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Identification of the gene homologous to HSV major DNA binding protein in the BHV-1 genome
Veterinary Microbiology, 22 (1990) 203-212 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
203
I d e n t i f i c a t i o n of the Gene H o m o l o g o u s to HSV Major D N A B i n d i n g P r o t e i n in the B H V - 1 G e n o m e S.K. BANDYOPADHYAY*, S.K. MITTAL** and H.J. FIELD***
Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES (Great Britain) (Accepted 24 October 1989)
ABSTRACT Bandyopadhyay, S.K., Mittal, S.K. and Field, H.J., 1990. Identification of the gene homologous to HSV major DNA binding protein in the BHV-1 genome. Vet. Microbiol., 22: 203-212. By means of Southern blot hybridisation using a cloned herpes simplex virus (HSV) major DNA binding protein (MDBP) gene as probe, the putative MDBP gene of BHV-1 was located within the Hind III G fragment which mapped between 0.352 and 0.381 map units of the BHV-1 genome. Moreover, an antiserum raised to HSV MDBP precipitated a 120kD polypeptide in a radio-immunoprecipitationtest.
INTRODUCTION
Bovine herpesvirus i (BHV-1) causes a variety of diseases in cattle including rhinotracheitis, conjunctivitis, encephalitis, abortion and genital lesions (Kahrs, 1977). Approximately 40 polypeptides have been identified in BHV1 infected cells of which 18 to 33 have variously been assigned as structural components of the virus (Misra et al., 1981; Bolton et al., 1983; Trapanier et al., 1986). The genome of BHV-1 is a double-stranded linear DNA molecule divided into a long and a short unique region with inverted repeat sequences which bracket the short region (Mayfield et al., 1983; Hammerschmidt et al., 1986). During virus replication, certain proteins are required to interact with virus DNA performing catalytic or regulatory functions in the synthesis of viral DNA (reviewed by McGeoch, 1987). One such protein, the MDBP has been implicated in important regulatory functions in HSV replication apart from its sup*Present address: Indian Veterinary Research Institute, Mukteswar-Kumanon: 263138, Nainital (UP), India **Present address: Department of Biology, McMaster Univeristy Hamilton, Ont., Canada. ***To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
204
S.K. BANDYOPADHYAY ET AL.
posed function concerning stabilisation of the replicative DNA complex (Conley et al., 1981; Lee and Knipe, 1983; Littler et al., 1983; Ruyechan and Weir, 1984; Godowski and Knipe, 1986). The identification of genes in the BHV-1 genome has recently been commenced. The genes homologous to HSV gB, DNA polymerase and thymidine kinase have been localised in the BHV-1 genome (Lawrence et al., 1986; Owen and Field, 1988; Mittal and Field, 1989). In the present communication we report the identification of the putative MDBPgene of BHV-1 using the strain BHV-1 (6660). MATERIALSAND METHODS The virus strain (6660) is of the 'Cooper' type with respect to its restriction endonuclease map (Mayfield et al., 1983; Owen and Field, 1988). Virus was grown in Madin Darby bovine kidney (MDBK) cells in the presence of Eagle's minimum essential medium (EMEM). For virus purification, confluent monolayers of MDBK cells were infected with BHV-1 (6660) using m.o.i, of 0.1 pfu/ cell. When the cytopathic effect was complete (48 to 72h post-infection) the virus was purified from the supernatant by a single cycle of 10-50% potassium tartrate density gradient centrifugation (Misra et al., 1981 ). For Southern blotting and hybridisations, virus DNA was extracted from purified BHV-1 virions. Restriction endonuclease digestion, agarose gel electrophoresis of digested DNA fragments and Southern blot transfer of DNA fragments to Hybond-N membranes were carried out using standard methods (Owen and Field, 1988). The Hind III 'G' fragment of BHV-1 (6660) DNA was cloned in pEMBL8 (Dente et al., 1983) and named pSKM7. The HSV-1 MDBP gene contained in the Eco RI 'F' fragment (pSG18), cloned in plasmid pBR325 (Goldin et al., 1981) was obtained from Dr. M. Levine of the University of Michigan, U.S.A., through Dr. A. Minson of the Department of Pathology, University of Cambridge, Great Britain. Probes for Southern-blot hybridisation were prepared either from the Sal I 'a' fragment directly (probe A) or this fragment was further digested with Barn HI (in order to remove the unwanted portion of the adjacent DNA polymerase gene). The resultant Bam HI 'a' and Barn HI 'b' fragments, which together contained most of the M D B P gene of HSV-1 (Fig. 2), were used for preparing probes (probes B and C respectively). DNA probes used in the Southern-blot hybridisation experiments were labelled with 32[p ]dCTP (Amersham; 3000 Ci/mmol) using an oligonucleotide labelling kit (Pharmacia). Hybridisation was carried out using standard methods (Owen and Field, 1988). For radio-immunoprecipitation, confluent monolayers of MDBK cells were grown in 35-mm Petri dishes and infected with BHV-1 at a multiplicity of infection of 10 pfu/cell. The cells were labelled with 35[S]-methionine (approx. 980 Ci/mmol) between 1 and 10h post-infection at a concentration of 50
