Haruki Otsuka

Nucleotide sequence of the marmoset herpesvirus thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide

The nucleotide sequence of a 2549-bp DNA fragment containing the entire coding region of the marmoset herpesvirus (MarHV) thymidine kinase gene (tk) and the flanking sequences was determined by the dideoxynucleotide chain termination method. The MarHV thymidine kinase polypeptide predicted from the nucleotide sequence contained 376 amino acids and had a molecular weight of 41,281. The sequencing data also reveal that the coding portion of another MarHV gene probably begins only 292 nucleotides downstream from the stop codon of the MarHV tk gene. There was relatively little nucleotide sequence homology between the MarHV tk gene and that of the herpes simplex virus (HSV) types 1 and 2 tk genes. Comparisons of the predicted amino acid sequences of the MarHV thymidine kinase polypeptide with that of the HSV-1 and HSV-2 thymidine kinase polypeptides, however, revealed clear, but interrupted, homology within several regions of the polypeptide chains. Amino acid sequence homology was particularly striking at residues 10 to 27 of the MarHV thymidine kinase polypeptide and residues 49 to 66 of the HSV-1 and HSV-2 thymidine kinase polypeptides. These same amino acid residues exhibit noticeable sequence homology to the mitochondrial beta subunit ATPase, oncogene p21 protein, adenylate kinase, and to other nucleotide-binding proteins. It has been proposed that the indicated regions of homology are elements of a nucleotide-binding pocket in ATPase, p21, and adenylate kinase, raising the possibility that amino acid residues 15 to 25 of the MarHV thymidine kinase and 54 to 64 of the HSV-1 and HSV-2 enzymes are likewise parts of nucleotide-binding sites.

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.

Expression of porcine pseudorabies virus genes by a bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) vector

SummaryRecombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 [infectious bovine rhinotracheitis virus (IBRV)] vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene. In one recombinant, the regulatory and coding sequences of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene. This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene. IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and theE. coli β-galactosidase gene were ligated to the late IBRV gIII promoter. Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, β-gal plaque assays, and by immunoprecipitation experiments on extracts from3H-mannose-labelled cells. The recombinant IBRV expressing β-gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences. The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII. The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit.

Blocking ELISA to distinguish pseudorabies virus-infected pigs from those vaccinated with a glycoprotein gIII deletion mutant

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV)-infected pigs from those immunized with a glycoprotein g92(gIII) deletion mutant, PRV(dlg92dltk). The blocking ELISA utilizes 96-well microtiter test plates coated with a cloned PRV g92(gIII) antigen, a mouse monoclonal antibody against gIII antigen (moMCAgIII): horseradish peroxidase (HRPO) conjugate, and undiluted test sera. Analyses can be completed in less than 3 hours with results printed out by an automated plate reader. Analyses on over 300 pig sera from PRV-free farms, on sera from other species, and on control sera containing antibodies to microorganisms other than PRV showed that the ratio of the optical density at 405 nm for the test sample to the optical density at 405 nm for the negative control (S/N value) was >0.7 for all sera. No false positives were identified. Likewise, the S/N values were >0.7 for over 400 sera obtained from pigs vaccinated twice with more than 1,000 times the standard PRV (dlg92dltk) dose or 1–4 times with the standard dose (2 × 105 TCID50/pig). Following challenge exposure to virulent PRV, the S/N values of the vaccinates were 0.1, showing that g92(gIII) antibodies in the sera of experimentally challenged pigs strongly blocked the binding of the moMCAgIII: HRPO conjugate to the antigen-coated wells. Sera of 233 pigs from PRV-infected herds with virus neutralization (VN) titers of 1:4 or greater were tested. All except 2 of these sera had S/N values <0.7 and more than 175 had S/N values <0.1. Sixteen sera from feral pigs with VN titers of 1:4 or greater had S/N values of 0.24 or less, but 2 sera with VN titers of 1:4 when tested 5 years prior to the PRV g92(gIII) blocking ELISA test gave false negative S/N values. Twenty-four of 29 pig sera from PRV-infected herds with VN titers < 1:4 were positive for g92(gIII) antibodies, illustrating the sensitivity of the PRV g92(gIII) blocking ELISA test. Analyses on 7 sera with VN titers of 1:4–1:64 showed that titers obtained with the PRV g92(gIII) blocking ELISA test were from 2- to 16-fold greater than the VN titers. The accuracy and sensitivity of the PRV g92(gIII) blocking ELISA test was further demonstrated by analyses of 40 unknown sera supplied in the National Veterinary Services Laboratories 1988 PRV check test kit.

