Biomedical subjects

H J Buhk

Publications and source records attributed to H J Buhk.

At least 19 recordsLinked to original sources

PubMed1999

Structure and function of the prDNA and the genomic termini of the gamma2-herpesvirus bovine herpesvirus type 4.

The linear virion DNA of bovine herpesvirus type 4 (BHV-4) is flanked by tandem repeats designated polyrepetitive DNA (prDNA). To investigate the structure and functional role of the prDNA for cleavage/packaging of progeny viral DNA, the complete nucleotide sequence (2267 bp) of a cloned prDNA unit of BHV-4 was determined. Moreover, the terminal fragments of the genome and the junctions between prDNA and the central unique DNA were analysed. In order to characterize the function of the prDNA of BHV-4, a transient packaging assay was developed. The prDNA has a G+C content of 71.1%. Its structure is composed of numerous internal repeats and every unit contains the conserved sequence of the cleavage/packaging signal. A fragment of 443 bp comprising the cleavage/packaging signal was found to be sufficient for cleavage and encapsidation of replicated concatemeric viral DNA. These results suggest that prDNA is a functionally important region of the genome of BHV-4.

Animals↗

PubMed1998

Transcription analysis of porcine circovirus (PCV).

This study focuses on gene expression of porcine circovirus (PCV) in order to identify viral genes and their corresponding mRNA transcripts. By northern blot analysis, the existence of three mRNAs could be demonstrated. Two mRNAs are encoded by the viral (-)-strand and one is encoded by the viral (+)-strand. The (+)-strand encoded mRNA transcript is 990 nucleotides (nt) long and corresponds to the open reading frame (ORF) 1, as shown by S1 mapping. The start point of this transcript is located at pos. 1238, as determined by primer extension analysis and rapid amplification of cDNA ends (RACE). The transcript is spliced as shown by direct reverse sequencing and RACE. It contains an untranslated "leader"-sequence 119 nt in size (pos. 1238 to 1120) which is joined to exon 2 of the ORF 1 transcript at pos. 737. The transcriptional regulatory elements have been identified functionally by CAT assays. They are located within a 258 base points (bp) fragment (pos. 1168 to 1425).

Animals↗

PubMed1998

Identification of a protein essential for replication of porcine circovirus.

The largest open reading frame of porcine circovirus (ORF 4) encodes a protein of 312 amino acids. The predicted gene product of ORF 4 shows similarities to Rep proteins of other plant circoviruses and geminiviruses. Three motifs have been identified that are characteristic for proteins involved in rolling circle replication and the consensus sequence for a putative dNTP-binding box (GKS) has been found. In this paper, experimental evidence is presented which indicates that ORF 4 encodes the replication protein of porcine circovirus. After cloning of the ORF 4 gene product, it was supplied in trans in a transient replication assay. The ORF 4 gene product promoted the replication of plasmid pOP11, which carries the origin of DNA replication of porcine circovirus. Since pOP11 itself is unable to replicate in virus-free porcine kidney cells, the ORF 4 gene product must be essential for replication of porcine circovirus.

Amino Acid Sequence↗

PubMed1997

Mapping and characterization of the origin of DNA replication of porcine circovirus.

The origin of DNA replication of porcine circovirus (PCV) was mapped to a 111-bp fragment. On top of a hairpin, a nonanucleotide (TAGTATTAC) homologous to nonanucleotides of other viruses was identified. Mutation of this element abolishes replication. PCV may be related to a virus family characterized by single-stranded circular DNA genomes, rolling-circle replication, and homology of their rep proteins.

Amino Acid Sequence↗

PubMed1995

The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle.

Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.

Animals↗

PubMed1994

Glycoprotein B of bovine herpesvirus type 4: its phylogenetic relationship to gB equivalents of the herpesviruses.

In order to estimate the phylogenetic relationship of BHV-4 among the herpesviruses, we have cloned and sequenced its glycoprotein B (gB). The 2.6 kb open reading frame codes for a 874 amino acid long protein. The comparison of its deduced amino acid sequence with those of its counterparts in 19 distinct herpesviruses groups BHV-4 into the gamma-herpesvirinae. The calculation of an evolutionary tree emphasized that BHV-4 is more closely related to herpesvirus saimiri (HVS) than to Epstein-Barr virus (EBV). However, in contrast to EBV and HVS, the gB of BHV-4 contains a putative protease cleavage site and 20 potential N-glycosylation sites. The alignment of the amino acid sequences revealed that 10 cysteine and 7 proline residues, as well as the motifs SPF and GQLG, were completely conserved among the 20 investigated gBs.

