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Biomedical subjects

A J Davison

Publications and source records attributed to A J Davison.

At least 37 records · Page 2Linked to original sources
PubMed1996

Isolation and characterization of herpes simplex virus type 1 mutants defective in the UL6 gene.

Previous studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and is a minor component of virions and capsids. In this report, we describe the construction of UL6- HSV-1 mutants with a disrupted UL6 gene using complementing cells and show that they fail to synthesize the UL6 protein or produce infectious virus in noncomplementing cells. The mutants synthesized but failed to process and encapsidate viral DNA and accumulated only immature capsids which lacked the UL6 protein. Immunofluorescence analysis showed that the UL6 protein, when expressed transiently in transfected cells in the absence of other HSV-1 proteins, is localized exclusively to the nucleus. We also investigated an HSV-1 mutant with a defect in gene UL33, the product of which is also thought to be involved in viral DNA processing and packaging. The phenotype of this mutant on noncomplementing cells with regard to failure to process and encapsidate viral DNA, accumulation of immature capsids, and inability to produce infectious virus was the same as that of UL6- viruses. This mutant, however, produced capsids containing the UL6 protein, indicating that association of the UL6 protein with the capsid is independent of the UL33 protein.

Animals↗
PubMed1996

The capsid architecture of channel catfish virus, an evolutionarily distant herpesvirus, is largely conserved in the absence of discernible sequence homology with herpes simplex virus.

Although herpesviruses have a wide host range and their genomes vary substantially in size, the nucleocapsid appears to be a conservative element of viral design. The capsid shell is icosahedrally symmetric (T = 16), and 125 nm in diameter and 15nm thick in the case of herpes simplex virus 1 (HSV-1). Channel catfish virus (CCV) has the gross morphology of a herpesvirus, although no relationship to other herpesviruses is evident from the sequences of its proteins. To examine CCV capsid architecture more closely, we have determined its structure by cryoelectron microscopy and three-dimensional image reconstruction. The CCV capsid is smaller than that of HSV-1, but its 12% smaller genome is packed to essentially the same average density; its icosahedral facets are flatter, and its shell is about 20% thinner, consistent with the smaller size of its major capsid protein. Otherwise, their major features are remarkably similar: CCV has the same triangulation number; its hexons and pentons also have chimney-like protrusions with an axial channel through each capsomer; and there are "triplexes" on the outer surface at the sites of local threefold symmetry. The basic herpesvirus capsid architecture is, therefore, remarkably well conserved in CCV and implies a utilitarian basis to this design. The protein composition of CCV mirrors that of HSV-1, except for the absence of the 12-kDa protein, VP26, which is dispensable for assembly in the HSV-1 system and, apparently, wholly dispensable for CCV.

Animals↗
PubMed1996

Effects of metals, ligands and antioxidants on the reaction of oxygen with 1,2,4-benzenetriol.

1,2,4-Benzenetriol is an active metabolite of the human leukemogen benzene that reacts rapidly with molecular oxygen (O2). The mechanism of autoxidation of benzenetriol is scantily characterized, and little is known of the effects of metals, metal chelators, radical scavengers, and antioxidants on the rate of reduction of O2. Here, we report that catalytic amounts of Cu2+ and Fe3+ accelerated the oxidation of benzenetriol (250 mu M) in a dose-dependent manner. Fe3+ (50 mu M) increased the rate of autoxidation by 91%, and Cu2+ (10 mu M) increased it 11-fold. In the absence of added metals, superoxide dismutase inhibited and desferrioxamine stimulated the autoxidation. In the Cu2+ -catalyzed reaction, superoxide dismutase neither inhibited nor stimulated, while desferrioxamine abolished the catalysis by Cu2+. In the presence of Fe3+, superoxide dismutase slowed the reaction, but desferrioxamine, surprisingly, did not. The presence of both superoxide dismutase and desferrioxamine blocked the autoxidation, either in the presence or absence of metals. We conclude: (1) superoxide is a propagator of sequential one-electron transfer reactions in the absence of added metals; (2) addition of Cu2+, unlike Fe3+, removes the dependence of the reaction on propagation by superoxide, presumably changing the radical-propagated chain reaction to a concerted two-electron transfer; (3) the further addition of desferrioxamine restores superoxide-dependent propagation. Taken with our previous data on the genotoxicity of benzenetriol, these findings have implications regarding a role for transition metals in the carcinogenicity of benzene.

Antioxidants↗
PubMed1995

The DNA sequence of equine herpesvirus 2.

