Gene UL2 of Herpes Simplex Virus Type 1 Encodes a Uracil-DNA Glycosylase

J. gen. Virol. (1989), 70, 449-454. Printedin Great Britain

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Key words: HSV-1/uracil-DNA glycosylase/mutagenesis, insertional

Gene UL2 of Herpes Simplex Virus Type 1 Encodes a Uracil-DNA Glycosylase By J A M E S M U L L A N E Y , H E L E N W. M c L . M O S S t AND D U N C A N J. M c G E O C H * t Institute o f Virology, University o f Glasgow, Church Street, Glasgow G l l 5JR, U.K. (Accepted 20 October 1988)

SUMMARY

An insertion mutant of herpes simplex virus type 1 has been constructed which carries the lacZ gene from Escherichia coli within the coding sequence of gene UL2, which is in the long unique region of the genome. In a one-step growth curve experiment this recombinant (called inl601) grew as well as the wild-type (wt) parent virus, indicating that the UL2 gene is dispensable for growth in tissue culture. Analysis of in 1601 D NA with restriction endonucleases showed no detectable changes from the wt apart from the insertion. Extracts of cells infected with inl601 possessed levels of viral D N A polymerase and alkaline exonuclease activities similar to those infected with the wt, but unlike the wt had negligible uracil-DNA glycosylase activity, suggesting strongly that the product of the UL2 gene is the uracil-DNA glycosylase. The sequence of the uracil-DNA glycosylase gene of E. coli was recently published, and the encoded amino acid sequence of this shows clear similarity to that of UL2, confirming our results. Uracil D N A glycosylase is one of a class of enzymes involved in D N A repair (Lindahl, 1979), and acts to remove uracil residues in DNA. It has been isolated from sources as diverse as bacteria (Cone et al., 1977) and humans (Caradonna & Cheng, 1980). The pathways which result in the presence of uracil in D N A are the incorporation of dUMP into the D N A during replication (Bessman et al., 1958) and deamination of cytosine in the D N A (Shapiro et al., 1973). Any uracil residues in the D N A as a result of the latter process would lead to a G. C to A . T mutation in the next round of replication. Caradonna & Cheng (1981) reported induction of uracil-DNA glycosylase activity in cells infected with herpes simplex virus types 1 and 2 (HSV-1, -2), and recently Caradonna et al. (1987) described isolation of an HSV-2 c D N A which encoded uracil-DNA glycosylase activity. They constructed a cDNA library with m R N A from HSV-2-infected cells, and by use of pooled isolates from this library in hybrid-arrest and in vitro translation experiments were able to isolate a clone apparently encoding uracil-DNA glycosylase. Southern analysis mapped this c D N A to between 0.065 and 0.080 map units on the prototype HSV genome. It seems likely that the role of the HSV enzyme is removal of uracil residues created by the deamination of cytosine rather than by misincorporation of dUMP residues (Caradonna et al., 1987) as there is a dUTP-hydrolysing activity present in infected ceils (Preston & Fisher, 1984; Fisher & Preston, 1986; Caradonna & Adamkiewicz, 1984). The genome of HSV-1 is a large molecule of double-stranded DNA, which is approximately 152 000 bp in length. The sequence of the viral genome has recently been determined (McGeoch et al., 1988 ; Perry & McGeoch, 1988). This analysis shows that there are at least 72 genes in the genome, about half of which at present are of unknown function. Comparison of the mapping data for the glycosylase with the sequence data indicated that the primary candidate for the tMember of the MRC Virology Unit. 0000-8545 © 1989 SGM

