6C6CC6C CBAGTC6CBA 6CTGGCACA6 CCCCT6CT6C CGCT6CCCBC 6CTT6CT666 C6TCTT6A66 TTCC66666A A6C66CACBT CTT6ABCTC6 AT6AC6AABC ACA66T6C6G CCCCACCCCC AGCC6CACCA C6CACACBCA 6TC6666C66 CGCACCCC6A66TT6ACTTC AAA66CCA66 6TCAAGBAC6 CCTTCTTAAG C6TCTCfC66 6EiAA6CCCBA A6AGACTCTC 6CC6TAC6C6 6AC6GGTCGC 6GCGCAGGC61-1"CGTAGAA6 C66TT6'r66c ABC66ATCCC C6CCC66AA6 C6C8CCB6,6 AT6 C6C Arc Barn HI (A) H R I CTC A66 ATC TAC CTC 6AC 66C 6CC TAC 66C ACC 66C AA6 A6C ACCAC6 L R I Y L O 6 A Y 6(D) T 6 K S T T GCCC66 61"6 AT6 6C6 CTC 66C 666 6C6 CT6 TAC 6T6 CCC 6A6 CC6 AT6 A R V H A L 6 6 A L Y V P E P H 6C6 TAC T66 C6C ACT CT6 TTC 6AC/kC66A,C ACB6"[6 6CC £~T ~TT TkC A Y W R T L F O T D T V A 6 I Y 6AT 6C6 CA6 ACC C66 AA6 CAG AAC 66C AGCCT6 AGC GAG6A6 6AC 6C6 O A Q T R K Q N 6 S L S E E 0 A 6CC CTC 6TC AC6 6C6 CAC 6AC CA6 6CC 6CC TTC 6C6 AC6 CC6TAC CT6 A L V T A H D Q A A F A T P Y L CT6 CT6 CAC ACEC6C CT6 6TC CCGCTCTTC 66G CCC6C6 6TC 6A6 66C L L H T R L V P L F G P A V E 6 CC6 CCCGAGAT6 AC6 6TC 6TC TIT GACC6C CAC CC6 61"6 6CC 6C6 AC6 P P E H T V V F D R H P V A A T 61"6 T6C TTC CC6 CT6 6C6 C6CTTC ATC 6TC 66G 6AC ATC A6C 6C6 6C6 V C F P L A R F I V 6 D I S A A 6CCTTC 61"6 66C CTGGCGGCCAC6 CT6 CCC 6GGGAGCCC CCCGGCGGC A F V 6 L A A T L P G E P P 6 G AAC CT6 b'T6 61"6 6CC TC6 CT6 6AC CCG6AC 6A6 CAC CT6 CGGC6C CT6 N L V V A S L D P D E H L R R L C6C 6CC C6C 6C6 C6C 6CC GGG6A6 CACGTG6AC GC6 CGCCT6 CTC ACG R A R A R A 6 E H V D A R L L T 6CC CT6 C6C AAC 6TC TAC 6CC AT6 CT6 6TC AAC ACBTC6 C6C TAC CT6 A L R N V V A 11 L V N T S R Y L AGCTC6 666 C6C CGCT66 CGC6AC 6AC T66 666 CGC6C6 CCGCGCTTC S S 6 R R '# R D D "H B R A P R F 6AC CA6 ACC AC6 C66 6AC T6C CTC 6C6 CTC AAC 6AG CTC TGCC6C CC6 0 Q T T R D C L A L N E L C R P C6C 6AC 6AC CCC6A6 CTC CA6 6AC ACC CTCTTT 66C 6C6 TAC AA6 GC6 R O O P E L Q D T L F B A Y K A CCC6A6 CTCTGC 6AC C6G CGC666 C6C CCGCTC 6A6 GT6 CAC 6C6 T6G P E L C O R R 6 R P L E V H A W GC6AT6 GAC6C6 CTC GTG6CC AA6 CTGCT6 CCGCTGC6C 6TC TCC ACC A I't O A L V A K L L P L R V S T 6TC BAC CT6 6BB CCCTC6 CC6 C6C 6TC TfiC 6CC GC66CC 6T6 BCG6C6 V O L 6 P S P R V C A A A V A A CAB AC6 C6C 66C AT6 BAG 6"1"6AC6 6A6 TCC GC6TAC 66C 6AC CAC ATC Q T R 6 tt E V T E $ A ¥ 6 I) H ! C66 CA6 T6C 61"6TGC 6CC TTC AC6 TC6 6A6 AT6 B66 6T6 TBA R Q C V C A F T S E H 6 V Ter CCCTC6CCCC TCCCACCCGC 6CC6C66CC6 6AT66AGACC 6CfACB6A6G CAACBACGAC 66CGT6GGA6 666GCTC66G 6CGC6TATAA AC6CAT6TBT AT61"CATCCC AATAA/~GTTT 6CC6T6CCC6 TCACCATGCC