IDENTIFICATION OF THE GENE HOMOLOGOUS TO HSV MDBP IN THE BHV-1 GENOME
205
Ci/ml in E M E M containing one-fifth the normal concentration of labelled methionine. After labelling, the monolayers were briefly washed with phosphate buffered saline and the cells were solubilised with 2 ml/dish of RIPA buffer (10 mM Tris-HC1 [pH 7.4], 150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS, 1% NP40, 1 mM PMSF) on ice for 0.5h. The solubilised suspension was centrifuged at 35 000 rpm for l h at 4°C and the supernatant was stored at - 7 0 ° C until required for analysis. A 150 ~l portion of this solubilised cell preparation was incubated at 37 °C for 2h with 20 ttl of either non-immune rabbit serum or rabbit monospecific antiserum to HSV MDBP (provided by Dr. K. N. Powell, Wellcome Research Laboratory, Beckenham, Kent, Great Britain) and 130 ~tl of RIPA buffer. To this was added 100 ttl of a 25% suspension of protein A coupled to Sepharose 6MB (Sigma) and the mixture was incubated for a further period of l h at 37 ° C with occasional shaking. The protein A beads were washed thoroughly with RIPA buffer and resuspended in 40 zl sample buffer (62.5 mM Tris-HC1 [pH 6.8], 2% SDS, 10% glycerol, 5% flmercaptoethanol, 0.0001% bromophenol blue) for analysis by polyacrylamide gel electrophoresis. The samples were analysed by SDS polyacrylamide electrophoresis following the method described by Laemmli (1970) and autoradiographed using Fugi RX X-ray films. RESULTS Localisation of the putative major DNA-binding protein gene in the BHV-1 genome BHV-1 DNA extracted from purified virions was digested with Hind III, Bam HI, Eco Ri or Hpa I and following agarose gel electrophoresis (Fig. la), the DNA fragments were Southern-blotted onto Hybond-N nylon filters. Under conditions of high stringency (washing with 0.1 X SSC, 0.1% SDS at 68 ° C ), all three probes, A, B, and C (Fig. 2), hybridised to a single band in each of the Barn HI, Eco RI and Hpa I digests of BHV-1 DNA. The probe B, containing approximately 65% of the HSV MDBP gene starting from the 5' end of the gene, hybridised only to the G fragment of Hind III digest of BHV-1 (Fig. lb). However, probe A and C containing respectively the whole and the last 35% gene from the 3' end of the HSV MDBP gene, both gave a positive, though somewhat weak signal with fragment A, in addition to strong signal obtained with fragment G, of Hind III digest of BHV-1 DNA (Fig. lb). To further define the region of hybridisation, the recombinant plasmid (pSKM7) DNA was electrophoresed after digestion with Hind III alone (which released the intact Hind III G fragment) and with Hind III and Bam HI combined to release a 4 kb sub-fragment (Fig. 3). Southern-blot hybridisation analysis of these DNA fragments revealed that the probe B hybridised only to the 4 kb fragment (Fig. 3B). Hybridisation to the 4 kb and not to the 7.8 kb
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IDENTIFICATION OF THE GENE HOMOLOGOUS TO HSV MDBP IN THE BHV-1 GENOME
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subfragment suggests that the region of homology starts from map coordinates of 0.35 of the BHV-1 genome and not ahead (Fig. 4). Radio-immunoprecipitation test Normal rabbit serum did not specifically precipitate any polypeptide from the solubilised preparation of BHV-1 infected MDBK cells, although traces of minor contaminating polypeptides could be detected suggesting non-specific precipitation. However, the specific rabbit antiserum to MDBP of HSV was found to precipitate a polypeptide from the solubilised preparation of BHV-1
Fig. 1. (a) Tris acetate agarose gel (0.7%) stained with ethidium bromide used for Southern blotting. BHV-1 virion DNA, was digested with: Hind III (lane 1 ), Bam HI (lane 2), Eco RI (lane 3 ) and HPa I (lane 4). The plasmid pSG18 containing the HSV MDBP gene was digested with: Sal I (lane 5) or with Barn HI (lanes 6 and 7). (b) Autoradiograph of Southern blot hybridisation of the agarose gel; Fig. 1 (a) probed with the HSV MDBP gene. The probes A, B, and C, were derived as described in Fig. 1. All three probes hybridised to Eco RI A, Bam HI G, Hind III G and Hpa I E fragments of BHV-1 genome as indicated by solid arrow-heads. In addition to the above, probe A and C but not probe B also hybridised to the Hind I I I A fragment (faintly represented in the autoradiograph and indicated by empty arrow heads). Lanes 5, 6 and 7 were cut out from shorter autoradiographic exposures of the same blots.
