Bovine herpesvirus-1 US3 protein kinase: critical residues and involvement in the phosphorylation of VP22.

The US3 gene product of bovine herpesvirus-1 (BoHV-1) is a protein kinase that is expressed early during infection and capable of autophosphorylation. By examining differentially labelled US3 moieties by co-immunoprecipitation, we demonstrated that the protein kinase interacts with itself in vitro, which supports autophosphorylation by US3. Based on its homology to other serine/threonine protein kinases, we defined two highly conserved lysines in US3, at position 195 within the ATP-binding pocket and at position 282 within the catalytic loop; altering either residue resulted in kinase-dead mutants, demonstrating that these two residues are critical for the catalytic activity of BoHV-1 US3. During immunoprecipitation experiments, US3 interacted weakly with VP22, another tegument protein of BoHV-1. Furthermore, VP22 co-localized with US3 inside the nucleus in BoHV-1-infected cells. In vitro kinase assays demonstrated that VP22 is phosphorylated not only by US3, but also by the cellular casein kinase 2 (CK2) protein. The selective CK2 protein kinase inhibitor, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and the less specific CK2 inhibitor Kenpaullone reduced VP22 phosphorylation, while CK1, protein kinase C or protein kinase A inhibitors did not affect phosphorylation. When US3 was included with VP22 in the kinase assay in the presence of DMAT, a low level of VP22 phosphorylation was observed. These data demonstrate that BoHV-1 VP22 interacts with both CK2 and US3, and that CK2 is the major kinase phosphorylating VP22, with US3 playing a minor role.

Inhibition of apoptosis in BHV-1-infected cells depends on Us3 serine/threonine kinase and its enzymatic activity.

Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers. To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors.

Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in Escherichia coli hosts.

In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene (tk), a well regulated viral gene has been chosen for this study. A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this cosmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then used to transform E. coli tdk- mutant strains, Ky895 or C600tdk- for the selection of the IBRV tk gene. The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning. The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene. The E. coli mutant strain C600tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of C600tdk- mutant.

Identification of the US3 gene product of BHV-1 as a protein kinase and characterization of BHV-1 mutants of the US3 gene.

We have identified the product of the US3 gene of bovine herpes virus type 1 (BHV-1), which is homologous to the herpes simplex virus type 1 (HSV-1) US3 protein kinase (PK) gene. The antibodies against the BHV-1 US3 gene product reacted with a 58 kDa polypeptide in BHV-1 infected cells and the 58 kDa polypeptide purified by immuno-precipitation demonstrated PK activity. Recent reports indicating that the US3 gene of HSV-1 is involved in the blockage of apoptosis in virus infected cells. As to the apoptosis in BHV-1 infected cells, we found following: (1) no apoptosis was observed in cells infected with wild type BHV-1 and the US3 mutants (2) the apoptosis induced by the osmotic shock of sorbitol treatment was blocked when cells were infected by the wild type BHV-1 (3) the US3 mutants of BHV-1 blocked the apoptosis of sorbitol treated cell, but the suppressive effect was delayed relative to that of wild type BHV-1 (4) the other BHV-1 mutants, with the intact US3 gene but with some other non-essential gene (genes) deleted behaved similar way to the US3 mutant. It is concluded that the US3 gene of BHV-1 is not directly involved in the blockage of apoptosis in infected cells.

Construction and characterization of an attenuated bovine herpesvirus type 1 (BHV-1) recombinant virus.

A recombinant bovine herpesvirus type 1 (BHV-1) virus (Gal-TK) has been constructed. The Gal-TK virus contains a chimeric reporter/marker gene coding for bacterial beta-galactosidase (beta-gal gene) that was inserted stably within the viral TK gene. This resulted in inactivation of the TK gene. The beta-gal gene is under the regulation of a strong, human cytomegalovirus-immediate early (HCMV-IE) promoter and is expressed as an authentic viral-coded gene. Even though the one-step growth kinetics of the recombinant and parent viruses were similar, the recombinant virus yielded less than the parent virus on Madin-Darby bovine kidney cells. After intranasal inoculation, the engineered virus was virtually avirulent for colostrum-deprived new-born calves. Similar to the parent virus, the recombinant virus replicated in the upper respiratory tract of calves, but the amount of progeny viruses produced was reduced significantly. The progeny viruses recovered from nasal swabs of animals inoculated with the recombinant and the Cooper strains of BHV-1 were easily distinguishable based on the beta-gal marker.

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.

Purification of the infectious bovine rhinotracheitis virus alkaline deoxyribonuclease expressed in Escherichia coli.