The complete DNA sequence of equine herpesvirus 2 (EHV-2) strain 86/67 was determined. The genome is 184,427 bp in size and has a base composition of 57.5% G + C. Unusually for a herpesvirus, about a third of the sequence distributed in several large blocks appears not to encode proteins. The 79 open reading frames that were identified as probably polypeptide-coding are predicted to encode 77 distinct proteins. Amino acid sequence comparisons confirmed that EHV-2 is a gamma-herpesvirus that is genetically collinear with herpesvirus saimiri (HVS; a gamma 2-herpesvirus) and Epstein-Barr virus (EBV; a gamma 1-herpesvirus), with a closer relationship to the former. Moreover, EHV-2 specifies eight proteins that have counterparts in HVS but not in EBV and only a single protein that has a homologue in EBV but not in HVS (EBV BCRF1, which encodes an interleukin 10-like protein). EHV-2 also encodes three potential G protein-coupled receptors, one with a counterpart in HVS that is specific for alpha chemokines, another with a counterpart in human cytomegalovirus (a beta-herpesvirus), which is specific for beta chemokines, and a third that is assigned more tentatively and lacks detectable counterparts in other herpesviruses.

Amino Acid Sequence↗
PubMed1995

Identification of structural proteins of channel catfish virus by mass spectrometry.

Emerging methods that couple mass spectrometry of fragments generated from isolated proteins with database searching offer a powerful means of identifying proteins and the genes that encode them. We have applied this technology to a herpesvirus, channel catfish virus, and have identified 12 genes, 11 viral and 1 cellular, that encode 16 principal structural proteins. These proteins include three components of the mature capsid and a potential scaffolding protein present in immature capsids, three protein kinases, a C3HC4 zinc-binding protein and cellular actin located in the tegument, and a multiply hydrophobic protein associated with the envelope.

Amino Acid Sequence↗
PubMed1994

Equid herpesviruses 1 and 4 encode functional homologs of the herpes simplex virus type 1 virion transactivator protein, VP16.

The herpes simplex virus type 1 (HSV-1) tegument protein VP16 is a potent transcriptional inducer of immediate-early gene expression, comprising an N-terminal domain involved in binding DNA linked to an acidic transactivating C-terminal domain. The gene encoding the counterpart of this protein in equid herpesvirus 4 (EHV-4) was sequenced. Comparisons with VP16 and the homologous proteins of equine herpesvirus 1 (EHV-1) and varicella-zoster virus (VZV) showed that a region in the N-terminal domain involved in formation of a complex with cellular proteins is partially conserved in all four proteins. In contrast, the C-terminal regions of the EHV proteins, like that of VZV, are not particularly acidic and are not significantly conserved with respect to the C-terminal region of VP16. Nevertheless, transient expression experiments indicated that the EHV-1 and EHV-4 proteins are able to transactivate HSV-1 and EHV-1 immediate-early promoters in a dose-dependent manner, which suggests that this activity is not dependent on an acidic C-terminal domain.

Amino Acid Sequence↗
PubMed1994

Comparative studies of the structural proteins and glycoproteins of equine herpesviruses 2 and 5.

The structural proteins of equine herpesvirus 2 (EHV-2) and EHV-5, recently shown to be gammaherpesviruses, were identified and compared. Labelled proteins and glycoproteins were separated by SDS-PAGE and although EHV-2 and EHV-5 had similar protein profiles, bands in some positions were virus-specific. Six glycoproteins, with distinct profiles, were identified for both EHV-2 and EHV-5. Rabbit antisera to EHV-2 and EHV-5 and horse antiserum to EHV-2 were used in radioimmunoprecipitations, Western blot analysis and ELISA to investigate the immunogenicity and cross-reactivity of virus proteins. These analyses revealed that while EHV-2 and EHV-5 proteins share many common epitopes, they also possess type-specific epitopes. A 0.71 kb region of the EHV-2 glycoprotein B (gB) gene was expressed as a fusion protein in Escherichia coli. Antiserum raised in a rabbit to the EHV-2 fusion protein was used to identify a 64K EHV-2 protein as EHV-2 gB. Antiserum to EHV-2 gB was used to identify a 66K EHV-5 protein as EHV-5 gB. These proteins, which may represent subunits of gB rather than the entire molecule, appear the most immunodominant of the structural virion proteins as identified by Western blot.

Animals↗
PubMed1994

Delineation of a sequence required for nuclear localization of the protein encoded by varicella-zoster virus gene 61.