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infected M D B K cells (Fig. 5, lane 6). F r o m t h e p o s i t i o n of the molecular weight m a r k e r s , we have c a l c u l a t e d t h e m o l e c u l a r weight o f this p o l y p e p t i d e as 120 kD. H o w e v e r , in r e l a t i o n t o t h e relative m i g r a t i o n of t h e I C P 8 (128 k D ) of
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HSV-1 (Fig. 5, lane 1 ), the molecular weight of the cross-reacting polypeptide of BHV-1 is expected to be approximately between 132 and 135 kD. CONCLUSION
Thus, Southern blot hybridisation of the BHV-1 genome using a probe containing the MDBP gene from HSV-1, indicated a homology of DNA sequences located between map co-ordinates of 0.352 to 0.381 of BHV-1 genome which may code for the putative M D B P of BHV-1 (Fig. 4). From the observation that the probes containing the 3' end of the HSV MDBP gene produced additional signals in A fragment, we speculate that the orientation of the gene extends from the G fragment towards the A fragment of the Hind III digest of the BHV-1 DNA which, if true, will suggest an opposite orientation to the previously located DNA polymerase gene (Owen and Field, 1988). It is of interest that Southern-blot hybridisation using HSV MDBP gene as a probe has also been used recently to identify the putative gene coding for the analogous protein in BHV-2 (Maragos and May, 1987). The radio-immunoprecipitation data led to the identification of one 120 kD polypeptide in BHV-l-infected MDBK cells which precipitated with rabbit antiserum to HSV MDBP. Studies on the regulation of BHV-1 gene expression
210
S.K. BANDYOPADHYAYET AL.
Fig. 5. Autoradiograph showing the immunoprecipitates of infected cell lysates, labelled with 35[S]methionine, reacted with an antiserum to HSV MDBP, and analysed by SDS-PAGE. Lanes 1, 2 and 3 contain B H K cells infected with HSV-1; lanes 4, 5 and 6 contain BHV-l-infected M D B K cells. Infected-cell fractions were reacted with normal rabbit serum (lanes 2 and 5 ) or HSV M D B P antiserum (lanes 1 and 6). The immune serum precipitated one 120 kD polypeptide from BHV1 infected cells and one 115 kD polypeptide (thought to be the MDBP, ICP8) from HSV-1 infected B H K cells. The positions of the molecular weight markers are shown on the right. All tracks are from the same gel; for clarity some irrelevant tracks have been omitted.
have revealed that this 120 kD non-structural polypeptide belongs to the early class of proteins induced by BHV-1 (Bandyopadhyay, unpublished observation). The above fact together with the observed serological cross-reactivity of this polypeptide with the HSV MDBP, provide strong circumstantial evidence that the 120 kD polypeptide (although the actual molecular weight may be a little higher) induced by BHV-1 is analogous to the better characterised MDBP ofHSV (Bayliss et al., 1975; Powell and Purifoy, 1976; Powell et al., 1981; Lee and Knipe, 1983). Similar antigenic cross-activity between the HSV MDBP and a polypeptide induced by BHV-2, pseudorabies virus and equine herpes
IDENTIFICATION OF THE GENE HOMOLOGOUS TO HSV MDBP IN THE BHV-1 GENOME
211
virus-1 have already been well-documented {Littler et al., 1981; Yeo et al., 1981; Caughman et al., 1985; Maragos and May, 1987). We have attempted to identify BHV-1 polypeptides having DNA binding ability in protein blotting experiments. In this technique BHV-1 infected cell proteins were transferred to nitrocellulose membranes and such blots were exposed to single or double-stranded 32[p]_labelled BHV-1 DNA fragments. By this technique, 8 BHV-1 infected cell polypeptides having molecular weights of 110, 93, 66, 46, 30, 17 and 12 kD were identified as DNA-binding proteins (unpublished results). However, the 120 kD polypeptide (proposed MDBP) failed to bind to DNA in this test. Failure to demonstrate the DNA binding property of the putative MDBP was not unexpected since failure or inconsistent binding of DNA of MDBPs of HSV-1, VZV or Herpesvirus saimiri (HVS) in protein-blot assays have all been reported (Blair and Honess, 1983; Roberts et al., 1985). DNA-cellulose column chromatography should be helpful for further characterisation of DNA binding proteins induced by BHV-1. Moreover, sequencing of the putative MDBP gene of BHV-1 and its comparison with MDBP genes of other herpesviruses would assist in identifying domains important in performing DNA binding functions. ACKNOWLEDGEMENTS We are thankful to Dr. K.N. Powell for the gift of HSV antisera to MDBP and to Dr. A.C. Minson for the plasmid pSG18. S.K.B. was supported by a Commonwealth Scholarship in UK and S.K.M. was supported by a Cambridge Nehru Scholarship. This research was also funded in part by a grant from the Agriculture and Food Research Council of Great Britain.