Nucleotide sequence analysis within the unique long segment of the infectious bovine rhinotracheitis virus (IBRV) genome identified an open reading frame of 1461 base pairs whose deduced polypeptide of 487 amino acids exhibited homology to alkaline deoxyribonucleases of other herpesviruses. To determine whether this IBRV gene product has nuclease activity, the gene designated UL12 was inserted into the vector pET-28a(+) and expressed in Escherichia coli as an oligohistidine-tagged protein. Upon induction with isopropyl beta-D-thiogalactopyranoside E. coli BL21 (DE3)[pLysS] cells harboring this recombinant plasmid produced a 57-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using metal affinity chromatography resulted in a homogeneous preparation of this recombinant protein. The purified protein exhibited both exonuclease and endonuclease activities, each with an alkaline pH optimum.

Determination of ability of a thymidine kinase-negative deletion mutant of bovine herpesvirus-1 to cause abortion in cattle.

The Cooper isolate of bovine herpesvirus-1, which causes abortion in cattle, was used to construct a thymidine kinase-negative (TK-) deletion mutant virus. Twelve heifers were inoculated IV at 25 to 29 weeks of pregnancy with either TK- or thymidine kinase-positive (TK+) Cooper virus. All heifers developed fevers of 1 to 2 C during the first week after inoculation. Temperatures of TK+ inoculates were slightly higher and remained above normal a few days longer than in TK- inoculates. Viremia was detected in 5 of 6 TK+ inoculates and in all 6 TK- inoculates. More virus isolations were made from nasal and vaginal swab specimens of TK+ inoculates than from swab specimens of TK- inoculates. All heifers developed virus neutralizing antibody within 14 days after inoculation and antibody titers were similar between the 2 groups. None of the TK- inoculated heifers aborted and their calves did not have neutralizing antibody at birth. Abortion occurred in 5 of 6 heifers given TK+ virus. All aborted fetuses were infected with bovine herpesvirus-1, as demonstrated by virus isolation or detection of viral antigen in fetal tissues. These results indicate that inactivation of the TK gene reduces abortifacient activity of bovine herpesvirus-1.

Development and trial of a bovine herpesvirus 1-thymidine kinase deletion virus as a vaccine.

An Australian bovine herpesvirus 1 (BHV1) isolate with a defined (427 base pair) deletion in the protein coding region of the thymidine kinase gene was obtained by standard marker rescue procedures. After selection in the presence of the nucleotide analogue 5'-iodo-deoxy-uridine the virus was analysed by hybridisation with three differential oligonucleotide probes, restriction endonuclease profile studies and DNA sequence analysis. The virus elicited an immune response in recipient animals after either intramuscular or intravenous administration and produced no significant deleterious side-effects when administered at a dose sufficient to stimulate the host immune response. The safety and immunogenicity of the recombinant BHV1 virus 39B1 were similar to those reported for other registered BHV1 vaccines and the virus would appear to be suitable for the production of a vaccine seed lot and more exhaustive field trials as a prelude to commercial vaccine production and registration.

The conserved open reading frame overlapping the thymidine kinase gene of different herpesviruses exists also in bovine herpesvirus 1.

Similar open reading frames (ORF) overlapping the thymidine kinase (TK) gene on the opposite strand or being close to it have been identified in 8 herpesviruses (Jacobson et al., 1989). Study of the DNA sequence of BHV-1 TK gene published by Mittal and Field (1989) and Kit and Kit (1986) revealed the existence of a similar ORF in this virus genome, too.

Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene.

The bovine herpesvirus-1 (BHV-1) genome was analysed by Southern blot hybridization using the herpes simplex virus type 1 (HSV-1) DNA polymerase gene as a probe. A 2.5 kilobase region which hybridized specifically to the HSV-1 DNA polymerase gene was identified within the Hind III G fragment at approximate map units 0.334-0.352. In order to provide further evidence that this is the location of the BHV-1 DNA polymerase gene, the 2.5 kilobase region was cloned and part of it sequenced. An uninterrupted stretch of over 800 nucleotides was obtained and an open reading frame spanning the entire sequence was identified. The amino acid sequence that it encodes shows striking homology to a region within the C-terminal half of other herpesvirus DNA polymerases.

Nucleotide and amino acid sequence analysis of the thymidine kinase gene of a bovine encephalitis herpesvirus.

The nucleotide and predicted amino acid sequence of the thymidine kinase (TK) gene of N 569, a bovine encephalitis herpesvirus (BEHV), has been determined and compared with those of avian, bovine and other mammalian herpesvirus TK genes. Striking differences were observed between the nucleotide sequence of this BEHV TK gene and those reported for bovine herpesvirus 1 (BHV-1). A total of 118 base changes, 39 base deletions and 14 base insertions were identified relative to the TK sequence of a BHV-1.2a strain, resulting in a net loss of seven residues. Comparison of the TK sequences of BEHV and the BHV-1 Q 3932 strain with that reported for the BHV-1 6660 strain suggested that the latter may have contained sequencing errors. The most significant difference between the BEHV TK sequence and those of BHV-1 strains was the absence of a contiguous stretch of ten codons.