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Fig. I. Position of UL2 in the HSV-I genome. The upper part of the figure shows the prototypic arrangement of the HSV-1 genome. Repeat sequences are represented by open boxes and unique sequences are shown as solid lines. The lower part of the figureshows an expanded view of the left end of UL and the positions of the genes UL1, UL2 and UL3. The position of the XbaI site used to mutagenize UL2 is marked by X and the position of the second possible initiation codon in UL2 is marked by the dotted line. The scale bar indicates 0.5 kbp on the lower part of the figure. glycosylase gene was UL2, which is at the left end of the long unique (UL)segment of the genome (see Fig. 1). We have now analysed this gene by insertional mutagenesis. The plasmid pGX23 (supplied by V. G. Preston, MRC Virology Unit) contains the HSV-1 BamHI e fragment, in which gene UL2 resides. UL2 was mutagenized by inserting an XbaI fragment of the plasmid pFJ3 (supplied by F. Rixon and J. McLauchlan, MRC Virology Unit) into the unique XbaI site in the UL2 gene. This fragment contained the Escherichia colilacZ gene (encoding fl-galactosidase) under the direction of the simian virus 40 early promoter. This gave rise to the plasmid pJM3. pJM3 D N A was digested with BstEII, which cuts the plasmid at three sites, each of which is at least 4 kbp from gene UL2. This D N A was cotransfected with HSV-1 strain 17 D N A onto confluent monolayers of BHK C13 cells in 50 mm plates using the calcium phosphate technique followed by a dimethyl sulphoxide boost (Stow & Wilkie, 1976). The plates were overlaid with Glasgow modified Eagle's medium (GMEM) which contained 100 units/ml penicillin, 100 ~tg/ml streptomycin, 1.5~ methyl cellulose and was supplemented with 5 ~ calf serum. The plates were incubated at 37 °C for 2 to 3 days, until plaques appeared. The medium was then removed and the plates were overlaid with GMEM containing 0"64~o Noble agar and 1.5 mM-5-bromo-4-chloro-3-indolyl fl-D-galactopyranoside, a chromogenic substrate for flgalactosidase. After overnight incubation, plaques formed by recombinant progeny virus expressing the fl-galactosidase gene turned blue. These blue plaques were picked and purified to homogeneity by four successive rounds of plaque purification. An elite stock, made from a single blue plaque picked from the fourth round of purification, was designated in 1601, and used in all subsequent experiments. The genomic structure of the recombinant in1601 was examined by restriction analysis of virus D N A with the enzymes XbaI, BamHI and BglII, as described by Lonsdale (1979) (data not shown). This analysis indicated that the insert was in the expected location in the viral genome, and no other changes were seen in inl601 DNA. A one-step growth curve experiment was performed on inl601 and the wild-type (wt) parent virus. Confluent monolayers of BHK C 13 cells on 35 mm plates were infected with either wt or inl601 virus at an m.o.i, of 5 p.f.u./cell and left to incubate for 1 h. The plates were then washed twice with GMEM without serum, and overlaid with 1.5 ml of GMEM supplemented with 5 ~ tryptose phosphate and 10~o calf serum (ETC10). Plates were harvested at l, 2, 3, 4, 6, 8, 10, 12,

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Fig. 2. One-step growth curves of wt ([7) and inl601 (11) virus. 21 and 25 h post-infection (p.i.) by scraping into the medium. The harvests were disrupted ultrasonically and titrated on 50 mm plates of confluent BHK C13 cells. Fig. 2 shows the onestep growth profiles of the wt parent and in1601 progeny virus. The two isolates had very similar growth characteristics, with both curves being as expected for HSV-1 wt. This is consistent with the observation that in1601 formed plaques and grew as well as the wt throughout the rounds of purification (data not shown). Extracts were made and assayed for uracil-DNA glycosylase activity by measuring the release of radioactivity from D N A containing [3H]uracil as described by Caradonna & Cheng (1980). Plates (90 mm) of confluent monolayers of BHK C 13 cells were either mock-infected or infected with wt or inl601 virus at an m.o.i, of 10 p.f.u./cell and harvested at 3, 6, 9 and 12 h p.i. The extracts were also assayed for viral D N A polymerase activity (Keir et al., 1966) and alkaline exonuclease activity (Francke et al., 1978). The results of the uracil-DNA glycosylase assay (Fig. 3a) show that there is no significant glycosylase activity associated with inl601 in comparison with the levels seen with wt and the mock-infected control. Checks were made for the wt enzyme that the released radioactivity was actually in uracil, as tested by thin-layer chromatography. As controls for the efficiency of the infections, viral D N A polymerase and alkaline exonuclease activities were measured in all the extracts, and the results are shown in Fig. 3 (b,c). These figures show that in1601 induces levels of both viral D NA polymerase and alkaline exonuclease activities which are comparable to those of the wt, the mock-infected levels of both enzymes being negligible. In order to determine how the gene was regulated, i.e. whether it was an early or late gene, infections were performed on BHK C13 cells with wt virus in the presence or absence of 300 I~g/ml phosphonoacetic acid (PAA), which inhibits viral D N A synthesis in wt HSV-1 (Shipkowitz et al., 1973). Confluent monolayers were incubated in ETC10 supplemented with 300 l.tg/ml PAA for 1 h before being infected with virus at an m.o.i, of 10 p.f.u./cell in the presence or absence of 300 ~tg/ml PAA. The plates were incubated for 1 h before being overlaid with ETC10 containing 300 ~tg/ml PAA. The cells were harvested at 24 h p.i. and extracts were prepared and assayed for uracil-DNA glycosylase, viral D N A polymerase and alkaline exonuclease activities as previously described. There was no significant difference in the levels of enzyme activity between cells infected in the presence or absence of PAA (data not shown), indicating that UL2 is an early gene. Although the results presented here show that inactivation of gene UL2 leads to a complete loss of glycosylase activity, this in itself cannot be taken as proof that this gene encodes the

