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W C Lawrence

Publications and source records attributed to W C Lawrence.

At least 19 recordsLinked to original sources
PubMed1997

Construction of a viable BHV1 mutant lacking most of the short unique region.

The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotein E was still replication-competent.

Animals↗
PubMed1996

Identification and characterization of BICP27, an early protein of bovine herpesvirus 1 which may stimulate mRNA 3' processing.

Sequence analysis of the left genomic terminus of bovine herpesvirus 1 (BHV-1) revealed two convergently transcribed genes with 3' ends about 300 bp apart. The gene on the left is the previously described circ gene; that on the right was found to encode a protein of 400 amino acids which was designated BICP27 because of its homology to ICP27 (Vmw63) of herpes simplex virus 1 (HSV-1) and related proteins from other alpha- beta- and gammaherpesviruses. Rabbit antisera raised against a synthetic oligopeptide representing the amino terminus of the predicted polypeptide demonstrated the presence of BICP27 in the nuclei of infected cells by in situ immunoadsorbent assays. In Western immunoblots, BICP27 was detected as a 50 kDa BHV-1 specific protein expressed with early kinetics, in contrast to HSV-1 ICP27 which is an immediate early (IE) protein. A DNA fragment containing BICP27 coding sequences was inserted into a baculovirus genome. The recombinant BICP27 protein, identified by its reactivity with the antipeptide sera, exhibited the same electrophoretic mobility as BICP27 specified by BHV-1. Transient expression assays using target genes differing only in their poly(A) sites showed that BICP27, like its HSV-1 counterpart, may be involved in 3' processing of mRNA.

Amino Acid Sequence↗
PubMed1996

Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins.

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.

Amidohydrolases↗
PubMed1996

Recombinant bovine herpesvirus-1 (BHV-1) lacking transactivator protein BICPO entails lack of glycoprotein C and severely reduced infectivity.

The immediate-early transactivator protein BICPO is a key regulatory element of bovine herpesvirus 1 (BHV-1) replication based on transient expression assays. To examine BICPO function in the context of the viral genome, we created recombinant BHV-1 expressing beta-galactosidase instead of BICPO. To complement the defect, a neomycin resistant MDBK cell line (M164) expressing BICPO was established, permitting selection of a blue-staining BHV-1 recombinant (A2G2). Southern blot and PCR analysis confirmed that the BICPO gene was interrupted by the beta-galactosidase gene and that wt progeny was absent. Compared with wt BHV-1, A2G2 reached lower titers in M164 cells but replicated with similar kinetics. Once isolated, A2G2 also grew in MDBK cells although the titer was reduced a further 10-fold and the virus remained strongly cell-associated. Thus, BICPO is not absolutely required for replication in cell culture. Gene expression of A2G2 was investigated by Western blots and immunofluorescence. Surprisingly, not only was BICPO absent, but glycoprotein C (gC) was also missing. Other viral genes were expressed normally. Semiquantitative PCR showed that A2G2 produced similar amounts of viral DNA as wt but a much smaller number of infectious particles. Cotransfection of A2G2 DNA and a plasmid containing the BICPO gene yielded revertant virus with fully restored wt properties. We conclude that BICPO is required for gC expression, and that the missing gC partly accounts for the reduced A2G2 infectivity.

Animals↗
PubMed1995

Abortion in heifers inoculated with a thymidine kinase-negative recombinant of bovine herpesvirus 1.