Sequencing and 5'- and 3'-end transcript mapping of the gene encoding the small subunit of ribonucleotide reductase from bovine herpesvirus type-1.

The complete nucleotide sequence of the gene encoding the small subunit of ribonucleotide reductase (RNR) from bovine herpesvirus type-1 (BHV-1) was determined. The genomic DNA fragment sequenced also represented regions corresponding to the carboxy termini of RNR large subunit and of a virion protein causing host shut-off. The small subunit polypeptide was constituted of 314 amino acid residues totalling 35.25 kDa. The major transcription initiation and termination sites of the small subunit mRNA were located 95 bases upstream and 88 nucleotides downstream from the coding region, respectively. These findings indicate that the mRNA was 1128 bases long which correlated well with the size of the polyadenylated transcript detected in Northern blot analysis (1.3 kb). Within the RNR large subunit coding region, a TATA box and two CAAT box motifs were found 26, 104, and 190 nucleotides, respectively, upstream from the transcription initiation site of the small subunit mRNA. In contrast to previous studies (Slabaugh et al., J. Virol. 1988, 62, 519-527; Boursnell et al., Virology 1991, 184, 411-416), our comparative analysis of five herpesviruses, one iridovirus, and one poxvirus small subunit protein sequences suggested that the seven viruses arose from a common lineage.

Analysis of the bovine herpesvirus type 1 thymidine kinase (TK) gene from wild-type virus and TK-deficient mutants.

Five thymidine kinase (TK)-deficient mutants (B1 to B5) of bovine herpesvirus type 1 (BHV-1) were isolated by selection for resistance to the nucleotide analogue bromovinyldeoxyuridine. The genetic lesion in mutant B1 was localized in a 2.7 kb SalI-SalI subfragment (fTK2.7) which maps between 0.456 and 0.475 within the HindIII A fragment of the BHV-1 genome. The tk genes from wild-type and the TK-mutants B1 to B5 were cloned and sequenced using eight unique synthetic primers designed from a published sequence. The BHV-1 tk gene sequence for the strain 6660 contained some differences compared with that published previously for strain LA. Alignment of the predicted amino acid sequence of the BHV-1 TK polypeptide with different herpesvirus TKs revealed five strongly conserved regions and also identified putative functional relationships with other enzymes. Several interesting features were apparent in the tk gene sequences from the TK- mutants. The TK mutant B1 was a typical frameshift and chain termination mutant due to the deletion of a single base. The tk gene sequence of mutant B2 revealed the deletion of three bases resulting in the loss of valine at amino acid residue 174 of the TK polypeptide. The tk genes of mutants B3 to B5 contained an identical change of a single base addition resulting in frameshift and premature chain termination. In contrast to wild-type BHV-1, the TK-defective mutants were incapable of adsorbing TK-neutralizing antibodies from serum.

Map location of the thymidine kinase gene of bovine herpesvirus 1.

Bovine herpesvirus 1 has been reported to contain a thymidine kinase (tk) gene which is nonessential for virus replication. We have isolated a thymidine kinase-negative mutant of the virus and localized the mutation by marker rescue experiments to a 1.1-kilobase BglII-SalI fragment which maps at 0.47 to 0.48 on the bovine herpesvirus 1 genomic map. A thymidine kinase-negative bovine cell line isolated in our laboratory was used in these studies.

Intramuscular and intravaginal vaccination of pregnant cows with thymidine kinase-negative, temperature-resistant infectious bovine rhinotracheitis virus (bovine herpes virus 1).

To test the safety and efficacy of a thymidine kinase-negative (TK-), temperature-resistant (TR) mutant of bovine herpes virus-1 (BHV-1) in pregnant cows, seronegative cows, 2-5 months pregnant, were vaccinated intramuscularly (i.m.) or intravaginally (i.vag.) with this candidate vaccine virus. I.m. vaccinated cows did not shed virus i.vag. or intranasally (i.n.), but i.vag. vaccinated cows replicated virus i.vag. for 8-9 days postvaccination (p.v.) Some of the cows were challenge exposed i.n. at 46 days p.v. with virulent TK+ BHV-1(Cooper). Vaccinated cows showed no clinical disease signs p.v. or postchallenge and responded anamnestically postchallenge. All cows delivered live calves. Pre-colostrum sera of the calves were negative for BHV-1 antibodies.

The location and nucleotide sequence of the thymidine kinase gene of bovine herpesvirus type 1.2.