REFERENCES Bayliss, G.J., Marsden, H.S. and Hay, J., 1975. Herpes simplex virus proteins: DNA binding proteins in infected cells and in the virus structure. Virology, 68:124-134. Blair, E.D. and Honess, R.W., 1983. DNA binding proteins specified by herpesvirus saimiri. J. Gen. Virol., 64: 2697-2715. Bolton, D.C., Zee, Y.C. and Ardans, A.A., 1983. Identification of envelop and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis. Vet. Microbiol., 8: 57-68. Caugh~nan, G.B., Staczek, J. and O'Callaghan, D.J., 1985. Equine herpes virus type 1 infected cell polypeptides: Evidence for immediate early/early/late regulation of viral gene expression. Virology, 145: 49-61. Conley, A.J., Knipe, D.M., Jones, P.C. and Roizman, B., 1981. Molecular genetics of herpes simplex virus. VII. Characterisation of a temperature sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of 'gamma' polypeptides. J. Virol., 37: 191-206.
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Dente, L., Cesareni, G. and Cortese, R., 1983. pEMBL: a new family of single-stranded plasmids. Nucleic Acids Res,, 11: 1645-1655. Godowski, P.J. and Knipe, D.M., 1986. Transcriptional control of herpesvirus gene expression: Gene functions required for positive and negative regulation. Proc. Natl. Acad. Sci. U.S.A., 83: 256-260. Goldin, A.L., Sandri-Goldin, R.M., Levine, M. and Glorioso, J.C., 1981. Cloning of herpes simplex virus type 1 sequences representing the whole genome. J. Virol., 38: 50-58. Hammerschmidt, W., Ludwig, H. and Buhk, H., 1986. Short repeats cause heterogeneity at genomic terminus of bovine herpus virus 1. J. Virol., 58: 43-49. Kahrs, S.F., 1977. Infectious bovine rhinotracheitis: A review and update. J. Am. Vet. Med. Assoc., 171: 1055-1064. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-683. Lawrence, W.C., D'urso, R.C., Kundel, C.A., Whitbeck, J.C. and Bello, L.J., 1986. Map location of the gene for a 130,000-dalton glycoprotein of bovine herpesvirus 1. J. Virol., 60: 405-414. Lee, C.K. and Knipe, D.M., 1983. Thermolability in the DNA binding activity associated with a protein encoded by mutants of herpes simplex virus type 1. J. Virol., 46: 909-919. Littler, E., Yeo, J., Killington, R.A., Purifoy, D.J.M. and Powell, K.L., 1981. Antigenic and structural conservation of herpesvirus DNA binding proteins. J. Gen. Virol., 56: 409-419. Littler, E., Purifoy, D.J.M., Minson, A. and Powell, K.L., 1983. Herpes simplex virus non-structural proteins. III. Function of the major DNA-binding protein. J. Gen. Virol., 64: 983-995. Maragos, C. and May, J.T., 1987. Studies of the major D NA binding proteins of two bovine herpes mammillitis virus isolates. Acta. Virol., 31: 168-171. Mayfield, J.E., Good, P.J., Van Oort, H.J., Campbell, A.R. and Reed, D.E., 1983. Cloning and cleavage site mapping of DNA from bovine herpesvirus-1 (Cooper strain). J. Virol., 47:259 264. McGeoch, D.J., 1987. The genome of herpes simplex virus: structure, replication and evolution. J. Cell Sci., 7 (supplement): 5194-5222. Misra, V., Blumenthal, R.M. and Babiuk, L.A., 1981. Proteins specified by bovine herpesvirus type 1 (infectious bovine rhinotracheitis virus). J. Virol., 40: 367-378. Mittal, S.K. and Field, H.J., 1989. Analysis of the bovine herpesvirus-1 thymidine kinase gene from wild type and TK-deficient mutants. J. Gen. Virol., in press. Owen, L.J. and Field, ~H.J., 1988. Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene. Arch. Virol., 98: 27-38. Powell, K.L. and Purifoy, D.J.M., 1976. DNA binding proteins of cells infected by herpes simplex virus type 1 and type 2. Intervirology, 7: 225-239. Powell, K.L., Littler, E. and Purifoy, D.J.M., 1981. Non-structural proteins of herpes simplex virus II. Major virus-specific DNA-binding protein. J. Virol., 39: 894-902. Roberts, C.R., Weir, A.C., Hay, J., Straus, S.E. and Ruyechan, W.T., 1985. DNA binding proteins present in Varicella-Zoster virus infected cells. J. Virol., 55: 45-53. Ruyechan, W.T. and Weir, A.C., 1984. Interactions with nucleic acid and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein. J. Virol., 52: 727-733. Trepanier, P., Minocha, H.C., Bastien, Y., Nadon, F., Seguin, C., Lussier, S. and Trudel, M., 1986. Bovine herpes 1: Strain comparison of a neutralizing epitope on the 96-kilodalton haemagglutinin. J. Virol., 60: 302-306. Yeo, J., Killington, R., Watson, D.H. and Powell, K., 1981. Studies on cross reactive antigens in the herpesviruses. Virology, 108: 256-266.