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Fig. 3. Time course of enzyme assays performed on extracts of mock-infected cells (O) or cells infected with wt virus ([1]) or inl601 ( I ) . Extracts were prepared as described by Caradonna & Cheng (1981). (a) Uracil D N A glycosylase activity of extracts made at 3, 6, 9 and 12 h p.i. The assay was performed under the conditions described by Caradonna & Cheng (1980), using a 1 in 10 dilution of the extracts. (b) Time course for the alkaline exonuclease activity, performed at pH 9 and using radiolabelled BHK D N A as described by Francke et al. (1978). Extracts were assayed at a dilution of 1 in 10 and acid-soluble radioactivity was measured after incubation at 37 °C for 1 h. (c) Viral D N A polymerase activity through the time course. D N A polymerase activity was measured in undiluted cell extracts essentially by the method of Keir et al. (1966) but using activated calf thymus D N A (Weissbach et al., 1973) as template and with a KC1 concentration of 200 mM.

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Fig. 4. A m i n o acid sequence comparison between E. coli ung gene and UL2 and its homologues in VZV and EBV. The first sequence is part of the E. coli ung gene from amino acid 50 to 221. The second sequence is of U L 2 from 164 to 333 (taking the first methionine reside as 1). The third sequence is that of VZV gene 59 from 134 to 303 and the fourth sequence is EBV B K R F 3 from 77 to 254. Pads inserted by the program to achieve optimal alignment are shown as dots. On the consensus line amino acids conserved in all four sequences are indicated.

enzyme. The UL2 gene could, in principle, encode a regulator of the glycosylase. However, in the light of the mapping data already available (Caradonna et al., 1987), it seems essentially certain that the UL2 gene codes for the glycosylase. Gene UL2 has counterparts in the other herpesviruses whose genomes have been sequenced. These are gene 59 in varicella-zoster virus (VZV) (Davison & Scott, 1986; Davison & Taylor, 1987) and BKRF3 in Epstein Barr virus (EBV) (Baer et al., 1984; Perry & McGeoch, 1988). The amino acid sequences of all three genes are notably conserved, especially towards their 3' termini. Thus, although the gene is dispensable for growth of HSV-1 in tissue culture its function may be important for survival of the viruses in vivo. Perry & McGeoch (1988) pointed out the presence of two possible initiation codons for the UL2 open reading frame (ORF). The first opens an ORF encoding a polypeptide of 334 amino acids with a predicted M, of 36 300, and the second opens an ORF encoding a polypeptide of 244 amino acids with a predicted Mr of 27 300. From comparisons of the amino acid sequences of UL2 with the homologues in VZV and EBV the second candidate initiation codon may be more likely. Since this work was completed, Varshney et al. (1988) have published the sequence of the E. coli uracil-DNA glycosylase gene and show that it encodes a protein of 229 residues with an Mr of 25 664. We have compared the amino acid sequence of this protein with the amino acid sequence of HSV-I UL2 and its homologues in VZV and EBV. As shown in Fig. 4, there are several regions of complete identity (see consensus) and there are other regions which have partial identity between three of the four sequences. The level of similarity between the herpesvirus sequences and the E. coli sequence is very striking, and confirms that UL2 does encode the uracil-DNA glycosylase enzyme. J.M. is in receipt of a Medical Research Council postgraduate studentship.

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(Received 7 July 1988)