Fig 2. Nucleotide sequence of the PRV NIA3 TK gene and flanking regions. A 1.8 kbp DNA fragment between the SphI site and the KpnI site was digested with several restriction endonucleases and subcloned into pUCl8. The nucleotide sequence of 1493 bp obtained and the predicted amino acid sequence of TK protein are given. Ter, termination codon. Locations of cleavage sites for BamHI, XhoI, the distal transcription signals and the putative AATAAA polyadenylation signals are underlined. The single guanine to adenine transition in codon 13, which causes the substitution of aspartic acid for glycine in the TK polypeptide of TK- mutant ATK5, is indicated.

active TK (Table 2). The polypeptide synthesized had an Mr of about 34-6K (Fig 3a, lane 3), in good agreement with that predicted from the gene sequence. The in vitro translation of R N A using plasmid pGEMTK3a produced a polypeptide of the same size (Fig. 3 a, lane 4),

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Fig. 3. SDS-PAGE of the polypeptides synthesized using different RNA preparations as templates. Equal volumes of reticulocyte lysate programmed with different RNA preparations were subjected to electrophoresis on a 12% polyacrylamide slab gel and labelled polypeptides were visualized by autoradiography of the dried gel. (a) No added RNA (lane 5); RNAs transcribed by SP6 RNA polymerase from pGEMTK (lane 3) and from pGEMTK3A (lane 4), linearized with Kpnl. (b) RNAs transcribed by SP6 RNA polymerase from pGEMTK and translated in reticulocyte lysate (lane 6) or wheatgerm extract (lane 4), or from pGEMATK5 translated in reticulocyte lysate (lane 5) or wheatgerm extract (lane 3). Polypeptides used as standards were from uninfected MS cells (lanes 1), and cells infected with African swine fever virus (Tabar6s et al., 1980b) labelled with [3sS]methionine from 20 h to 22 h post-infection. (lane 2). IP, Infected cell polypeptides.

which confirms that the initiation codon is located in B a m H I fragment 11. The next ATG codon, located 219 nucleotides upstream on B a m H I fragment 9, cannot be the initiation codon because a termination codon is found 141 nucleotides downstream of it. The nucleotide sequence of the PRV TK gene revealed an ORF of 320 codons and the Mr of 34979 calculated from the deduced amino acid sequence corresponds closely to the value of 34.6K determined from PAGE of the TK polypeptide synthesized in reticulocyte cell-free and wheatgerm extracts. This differs slightly from the Mr of 32480 deduced for the TK of PRV strain Bucharest (BUK) when transcription was initiated at the same codon (Kit, 1985a, b). Alignment of the TK polypeptides from PRV strains NIA3 and BUK (with the same initiation codon being used in each) shows that the possible nucleoside-binding site at residues 111 to 127, which corresponds to residues 162 to 178 of the HSV-1 TK (Darby et al., 1986), is mutated in PRV BUK by the replacement of an alanine with a proline at residue 119, which is highly conserved in the TK polypeptides of equine herpesvirus type 1 (EHV- 1) (Robertson & Whalley, 1988), infectious bovine