All characterized alphaherpesviruses encode a protein whose N-terminal region contains a novel zinc-binding motif, the C3HC4 domain. Homology between the different proteins is in general limited to key residues in this domain. In order to identify a separate landmark site in the C3HC4 protein encoded by varicella-zoster virus gene 61, namely the region required for nuclear localization, we have analysed a range of mutants in transient expression and immunofluorescence experiments. A basic region (RGAKRR) at residues 387 to 392 was found to be required for nuclear localization, and residues 390 and 391 were critical.

Amino Acid Sequence↗
PubMed1994

Characterization of the putative protein kinases specified by varicella-zoster virus genes 47 and 66.

The proteins predicted to be encoded by varicella-zoster virus (VZV) genes 47 and 66 display sequence similarity to the serine/threonine family of protein kinases. Homologues of gene 47 exist in alpha-, beta- and gamma-herpesviruses but homologues of gene 66 are specific to the alpha-herpesviruses. Monospecific rabbit antisera were raised against two separate fusion proteins constructed from a portion of each protein fused to the carboxy terminus of beta-galactosidase. These antisera were used to characterize the 47 and 66 proteins in VZV-infected cells and in cells infected with vaccinia virus recombinants expressing each protein. The 47 proteins is a 54K phosphoprotein which is distributed between the cytoplasmic and nuclear compartments of VZV-infected cells and is associated with the capsid/tegument fraction of purified VZV particles. Gene 66 encodes a 48K phosphoprotein when expressed by VZV or a vaccinia virus recombinant, and, in the latter case, the 66 protein was located exclusively in the cytoplasm. The 47 protein immunoprecipitated from VZV-infected cells could be phosphorylated in vitro, but the same protein produced by in vitro transcription and translation could not. This and other evidence indicates that additional proteins induced or encoded by VZV may be involved in the phosphorylation of the 47 protein.

Base Sequence↗
PubMed1994

The equine herpesvirus type 1 glycoprotein homologous to herpes simplex virus type 1 glycoprotein M is a major constituent of the virus particle.

Glycoprotein 45 is a major envelope glycoprotein of equine herpesvirus type 1. The gene encoding this protein is located between map units 0.615 and 0.636 on the virus genome and evidence has suggested that it is encoded by gene 52, one of four genes within this region. Using PCR we have amplified gene 52 and subsequently cloned it into a mammalian expression vector under the control of the human cytomegalovirus immediate early gene promoter. The gene was expressed in COS-7 cells and its product was detected by immunofluorescence and Western blotting. The results indicate that glycoprotein 45 is encoded by gene 52, and that it is the homologue of herpes simplex virus type 1 glycoprotein M.

Base Sequence↗
PubMed1993

The DNA sequence of adenovirus type 40.

The 34,214 bp DNA sequence of adenovirus type 40 strain Dugan was determined directly from random fragments of virion DNA cloned into a bacteriophage M13 cloning vector. The gene layout is similar to that of other human adenoviruses, and in addition contains two potential protein-coding regions that are conserved, but have not been recognized previously, in other adenovirus genomes. One is oriented rightward, contained within the intron in the protein-coding region for the L4 33K gene, and would encode a protein sharing N-terminal sequence with 33K. The other is a leftward oriented exon located between the E3 and L5 IV (fibre) regions which would specify the N terminus of a novel protein. The region encoding the C terminus of this protein is not apparent from sequence data.

Adenoviruses, Human↗
PubMed1993

Benzene metabolite, 1,2,4-benzenetriol, induces micronuclei and oxidative DNA damage in human lymphocytes and HL60 cells.

The triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), is readily oxidized to its corresponding quinone via a semiquinone radical. During this process, active oxygen species are formed that may damage DNA and other cellular macromolecules. The ability of BT to induce micronuclei (MN) and oxidative DNA damage has been investigated in both human lymphocytes and HL60 cells. An antikinetochore antibody based micronucleus assay was used to distinguish MN containing kinetochores and potentially entire chromosomes (kinetochore-positive, K+) from those containing acentric chromosome fragments (kinetochore-negative, K-). BT increased the frequency of MN formation twofold in lymphocytes and eightfold in HL60 cells with the MN being 62% and 82% K+, respectively. A linear dose-related increase in total MN, mainly in K(+)-MN, was observed in both HL60 cells and lymphocytes. Addition of copper ions (Cu2+) potentiated the effect of BT on MN induction threefold in HL60 cells and altered the pattern of MN formation from predominantly K+ to K-. BT also increased the level of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker of active oxygen-induced DNA damage. Cu2+ again enhanced this effect. Thus, BT has the potential to cause both numerical and structural chromosomal changes in human cells. Further, it may cause point mutations indirectly by generating oxygen radicals. BT may therefore play an important role in benzene-induced leukemia.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Equine herpesviruses 2 and 5 are gamma-herpesviruses.