The Copper isolate of bovine herpesvirus 1 (BHV-1) was used to produce a thymidine kinase-negative (TK-) recombinant by insertion of a beta-galactosidase (bgal) expression cassette into the TK coding region. The recombinant virus (TK- bgal+) was tested for abortifacient activity in cattle by inoculation of 5 pregnant heifers at 25 to 29 weeks gestation. Five additional heifers were inoculated with the Cooper TK-positive (TK+) virus to serve as controls. After inoculation, both groups of heifers developed similar febrile responses and neutralizing antibody titers. Virus was isolated from blood of all heifers during the first postinoculation (PI) week, and isolation frequencies were similar for both groups. In contrast, whereas virus was isolated from many of the nasal and vaginal swab specimens of heifers inoculated with TK+ virus, only rare virus isolations were made from the heifers given TK- bgal+ virus. All heifers inoculated with TK+ virus aborted between PI days 19 and 35. The finding of characteristic microscopic lesions and viral antigen in fetal tissues indicated that the abortions were caused by BHV-1 infection. Virus was isolated from 3 fetuses, and all isolates were TK+ virus. Two heifers inoculated with TK- bgal+ virus aborted at PI days 25 and 39. Fetal tissues had typical BHV-1 microscopic lesions and viral antigen. Virus was isolated from blood of both fetuses, and the isolates were TK- bgal+. Results of this study indicate that inactivation of the TK gene reduces, but does not eliminate, the abortifacient activity of BHV-1.

Abortifacient Agents↗
PubMed1994

Characterization of the bovine herpesvirus 1 homolog of the herpes simplex virus 1 UL24 open reading frame.

An open reading frame (ORF) of 293 codons which partially overlaps the bovine herpesvirus 1 (BHV1) thymidine kinase (tk) ORF in a head-to-head orientation has been identified by nucleotide sequence analysis. The predicted amino acid sequence of this ORF exhibits an overall identity of 38.1% with that of the herpes simplex virus type 1 (HSV1) UL24 ORF as well as an 80.7% identity with the 57-amino-acid consensus sequence found in the UL24-like ORFs of eight herpesviruses (Jacobson et al., J. Virol, 49, 947-959, 1989). We have therefore designated this BHV1 ORF as the homolog of the HSV1 UL24 ORF with which it also shares a common position and orientation relative to the tk ORF. The BHV1 UL24-like ORF is contained within a 5.2-kb RNA transcript initiating within the tk ORF and is the largest in a group of four 3'-coterminal transcripts. This transcript pattern closely resembles that observed in HSV1-infected cells where five transcripts, the two largest of which contain the UL24 ORF, appear to be 3'-coterminal. In contrast to HSV1 mutants with lesions in the UL24 ORF, a BHV1 deletion mutant which failed to produce detectable levels of the 5.2-kb transcript grew nearly as well as wild-type virus.

Amino Acid Sequence↗
PubMed1992

Latency and reactivation of a thymidine kinase-negative bovine herpesvirus 1 deletion mutant.

A bovine herpesvirus 1 (BHV 1) mutant variant with a deletion in the thymidine kinase (TK) gene was assessed for its ability to establish latency and be reactivated in cattle. After treatment with dexamethasone, reactivated TK- BHV 1 was isolated from one of four cattle that received virus by intravenous inoculation only, and from four of four cattle that received virus by intranasal, intravaginal, and intravenous inoculation. Results prove that TK- BHV 1 will establish latency and can be reactivated in the natural host.

Animals↗
PubMed1992

Sequence and transcript analysis of the bovine herpesvirus 1 thymidine kinase locus.

A detailed sequence and transcript analysis of the thymidine kinase (tk) gene of bovine herpesvirus 1 (BHV1, Cooper strain) was carried out. A tk open reading frame (ORF) of 1077 bp was identified and compared with tk ORFs previously published for other strains of BHV1. The Cooper sequence was in good agreement with that of strain Q3932 but differed significantly from strains 6660 and LA. Reanalysis of the LA tk sequence, however, failed to confirm this difference. Except for five single base substitutions, our results indicate that the Cooper, Q3932, and LA strains share the same tk sequence. The size of the tk mRNA was determined by Northern blot analysis. In contrast to the size of other herpesvirus tk mRNAs (approximately 1.5 kb), the BHV1 tk transcript was 4.3 kb. Northern blot analysis indicated that the tk transcript was 3' coterminal with the downstream 3.1-kb transcript which encodes a BHV1 homologue of the HSV1 H glycoprotein (gH). The 5' ends of the tk and gH mRNAs were mapped by S1 analysis to positions 135 and 91 bp upstream of their respective translation start sites. The 5' end of the tk transcript was found to overlap a 5.2-kb transcript with opposite polarity to the tk mRNA.