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rhinotracheitis virus (Mittal & Field, 1989), bovine herpesvirus type 2 (Sheppard & May, 1989), feline herpesvirus type 1 (Nunberg et al., 1989), HSV-1 (McKnight, 1980; Wagner et al., 1981), HSV-2 (Swain & Galloway, 1983), varicella-zoster virus (Davison & Scott, 1986), marmoset herpesvirus (Otsuka & Kit, 1984), Marek's disease virus (Scott et al., 1989) and herpesvirus of turkeys (Scott et al., 1989). The TK polypeptide of PRV strain NIA3 has a longer carboxy terminus than PRV strain BUK with greater identity to the carboxy terminus of EHV-1. Spontaneous T K - mutants of viruses containing the TK gene can be selected by plaque formation in the presence of 5-bromodeoxyuridine (BUdR). To obtain PRV strains capable of growing in the presence of BUdR, cultures of BHK(TK-) (kindly supplied by B. Roizman, University of Chicago, I11., U.S.A.) were inoculated with PRV strain NIA3 and grown in Dulbecco's modified Eagle's medium supplemented with 5% foetal calf serum and 25 ~tg BUdR/ml. Five mutants were purified by plaque selection on PK 15 cells and their TK activity was assayed indirectly by measuring the uptake of [3H]thymidine ([3H]TdR 82.2 Ci/mmol; New England Nuclear) into BHK(TK-) cells; 5 x 104 BHK(TK-) cells were infected with PRV and labelled with 1 ~tCi [3H]TdR for 24 h after infection. Cultures were lysed with 0.5% SDS and 10 mM-NaOH, and precipitated with ice-cold 10% TCA. Precipitates were collected by filtration through glass-fibre filters and counted in toluene-based scintillation fluid. The TK genes of two of these mutants (ATK3 and ATK5) (Table 1) were cloned in pGEM3Z to give pGEMATK3 and pGEMATK5, respectively. These plasmids were sequenced as described for the wild-type virus. In both cases, a single guanine to adenine transition was found at codon 13 (Fig. 3). This produces a substitution of aspartic acid for glycine at position 13 of the TK polypeptide and the result is the loss of TK activity, as demonstrated by in vitro translation of RNA synthesized in vitro (Table 2). pGEMATK3 and pGEMATK5 were linearized at an internal KpnI site close to the T7 promoter and transcribed by the procedure described previously. The resulting RNA was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine and analysed by 12% SDS-PAGE (Tabar6s et al., 1980a). TK activity was measured in reticulocyte lysate reactions by following the conversion of [3H]thymidine to a negatively charged species able to bind to DEAEcellulose paper; unlabelled reticulocyte lysates were assayed for TK activity after 4 h translation in the presence of 50 pM-TdR to stabilize nascent TK activity (Weir et al., 1982). The polypeptide synthesized by the mutant plasmid had an Mr of about 34.6K (Fig. 3b, lanes 3 and 5) with the

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Table 1. Levels o f T K activity in extracts o f cells infected with P R V Source of extract

TK activity (c.p.m.)*

BHK(TK-) BHK(TK-) infected with PRV ATK5 BHK(TK-) infected with PRV ATK3 BHK(TK-) infected with PRV NIA3

5060 1705 3169 47011

* TK activity was assayed by measuring the incorporation of [3HITdR into DNA in a 24 h period.

Table 2. Cell-free translation o f R N A * RNA

Plasmid

TK activity (c.p.m.)