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I d e n t i f i c a t i o n of the Gene H o m o l o g o u s to HSV Major D N A B i n d i n g P r o t e i n in the B H V - 1 G e n o m e S.K. BANDYOPADHYAY*, S.K. MITTAL** and H.J. FIELD***
Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES (Great Britain) (Accepted 24 October 1989)
ABSTRACT Bandyopadhyay, S.K., Mittal, S.K. and Field, H.J., 1990. Identification of the gene homologous to HSV major DNA binding protein in the BHV-1 genome. Vet. Microbiol., 22: 203-212. By means of Southern blot hybridisation using a cloned herpes simplex virus (HSV) major DNA binding protein (MDBP) gene as probe, the putative MDBP gene of BHV-1 was located within the Hind III G fragment which mapped between 0.352 and 0.381 map units of the BHV-1 genome. Moreover, an antiserum raised to HSV MDBP precipitated a 120kD polypeptide in a radio-immunoprecipitationtest.
INTRODUCTION
Bovine herpesvirus i (BHV-1) causes a variety of diseases in cattle including rhinotracheitis, conjunctivitis, encephalitis, abortion and genital lesions (Kahrs, 1977). Approximately 40 polypeptides have been identified in BHV1 infected cells of which 18 to 33 have variously been assigned as structural components of the virus (Misra et al., 1981; Bolton et al., 1983; Trapanier et al., 1986). The genome of BHV-1 is a double-stranded linear DNA molecule divided into a long and a short unique region with inverted repeat sequences which bracket the short region (Mayfield et al., 1983; Hammerschmidt et al., 1986). During virus replication, certain proteins are required to interact with virus DNA performing catalytic or regulatory functions in the synthesis of viral DNA (reviewed by McGeoch, 1987). One such protein, the MDBP has been implicated in important regulatory functions in HSV replication apart from its sup*Present address: Indian Veterinary Research Institute, Mukteswar-Kumanon: 263138, Nainital (UP), India **Present address: Department of Biology, McMaster Univeristy Hamilton, Ont., Canada. ***To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
204
S.K. BANDYOPADHYAY ET AL.
posed function concerning stabilisation of the replicative DNA complex (Conley et al., 1981; Lee and Knipe, 1983; Littler et al., 1983; Ruyechan and Weir, 1984; Godowski and Knipe, 1986). The identification of genes in the BHV-1 genome has recently been commenced. The genes homologous to HSV gB, DNA polymerase and thymidine kinase have been localised in the BHV-1 genome (Lawrence et al., 1986; Owen and Field, 1988; Mittal and Field, 1989). In the present communication we report the identification of the putative MDBPgene of BHV-1 using the strain BHV-1 (6660). MATERIALSAND METHODS The virus strain (6660) is of the 'Cooper' type with respect to its restriction endonuclease map (Mayfield et al., 1983; Owen and Field, 1988). Virus was grown in Madin Darby bovine kidney (MDBK) cells in the presence of Eagle's minimum essential medium (EMEM). For virus purification, confluent monolayers of MDBK cells were infected with BHV-1 (6660) using m.o.i, of 0.1 pfu/ cell. When the cytopathic effect was complete (48 to 72h post-infection) the virus was purified from the supernatant by a single cycle of 10-50% potassium tartrate density gradient centrifugation (Misra et al., 1981 ). For Southern blotting and hybridisations, virus DNA was extracted from purified BHV-1 virions. Restriction endonuclease digestion, agarose gel electrophoresis of digested DNA fragments and Southern blot transfer of DNA fragments to Hybond-N membranes were carried out using standard methods (Owen and Field, 1988). The Hind III 'G' fragment of BHV-1 (6660) DNA was cloned in pEMBL8 (Dente et al., 1983) and named pSKM7. The HSV-1 MDBP gene contained in the Eco RI 'F' fragment (pSG18), cloned in plasmid pBR325 (Goldin et al., 1981) was obtained from Dr. M. Levine of the University of Michigan, U.S.A., through Dr. A. Minson of the Department of Pathology, University of Cambridge, Great Britain. Probes for Southern-blot hybridisation were prepared either from the Sal I 'a' fragment directly (probe A) or this fragment was further digested with Barn HI (in order to remove the unwanted portion of the adjacent DNA polymerase gene). The resultant Bam HI 'a' and Barn HI 'b' fragments, which together contained most of the M D B P gene of HSV-1 (Fig. 2), were used for preparing probes (probes B and C respectively). DNA probes used in the Southern-blot hybridisation experiments were labelled with 32[p ]dCTP (Amersham; 3000 Ci/mmol) using an oligonucleotide labelling kit (Pharmacia). Hybridisation was carried out using standard methods (Owen and Field, 1988). For radio-immunoprecipitation, confluent monolayers of MDBK cells were grown in 35-mm Petri dishes and infected with BHV-1 at a multiplicity of infection of 10 pfu/cell. The cells were labelled with 35[S]-methionine (approx. 980 Ci/mmol) between 1 and 10h post-infection at a concentration of 50