On the basis of their restriction endonuclease digestion patterns, four Australian bovine herpesvirus type 1 (BHV-1) isolates were classified as belonging to the BHV-1.2a subtypes. The thymidine kinase (TK) genes of all four BHV-1.2a isolates were located on a 3.5 kb SalI restriction fragment. This is in contrast to North American and European BHV-1.1 isolates whose TK genes are contained on a 2.6 to 2.8 kb SalI fragment. The restriction fragments containing the TK genes were cloned into phagemid vectors and their sequences determined using the dideoxynucleotide chain termination method. The BHV-1.2a isolates possessed identical TK gene sequences, which differed from previously published TK sequences for the LA and 6660 BHV-1.1 strains. In addition to five single base alterations, there were six separate base insertions which resulted in two major frameshifts which spanned an area of 72 amino acids or 20% of the expressed TK gene product. The predicted amino acid sequence exhibited a higher degree of similarity to other herpesvirus TKs, suggesting that previously published TK gene sequences may have been incorrect. The present nucleotide sequence and corresponding amino acid composition reinforces previous observations concerning regions of herpesvirus TK amino acid conservation and should assist in future studies into the evolution and functional domains of herpesvirus TKs.

Thymidine kinase-negative bovine herpesvirus type 1 mutant is stable and highly attenuated in calves.

Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk-) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk+ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk- BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk- BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk- BHV-1 vaccination reduced the multiplication of tk+ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk- BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without reverting in vivo to tk+. The results demonstrate that the tk- BHV mutant is attenuated and efficacious as a vaccine.

Expression kinetics of the late UL12 gene encoding the bovine herpesvirus 1 alkaline nuclease.

We characterized the expression kinetics of the transcript and protein generated from the bovine herpesvirus 1 (BHV1) homologue of the herpes simplex virus 1 (HSV1) UL12 gene that encodes a viral alkaline nuclease. The BHV1 UL12 coding sequence, which was previously shown to express in E. coli a protein exhibiting nuclease activity, is located at positions 82776-->84239 of the viral genome. Northern blot analysis of RNA from BHV1-infected cells with a single strand RNA probe complementary to UL12 detected four specific 3' coterminal viral transcripts of 4.2, 3.7, 2.2, and 0.7 kb that accumulated simultaneously from 6 to 24 hours post-infection (p.i.). S1 nuclease mapping of the UL12 capping site at position 82384 of the genome as well as the identification of a consensus polyadenylation signal at 84490-84495 allowed us to establish that the 2.2 kb transcript corresponds to that of UL 12. A UL 12 specific antiserum generated against a T7-Tag/UL12 fusion protein expressed in E. coli detected a 53 kDa protein in cell lysates from BHV1-infected cells, whose size correlated with that predicted (51,844 Da), which accumulated from 12 to 30 h p.i. Differences observed between the transcriptional and translational expression profiles suggest that the UL12 of BHV1 is regulated at both the translational and posttranslational levels. Surprisingly, the protein expression was strictly dependent on viral DNA synthesis, unambiguously demonstrating that BHV1 UL12 belongs to viral genes of the gamma2 class. This is in contrast to the HSV1 and pseudorabies homologues that are classified as early (beta) genes. Further studies will be required to determine whether these kinetic differences have any functional implications.

Thymidine kinase (TK) induction after infection of TK-deficient rabbit cell mutants with bovine herpesvirus type 1 (BHV-1): isolation of TK- BHV-1 mutants.

Cytosol thymidine kinase (TK) activity is enhanced at 6 hr after bovine embryo tracheal (EBTr) and rabbit skin fibroblast (RAB-9) cells are infected with the Los Angeles and Cooper strains of bovine herpesvirus type 1 (BHV-1). To learn whether this enhancement resulted from the induction of a virus-specific TK activity, biochemical and genetic studies were carried out. The biochemical experiments demonstrated that: (i) the BHV-1-induced TK activity had a relative disc PAGE mobility (Rm) characteristic of other herpesvirus-encoded TKs and distinctly different from the Rm value of the cytosol TK of host cells; and (ii) the BHV-1-induced TK was significantly more sensitive to competitive inhibition by arabinosylthymine (araT) than the cytosol TKs of EBTr and RAB-9 cells. The genetic experiments entailed the isolation of a bromodeoxyuridine (BrdUrd)-resistant rabbit cell line [RAB(BU)] deficient in cytosol TK activity and of BrdUrd- and araT-resistant BHV-1 mutants. RAB(BU) cells acquired TK activity after they were infected by wild-type, TK+ BHV-1, but not drug-resistant BHV-1 mutants. The experiments strongly suggest that wild-type BHV-1 induces a virus-specific TK activity.