Equine herpesviruses 2 and 5 (EHV-2 and EHV-5) have biological properties and genome structures that support their classification as members of the Betaherpesvirinae. In order to investigate whether this is supported by genetic data, we analysed the sequences of random DNA fragments and identified 25 EHV-2 and 28 EHV-5 genes that encode amino acid sequences with significant homology to proteins from other herpesviruses. Greatest similarity was to proteins specified by the gamma-herpesviruses Epstein-Barr virus (a gamma 1-herpesvirus) and herpesvirus saimiri (a gamma 2-herpesvirus), and the level of similarity was marginally greater to the latter. Also, like other gamma-herpesviruses, the EHV-2 and EHV-5 genomes are deficient in the CG dinucleotide, suggesting that latent genomes are methylated. EHV-2 and EHV-5 are related to each other more closely than they are to other herpesviruses, but are clearly distinct gamma-herpesviruses. The data support the establishment of at least one more subdivision of the gamma-herpesviruses (the gamma 3-herpesviruses).

Amino Acid Sequence↗
PubMed1993

A cosmid-based system for constructing mutants of herpes simplex virus type 1.

Cosmids containing large fragments of herpes simplex virus type 1 DNA were prepared using a vector that allows intact inserts to be excised using the restriction endonuclease PacI. Two independent sets (A and B) of five cosmids were identified whose inserts overlap and represent the entire viral genome, and set C was obtained by replacing two cosmids in set B. Each set gave rise to viral plaques when digested with PacI and transfected into cells in culture. Two cosmids common to sets B and C ostensibly contain one of the origins of viral DNA replication (oriL) in a region of overlap between inserts, but both actually consist of a minority of apparently intact (ori+L) forms and a majority of deleted (ori-L) forms. These sets yielded exclusively ori+L viral progeny. When either of these cosmids was replaced by a derivative comprising only ori-L forms, ori+L and ori-L progeny were obtained, and only ori-L progeny were produced when both were replaced. One cosmid in set A contains the oriL locus in a nonoverlapping region and lacks ori+L forms. This set generated only ori-L virus. Viral mutants with lesions in either or both of genes UL2 and UL44, which are not essential for growth in cell culture, were constructed using cosmids containing specifically introduced frameshift mutations. A mutant with a frameshift mutation in an essential gene (UL33) was isolated by transfecting a complementing cell line. These results indicate that a cosmid-based system will facilitate isolation of large numbers of defined viral mutants.

Animals↗
PubMed1993

The DNA sequence of the equine herpesvirus 4 gene encoding glycoprotein gp17/18, the homologue of herpes simplex virus glycoprotein gD.

The nucleotide sequence of the gene to the left of the gI gene of equine herpesvirus 4 (EHV-4) was determined. The gene encodes a peptide of 402 amino acids with an unprocessed M(r) of 45,323. The predicted polypeptide has several features of a glycoprotein including a hydrophobic signal sequence, a membrane spanning domain and four potential N-linked glycosylation sites within the proposed external domain. The predicted amino acid sequence of EHV-4 gD shows 83% identity with that of equine herpesvirus 1 gD. Conservation of the tertiary structure is suggested by the alignment of six cysteine residues with those of the gD of six other alphaherpesviruses. Screening a lambda gt11/EHV-4 expression library with monoclonal antibodies against several of the most abundant EHV-4 glycoproteins unequivocally identified the protein encoded by the EHV-4 gD gene as gp17/18.

Amino Acid Sequence↗
PubMed1992

Effects of ligands on reduction of oxygen by vanadium(IV) and vanadium(III).

V(IV) and V(III) reduce molecular oxygen with increasing rates as the pH is raised from 6.0 to 7.4. Under all conditions tested, V(IV) is the more efficient reductant. EDTA and ATP generally inhibit the reduction of oxygen by V(III) and V(IV). In contrast, desferrioxamine accelerates the reduction of oxygen by V(IV) but with decreasing effectiveness at pH 7.4 compared to pH 6.0, while desferrioxamine accelerates the reduction of oxygen by V(III) only at pH 6.0. Histidine enhances the reduction of oxygen by V(IV) at pH 7.0 and 7.4. The observed rates of oxygen reduction by V(III) and V(IV) imply that the intracellular distribution of vanadium among its redox states reflects not an equilibrium but a steady state.

Adenosine Triphosphate↗