IDENTIFICATION OF THE GENE HOMOLOGOUS TO HSV MDBP IN THE BHV-1 GENOME
205
Ci/ml in E M E M containing one-fifth the normal concentration of labelled methionine. After labelling, the monolayers were briefly washed with phosphate buffered saline and the cells were solubilised with 2 ml/dish of RIPA buffer (10 mM Tris-HC1 [pH 7.4], 150 mM NaCl, 0.5% sodium deoxycholate, 1% SDS, 1% NP40, 1 mM PMSF) on ice for 0.5h. The solubilised suspension was centrifuged at 35 000 rpm for l h at 4°C and the supernatant was stored at - 7 0 ° C until required for analysis. A 150 ~l portion of this solubilised cell preparation was incubated at 37 °C for 2h with 20 ttl of either non-immune rabbit serum or rabbit monospecific antiserum to HSV MDBP (provided by Dr. K. N. Powell, Wellcome Research Laboratory, Beckenham, Kent, Great Britain) and 130 ~tl of RIPA buffer. To this was added 100 ttl of a 25% suspension of protein A coupled to Sepharose 6MB (Sigma) and the mixture was incubated for a further period of l h at 37 ° C with occasional shaking. The protein A beads were washed thoroughly with RIPA buffer and resuspended in 40 zl sample buffer (62.5 mM Tris-HC1 [pH 6.8], 2% SDS, 10% glycerol, 5% flmercaptoethanol, 0.0001% bromophenol blue) for analysis by polyacrylamide gel electrophoresis. The samples were analysed by SDS polyacrylamide electrophoresis following the method described by Laemmli (1970) and autoradiographed using Fugi RX X-ray films. RESULTS Localisation of the putative major DNA-binding protein gene in the BHV-1 genome BHV-1 DNA extracted from purified virions was digested with Hind III, Bam HI, Eco Ri or Hpa I and following agarose gel electrophoresis (Fig. la), the DNA fragments were Southern-blotted onto Hybond-N nylon filters. Under conditions of high stringency (washing with 0.1 X SSC, 0.1% SDS at 68 ° C ), all three probes, A, B, and C (Fig. 2), hybridised to a single band in each of the Barn HI, Eco RI and Hpa I digests of BHV-1 DNA. The probe B, containing approximately 65% of the HSV MDBP gene starting from the 5' end of the gene, hybridised only to the G fragment of Hind III digest of BHV-1 (Fig. lb). However, probe A and C containing respectively the whole and the last 35% gene from the 3' end of the HSV MDBP gene, both gave a positive, though somewhat weak signal with fragment A, in addition to strong signal obtained with fragment G, of Hind III digest of BHV-1 DNA (Fig. lb). To further define the region of hybridisation, the recombinant plasmid (pSKM7) DNA was electrophoresed after digestion with Hind III alone (which released the intact Hind III G fragment) and with Hind III and Bam HI combined to release a 4 kb sub-fragment (Fig. 3). Southern-blot hybridisation analysis of these DNA fragments revealed that the probe B hybridised only to the 4 kb fragment (Fig. 3B). Hybridisation to the 4 kb and not to the 7.8 kb
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IDENTIFICATION OF THE GENE HOMOLOGOUS TO HSV MDBP IN THE BHV-1 GENOME
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subfragment suggests that the region of homology starts from map coordinates of 0.35 of the BHV-1 genome and not ahead (Fig. 4). Radio-immunoprecipitation test Normal rabbit serum did not specifically precipitate any polypeptide from the solubilised preparation of BHV-1 infected MDBK cells, although traces of minor contaminating polypeptides could be detected suggesting non-specific precipitation. However, the specific rabbit antiserum to MDBP of HSV was found to precipitate a polypeptide from the solubilised preparation of BHV-1
Fig. 1. (a) Tris acetate agarose gel (0.7%) stained with ethidium bromide used for Southern blotting. BHV-1 virion DNA, was digested with: Hind III (lane 1 ), Bam HI (lane 2), Eco RI (lane 3 ) and HPa I (lane 4). The plasmid pSG18 containing the HSV MDBP gene was digested with: Sal I (lane 5) or with Barn HI (lanes 6 and 7). (b) Autoradiograph of Southern blot hybridisation of the agarose gel; Fig. 1 (a) probed with the HSV MDBP gene. The probes A, B, and C, were derived as described in Fig. 1. All three probes hybridised to Eco RI A, Bam HI G, Hind III G and Hpa I E fragments of BHV-1 genome as indicated by solid arrow-heads. In addition to the above, probe A and C but not probe B also hybridised to the Hind I I I A fragment (faintly represented in the autoradiograph and indicated by empty arrow heads). Lanes 5, 6 and 7 were cut out from shorter autoradiographic exposures of the same blots.