Base Sequence↗
PubMed1992

Relationship of bovine herpesvirus 1 immediate-early, early, and late gene expression to host cellular gene transcription.

Bovine herpesvirus 1 (BHV-1) gene expression was examined by RNA blot hybridization using clones representing immediate-early, early, and late genes. An immediate-early protein gene probe hybridized with two transcripts, 3.4 and 5.8 kb, expressed by infected cells in the presence of cycloheximide (CH). During infection of cells without metabolic inhibitors these transcripts were detected as early as 2 hr postinfection (p.i.) and accumulated to 8 hr p.i. The early gene probe, thymidine kinase, hybridized with a 4.3-kb RNA that was detected in the presence of phosphonoacetic acid (PAA), but not in the presence of CH. The late gene probe, glycoprotein III, (gIII) hybridized with a 1.6-kb transcript that was not expressed by infected cells treated with CH and only in very reduced amounts by infected cells treated with PAA. The gIII RNA was not detected until 4 hr p.i. in total cell RNA. Transcripts for the bovine actin and beta-galactosyltransferase genes did not decrease in BHV-1-infected cells until 6 hr p.i., coincident with the increase of BHV-1 DNA and RNA synthesis. Consequently, shutoff of host cell transcription by BHV-1 may be different than what has been described for herpes simplex virus.

Animals↗
PubMed1992

Bovine herpesvirus 1 as a live virus vector for expression of foreign genes.

DNA sequence analysis and 5'-end mapping of mRNA were used to identify and clone DNA fragments which contain the presumptive promoter (Pgl) and poly(A) site (An) of the bovine herpesvirus 1 (BHV1) gl glycoprotein. To confirm the presence of these regulatory regions in the above fragments, they were cloned together with a chloramphenicol acetyltransferase (CAT) reporter gene into pUC19. The recombinant plasmid formed, pEC3, was capable of inducing CAT activity when transfected into bovine cells demonstrating that the Pgl-CAT-An sequence constituted a functional CAT expression cassette. The cassette was excised from pEC3, transferred to a plasmid insertion vector (pIV3A) and subsequently inserted into the thymidine kinase (tk) gene of BHV1. Insertion in either orientation, relative to the tk gene, gave rise to BHV1 recombinants which expressed CAT activity in infected cells. Analysis of RNA from infected cells indicated that CAT transcripts were present in multiple species of RNA. This unexpected result was found to reflect temporal shifts in promoter and poly(A) site usage during infection. Although the poly(A) site which forms part of the expression cassette was used extensively early in infection, most CAT transcripts synthesized at late times read through this promoter-proximal site and terminated at the distal site normally used for tk mRNA synthesis.

Base Sequence↗
PubMed1991

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.

Abortion, Veterinary↗
PubMed1988

Comparison of the bovine herpesvirus 1 gI gene and the herpes simplex virus type 1 gB gene.

In a previous report, we localized the gene for a 130-kilodalton envelope glycoprotein (gI) of bovine herpesvirus 1 (BHV-1) to a 3.6-kilobase HpaI-KpnI restriction endonuclease fragment from the long unique region of the BHV-1 genome (map position 0.405 to 0.432) and showed that a herpes simplex virus 1 (HSV-1) glycoprotein B (gB) probe uniquely hybridized to this BHV-1 restriction fragment. Here we present the complete nucleotide sequence of the BHV-1 gI gene and the predicted 932-amino-acid sequence of the gI primary translation product. Comparison with the published nucleotide sequence of the HSV-1 (KOS) gB gene (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984) reveals a similarity of 56.3% at the nucleotide level and 45.9% at the amino acid level. Upstream of the proposed gI coding region are potential mRNA transcriptional promoter elements including a TATA box and multiple Sp1 binding sites (GC boxes). Downstream of the gI coding region are two sequence elements associated with mRNA cleavage and polyadenylation (AATAAA and a GT-rich region roughly 30 nucleotides further downstream). Like HSV-1 gB, the predicted gI amino acid sequence exhibits two broad hydrophobic regions likely to represent a transient amino-terminal signal sequence and a transmembrane anchor domain (near the carboxyl terminus). Additional features shared with gB include 6 potential N-linked glycosylation sites and 10 highly conserved cysteine residues in the gI extracellular domain. Two regions of nonsimilarity between gI and gB are a centrally located 22-amino-acid region of gI for which there is essentially no gB counterpart and the transient amino-terminal leaders which differ in both size and sequence. The hydrophobic signal sequence of the gI leader, unlike that of gB, is preceded by an unusually large region of predominantly hydrophilic amino acids. The unusual length of the gI leader may result from an overlap between that portion of the gI coding region and a potential upstream coding region.