Lysate without RNA RNA (in vitro) RNA (in vitro)

pGEMATK5 pPGEMTKt

2900 3474 24607

* The TK activity was assayed by measuring the conversion of [3H]thymidine into [3H]thymidilate. RNAs were translated in a micrococcal nuclease-treated reticulocyte lysate and an aliquot was assayed. t pGEMTK and pGEMATK5 were linearized with KpnI.

s a m e m o b i l i t y as the p e p t i d e d e r i v e d f r o m the w i l d - t y p e gene (Fig. 3b, lanes 4 a n d 6). In a d d i t i o n , B a m H I f r a g m e n t 11 f r o m p R A 3 a n d the T K - virus A T K 5 was cloned into p D S 1 2 / R B S I I - 2 ( k i n d l y s u p p l i e d b y R. P e r o n a , C.S.I.C., M a d r i d , S p a i n ) (Bujard et al., 1987) to yield p l a s m i d s p D S I I - 1 1 T K a n d p D S I I - 1 1 A T K 5 a n d t r a n s f o r m a n t s were p l a t e d on drug selection plates. F o r selection o f T K expression, the T K - K Y 8 9 3 s t r a i n ( H i r a g a et al., 1967) o f Escherichia coli was used. U n t r a n s f o r m e d E. coli s t r a i n K Y 8 9 3 cells a n d those t r a n s f o r m e d w i t h p l a s m i d s p D S 1 2 / R B S I I - 2 or p D S I I l l A T K 5 were u n a b l e to g r o w on t h e selection m e d i a ; only clones c o n t a i n i n g w i l d - t y p e viral T K c o m p l e m e n t ing the cellular T K d e f e c t s u r v i v e d on the drug selection plates. T h e h e r p e s v i r u s T K has served as a selectable m a r k e r for genetic e n g i n e e r i n g ( R o i z m a n & J e n k i n s , 1985) a n d the T K gene o f P R V has b e e n used for g e n e t i c m a n i p u l a t i o n o f this virus ( T h o m s e n et al., 1987). M u t a t i o n in T K genes has b e e n d e s c r i b e d for v a c c i n i a virus (Bajszar et al., 1983) a n d h e r p e s v i r u s ( D a r b y et al., 1986; M i t t a l & F i e l d , 1989). I n v a c c i n i a virus, three spontaneous TK- mutants with nonsense phenotypes were isolated a n d the m u t a t i o n s were a n a l y s e d b y n u c l e o t i d e s e q u e n c i n g ( W e i r & Moss, 1983); in e a c h case, a n e x t r a nucleotide, i d e n t i c a l to the one p r e c e d i n g it, was found. T h i s e x t r a n u c l e o t i d e results in a f r a m e s h i f t a n d the o c c u r r e n c e o f a t e r m i n a t i o n c o d o n d o w n s t r e a m . I n H S V , two o f the o b s e r v e d s u b s t i t u t i o n s were l o c a t e d close to one a n o t h e r in the p r i m a r y

sequence, affecting the apparent affinity of TK for nucleoside substrates. A third substitution was located some distance away, towards the C terminus, at residue 336 and affected markedly ATP and nucleoside binding. In the mutant of PRV NIA3 described here, a single transition was found at the ATP-binding site (Darby et al., 1986; Gentry, 1985) close to the amino-terminal, resulting in the loss of TK activity. This result is consistent with those obtained from site-directed mutagenesis of the ATP-binding site of HSV-1 TK (Liu & Summers, 1988). The substitution of aspartic acid for glycine in the PRV TK- polypeptide could result in repulsion between the carboxyl group of the aspartyl and the phosphate groups of ATP, and/or perturb the protein structure of the enzyme (Liu & Summers, 1988), thus explaining the loss of TK activity. This replaced glycine is conserved in all alphaherpesvirus and gammaherpesvirus TK polypeptides (lioness et al., 1989), whereas in poxvirus and cellular TK it is replaced by phenylalanine (Boyle et al., 1987). We are grateful to S. Fernandez and J. Alvarez for excellent technical assistance, and to Dr E. Domingo (C.S.I.C., Madrid) and Dr R. Marco (Facultad de Medicina, U.A.M., Spain) for help with the manuscript. This work was supported by a grant from Laboratorios Ovejero S.A. (Spain).

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(Received 4 December 1990; Accepted 18 February 1991)

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