208
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infected M D B K cells (Fig. 5, lane 6). F r o m t h e p o s i t i o n of the molecular weight m a r k e r s , we have c a l c u l a t e d t h e m o l e c u l a r weight o f this p o l y p e p t i d e as 120 kD. H o w e v e r , in r e l a t i o n t o t h e relative m i g r a t i o n of t h e I C P 8 (128 k D ) of
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HSV-1 (Fig. 5, lane 1 ), the molecular weight of the cross-reacting polypeptide of BHV-1 is expected to be approximately between 132 and 135 kD. CONCLUSION
Thus, Southern blot hybridisation of the BHV-1 genome using a probe containing the MDBP gene from HSV-1, indicated a homology of DNA sequences located between map co-ordinates of 0.352 to 0.381 of BHV-1 genome which may code for the putative M D B P of BHV-1 (Fig. 4). From the observation that the probes containing the 3' end of the HSV MDBP gene produced additional signals in A fragment, we speculate that the orientation of the gene extends from the G fragment towards the A fragment of the Hind III digest of the BHV-1 DNA which, if true, will suggest an opposite orientation to the previously located DNA polymerase gene (Owen and Field, 1988). It is of interest that Southern-blot hybridisation using HSV MDBP gene as a probe has also been used recently to identify the putative gene coding for the analogous protein in BHV-2 (Maragos and May, 1987). The radio-immunoprecipitation data led to the identification of one 120 kD polypeptide in BHV-l-infected MDBK cells which precipitated with rabbit antiserum to HSV MDBP. Studies on the regulation of BHV-1 gene expression
210
S.K. BANDYOPADHYAYET AL.
Fig. 5. Autoradiograph showing the immunoprecipitates of infected cell lysates, labelled with 35[S]methionine, reacted with an antiserum to HSV MDBP, and analysed by SDS-PAGE. Lanes 1, 2 and 3 contain B H K cells infected with HSV-1; lanes 4, 5 and 6 contain BHV-l-infected M D B K cells. Infected-cell fractions were reacted with normal rabbit serum (lanes 2 and 5 ) or HSV M D B P antiserum (lanes 1 and 6). The immune serum precipitated one 120 kD polypeptide from BHV1 infected cells and one 115 kD polypeptide (thought to be the MDBP, ICP8) from HSV-1 infected B H K cells. The positions of the molecular weight markers are shown on the right. All tracks are from the same gel; for clarity some irrelevant tracks have been omitted.
have revealed that this 120 kD non-structural polypeptide belongs to the early class of proteins induced by BHV-1 (Bandyopadhyay, unpublished observation). The above fact together with the observed serological cross-reactivity of this polypeptide with the HSV MDBP, provide strong circumstantial evidence that the 120 kD polypeptide (although the actual molecular weight may be a little higher) induced by BHV-1 is analogous to the better characterised MDBP ofHSV (Bayliss et al., 1975; Powell and Purifoy, 1976; Powell et al., 1981; Lee and Knipe, 1983). Similar antigenic cross-activity between the HSV MDBP and a polypeptide induced by BHV-2, pseudorabies virus and equine herpes
IDENTIFICATION OF THE GENE HOMOLOGOUS TO HSV MDBP IN THE BHV-1 GENOME
211
virus-1 have already been well-documented {Littler et al., 1981; Yeo et al., 1981; Caughman et al., 1985; Maragos and May, 1987). We have attempted to identify BHV-1 polypeptides having DNA binding ability in protein blotting experiments. In this technique BHV-1 infected cell proteins were transferred to nitrocellulose membranes and such blots were exposed to single or double-stranded 32[p]_labelled BHV-1 DNA fragments. By this technique, 8 BHV-1 infected cell polypeptides having molecular weights of 110, 93, 66, 46, 30, 17 and 12 kD were identified as DNA-binding proteins (unpublished results). However, the 120 kD polypeptide (proposed MDBP) failed to bind to DNA in this test. Failure to demonstrate the DNA binding property of the putative MDBP was not unexpected since failure or inconsistent binding of DNA of MDBPs of HSV-1, VZV or Herpesvirus saimiri (HVS) in protein-blot assays have all been reported (Blair and Honess, 1983; Roberts et al., 1985). DNA-cellulose column chromatography should be helpful for further characterisation of DNA binding proteins induced by BHV-1. Moreover, sequencing of the putative MDBP gene of BHV-1 and its comparison with MDBP genes of other herpesviruses would assist in identifying domains important in performing DNA binding functions. ACKNOWLEDGEMENTS We are thankful to Dr. K.N. Powell for the gift of HSV antisera to MDBP and to Dr. A.C. Minson for the plasmid pSG18. S.K.B. was supported by a Commonwealth Scholarship in UK and S.K.M. was supported by a Cambridge Nehru Scholarship. This research was also funded in part by a grant from the Agriculture and Food Research Council of Great Britain.