Amino Acid Sequence↗
PubMed1987

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.

Animals↗
PubMed1986

Map location of the gene for a 130,000-dalton glycoprotein of bovine herpesvirus 1.

A bovine herpesvirus 1 variant (mar6) containing a mutation in a viral glycoprotein with a molecular weight of 130,000 (g130) was isolated by selecting for resistance to a neutralizing monoclonal antibody (130-6) directed against g130. Mar6 was completely resistant to neutralization by monoclonal antibody 130-6 in the presence and absence of complement, but was neutralized by polyvalent immune sera. The mar6 mutant synthesized and processed g130, but produced plaques which failed to react with monoclonal antibody 130-6 in an in situ immunoassay (black plaque). However, monoclonal antibody 130-6 was capable of binding and immunoprecipitating g130 from infected-cell extracts produced by lysis of mar6-infected cells with nonionic detergents. The mutation in mar6 was mapped by marker rescue with cloned bovine herpesvirus 1 restriction enzyme fragments to a 3.8-kilobase fragment at approximate map units 0.405 to 0.432. In addition, it was found that a DNA probe containing the glycoprotein B gene of herpes simplex type 1 hybridized uniquely to the same 3.8-kilobase fragment which was shown by marker rescue to contain the mutation site in the gene for bovine herpesvirus 1 g130.

Antibodies, Monoclonal↗
PubMed1981

Herpes simplex type 1 DNA in human brain tissue.

Herpes simplex virus type 1 (HSV-1) is known to reside latently in the trigeminal ganglia of man. Reactivation of this virus causes skin lesions and may occasionally infect other tissues, including the brain. To determine whether the brain tissue of humans free of clinical signs of HSV-1 infection contains any trace of HSV-1, we examined the DNA from brain tissue by endonuclease digestion, separation of the fragments by gel electrophoresis, and hybridization with labeled HSV-1 DNA probes. Hybrid bands were detected autoradiographically in experiments using cloned and virion-purified fragments of the HSV-1 genome. HSV-1 DNA sequences were found in 6 of 11 human brain DNA samples tested. In some cases, these bands corresponded to the bands expected for the complete viral genome, whereas others contained bands representing only a part of the genome. In some cases, the terminal fragments could be found, suggesting that the DNA was in a linear, nonintegrated form.

Brain↗
PubMed1980

Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2.

Capsids of herpes simplex virus (HSV) types 1 and 2 contain seven polypeptides ranging in molecular weight from 154,000 to 12,000 (termed NC-1 through NC-7 in order of descending molecular weight). Antibodies prepared to HSV-1 capsid polypeptides isolated from sodium dodecyl sulfate-polyacrylamide gels reacted in an immunofluorescence assay against HSV-1-infected KB cells. Three of the antibodies (anti-NC-1, anti-NC-2, and anti-NC-3,4) also reacted with HSV-2-infected cells. Tryptic peptide analysis showed that each of the HSV-1 capsid polypeptides had a unique methionine peptide profile, and none appeared to be derived from the major capsid polypeptide. Comparative peptide analysis of HSV-1 and HSV-2 showed that one polypeptide (NC-7, 12,000 molecular weight) had an identical methionine peptide profile and a very similar arginine peptide profile in both virus types. The arginine peptide profile of NC-7 of HSV-1 was very different from the arginine profile of KB histone H4. Although there were certain intertypic similarities in the methionine peptide profiles of the other capsid components especially in NC-1 (the major capsid protein), there was no case where the tryptic peptides were identical in the two virus types.

Capsid↗