Biochemical transformation of LM(TK−) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Abstract Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique Pvu II cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the Bam HI to the Pvu II cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the Pvu II to the Bgl II cleavage site. Plasmid pMH110 biochemically transformed LM(TK − )cells to the TK + phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 m M thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK − )/TF pMH110 E2 and LM(TK − )/TF pMH110 Hc2] with [ 35 S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK − ) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the Bgl II cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by Hinc II- and Eco RI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA Bgl II cleavage site.

Cloning of the marmoset herpesvirus thymidine kinase gene and analyses of the boundaries of the coding region.

Abstract In order to delimit the approximate boundaries of the marmoset herpesvirus (MarHV) thymidine kinase (TK) gene, Hind III and Bam HI digests of MarHV DNA were cloned in plasmid pBR322. Several recombinant plasmids which transformed E. coli K12 strain RR1 to ampicillin resistance were isolated. The MarHV DNA inserts in these plasmids accounted for about half of the MarHV genome. One of the plasmids, pMAR4, contained a 9.1-kbp fragment of MarHV DNA ( Hind III-G), transformed LM(TK − ) cells to TK + , and hybridized to the Bam HI-I fragment of MarHV DNA, which had previously been shown to have TK-transforming activity. pMAR4 DNA had little or no homology to the 2-kbp Puv II fragment of HSV-1 DNA, which contains the HSV-1 TK gene. Cleavage with Pvu II, Sac I, Sma I, and Kpn I inactivated the TK-transforming activity of pMAR4, but cleavage with Hind III, Pst I, Eco RI, Xho I, Xba I, and Bam HI did not. Deletion mutants pMAR401 and pMAR420, which lacked the 2.6-kbp Kpn I and the 2.75-kbp Eco RI fragments, respectively, of pMAR4, lost transforming activity, whereas pMAR410, which lacked a 2.9kbp Xho I fragment of pMAR4 did not. Recombinant plasmid pMAR430, which contained a 3-kbp Pst I fragment of pMAR4, also transformed LM(TK − ) cells to TK + . The results strongly suggest that the coding region of the MarHV TK gene was within a 2.4-kbp pMAR4 sequence extending from the Pst I (0.33 kbp) to the Eco RI (2.7 kbp) cleavage sites.

Biochemical transformation of mouse cells by a purified fragment of marmoset herpesvirus DNA.

Although the size of marmoset herpesvirus (MarHV) DNA, estimated by velocity sedimentation in sucrose gradients, was similar to that of herpes simplex virus type 1 (HSV-1) DNA, the restriction endonuclease sites of MarHV and HSV-1 DNAs were quite different. A specific BamHI restriction fragment (6.2 x 10(6) daltons) of MarHV DNA biochemically transformed LM(TK-) mouse fibroblasts to the thymidine kinase(TK)-positive phenotype. Rabbit antisera, prepared against MarHV TK, inhibited MarHV-induced TK, but not HSV-1, HSV-2, or cellular TKs. Disc PAGE analyses and enzyme neutralization experiments with the anti-MarHV TK sera demonstrated that the TK expressed in MarHV transformants was MarHV-specific.

Blocking ELISA for distinguishing infectious bovine rhinotracheitis virus (IBRV)-infected animals from those vaccinated with a gene-deleted marker vaccine

Abstract A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of >0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N <0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values >0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (<0.8). One serum with a VN titer of 1:2 had a borderline (±) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115 116 sera with VN titers of 1:2 to 1:256 had positive S/N values (<0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48.

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

Abstract Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK−)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK−) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA+, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK−) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA+ as well as TK+. The HANA+ cells included LM(TK−)/TF pAGO PP and LM(TK−)/TF pAGO PS clones. The latter are clones of LM(TK−) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 106 daltons) and a PvuII-SmaI fragment (0.9 × 106 daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.