REFERENCES Bayliss, G.J., Marsden, H.S. and Hay, J., 1975. Herpes simplex virus proteins: DNA binding proteins in infected cells and in the virus structure. Virology, 68:124-134. Blair, E.D. and Honess, R.W., 1983. DNA binding proteins specified by herpesvirus saimiri. J. Gen. Virol., 64: 2697-2715. Bolton, D.C., Zee, Y.C. and Ardans, A.A., 1983. Identification of envelop and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis. Vet. Microbiol., 8: 57-68. Caugh~nan, G.B., Staczek, J. and O'Callaghan, D.J., 1985. Equine herpes virus type 1 infected cell polypeptides: Evidence for immediate early/early/late regulation of viral gene expression. Virology, 145: 49-61. Conley, A.J., Knipe, D.M., Jones, P.C. and Roizman, B., 1981. Molecular genetics of herpes simplex virus. VII. Characterisation of a temperature sensitive mutant produced by in vitro mutagenesis and defective in DNA synthesis and accumulation of 'gamma' polypeptides. J. Virol., 37: 191-206.
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Dente, L., Cesareni, G. and Cortese, R., 1983. pEMBL: a new family of single-stranded plasmids. Nucleic Acids Res,, 11: 1645-1655. Godowski, P.J. and Knipe, D.M., 1986. Transcriptional control of herpesvirus gene expression: Gene functions required for positive and negative regulation. Proc. Natl. Acad. Sci. U.S.A., 83: 256-260. Goldin, A.L., Sandri-Goldin, R.M., Levine, M. and Glorioso, J.C., 1981. Cloning of herpes simplex virus type 1 sequences representing the whole genome. J. Virol., 38: 50-58. Hammerschmidt, W., Ludwig, H. and Buhk, H., 1986. Short repeats cause heterogeneity at genomic terminus of bovine herpus virus 1. J. Virol., 58: 43-49. Kahrs, S.F., 1977. Infectious bovine rhinotracheitis: A review and update. J. Am. Vet. Med. Assoc., 171: 1055-1064. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-683. Lawrence, W.C., D'urso, R.C., Kundel, C.A., Whitbeck, J.C. and Bello, L.J., 1986. Map location of the gene for a 130,000-dalton glycoprotein of bovine herpesvirus 1. J. Virol., 60: 405-414. Lee, C.K. and Knipe, D.M., 1983. Thermolability in the DNA binding activity associated with a protein encoded by mutants of herpes simplex virus type 1. J. Virol., 46: 909-919. Littler, E., Yeo, J., Killington, R.A., Purifoy, D.J.M. and Powell, K.L., 1981. Antigenic and structural conservation of herpesvirus DNA binding proteins. J. Gen. Virol., 56: 409-419. Littler, E., Purifoy, D.J.M., Minson, A. and Powell, K.L., 1983. Herpes simplex virus non-structural proteins. III. Function of the major DNA-binding protein. J. Gen. Virol., 64: 983-995. Maragos, C. and May, J.T., 1987. Studies of the major D NA binding proteins of two bovine herpes mammillitis virus isolates. Acta. Virol., 31: 168-171. Mayfield, J.E., Good, P.J., Van Oort, H.J., Campbell, A.R. and Reed, D.E., 1983. Cloning and cleavage site mapping of DNA from bovine herpesvirus-1 (Cooper strain). J. Virol., 47:259 264. McGeoch, D.J., 1987. The genome of herpes simplex virus: structure, replication and evolution. J. Cell Sci., 7 (supplement): 5194-5222. Misra, V., Blumenthal, R.M. and Babiuk, L.A., 1981. Proteins specified by bovine herpesvirus type 1 (infectious bovine rhinotracheitis virus). J. Virol., 40: 367-378. Mittal, S.K. and Field, H.J., 1989. Analysis of the bovine herpesvirus-1 thymidine kinase gene from wild type and TK-deficient mutants. J. Gen. Virol., in press. Owen, L.J. and Field, ~H.J., 1988. Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene. Arch. Virol., 98: 27-38. Powell, K.L. and Purifoy, D.J.M., 1976. DNA binding proteins of cells infected by herpes simplex virus type 1 and type 2. Intervirology, 7: 225-239. Powell, K.L., Littler, E. and Purifoy, D.J.M., 1981. Non-structural proteins of herpes simplex virus II. Major virus-specific DNA-binding protein. J. Virol., 39: 894-902. Roberts, C.R., Weir, A.C., Hay, J., Straus, S.E. and Ruyechan, W.T., 1985. DNA binding proteins present in Varicella-Zoster virus infected cells. J. Virol., 55: 45-53. Ruyechan, W.T. and Weir, A.C., 1984. Interactions with nucleic acid and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein. J. Virol., 52: 727-733. Trepanier, P., Minocha, H.C., Bastien, Y., Nadon, F., Seguin, C., Lussier, S. and Trudel, M., 1986. Bovine herpes 1: Strain comparison of a neutralizing epitope on the 96-kilodalton haemagglutinin. J. Virol., 60: 302-306. Yeo, J., Killington, R., Watson, D.H. and Powell, K., 1981. Studies on cross reactive antigens in the herpesviruses. Virology, 108: 256-266.