Amino Acid Sequence↗

PubMed1994

Risk evaluation.

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Bacteria↗

PubMed1991

Conserved domains of glycoprotein B (gB) of the monkey virus, simian agent 8, identified by comparison with herpesvirus gBs.

The herpesvirus simian agent 8 (SA8) gene which corresponds to the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) was localized, cloned and sequenced. Comparison of its deduced amino acid sequence with those of its counterparts in 12 other distinct herpesvirus was used to evaluate their homology and phylogenetic relationship. The results emphasized that SA8 gB is more closely related to those of HSV-1 and -2, and bovine herpesvirus 2 than to the homologous proteins of other herpesviruses. Furthermore, the alignment showed several regions of domains conserved in the closely related sequences, including four conserved in all the herpesvirus gB sequences examined. The conservation of 10 cysteine residues and most of the proline residues, as well as several potential N-glycosylation sites, suggested that the secondary and tertiary structures of these gBs were similar.

Amino Acid Sequence↗

PubMed1990

Recombination of genomic terminus of bovine herpesvirus type 1 with cellular DNA.

Bovine herpesvirus 1 (BHV-1) has a linear DNA genome of about 135 kb which appears as two isomers, resulting from its short unique segment being present in the two possible orientations with respect to the large unique segment. BHV-1 also circularizes its DNA to form replicative molecules. Definition of the target sequences at the genomic termini involved in the recombination events during genomic replication and isomerization, as well as virus maturation, led to the discovery that 10% of the genome molecules have additional DNA sequences attached to the right-hand terminus, as shown by electron microscopy. Three such tails have been cloned molecularly; they differ in length and nucleotide sequence, and hybridization experiments demonstrate the cellular origin of two of the three tails. The evidence presented here is consistent with a proportion of the BHV-1 genomes recombining their DNA with cellular DNA during lytic infection.

Animals↗

PubMed1990

Genomic termini of equine herpesvirus 1.

After cell infection with the equine herpesvirus 1 (EHV-1), the termini of the linear double-stranded DNA genome fuse to form circular forms. To investigate the mechanisms in the generation and cleavage of such replicative-form DNAs, the genomic termini, the fusion of termini from replicative-form molecules, and the junction between the short and long genome segments have been analyzed by restriction mapping, blot hybridizations, cloning, and sequencing. The data suggest that the genome ends are not redundant and that the genomic termini are fused in replicative intermediates via 3' single-base extensions at the termini of the unique long segment (UL) and terminal repeat (TR). Adjacent to the EHV-1 termini are AT and gamma sequence elements highly conserved among different herpesviruses. We propose that both of these sequence elements are important for the cleavage of EHV-1 replicative forms.

Animals↗

PubMed1989

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver.

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.

Animals↗

PubMed1988

Replication of single-stranded porcine circovirus DNA by DNA polymerases alpha and delta.

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64-66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39-57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase alpha and delta as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase alpha and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase alpha holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789-4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase alpha is blocked with the DNA polymerase alpha specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase delta can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.

Animals↗

PubMed1988

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1).

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.

Amino Acid Sequence↗

PubMed1988

Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1).

Bovine herpesvirus 2 (BHV-2) specifies a glycoprotein of 130 kDa (gB BHV-2) which shows extensive homology to glycoprotein B (gB-1) of herpes simplex virus 1 (HSV-1). The BHV-2-specific 130-kDa glycoprotein is able to induce cross-reacting antibodies, some of which even cross-neutralize HSV-1. In order to determine the genome localization of gB BHV-2 and in order to identify conserved antigenic domains in both glycoproteins, we established libraries of subgenic fragments of BHV-2 and HSV-1 DNA in the prokaryotic expression vector lambda gt11 and screened them with cross-reacting monoclonal antibodies which allowed us to identify recombinant lambda gt11 clones expressing gB fusion protein. Nucleotide sequencing of inserted DNA fragments within these recombinant lambda gt11 clones revealed that they originated from the carboxy-terminal part of the major DNA-binding proteins (dbp) of BHV-2 (dbp BHV-2) and its counterpart ICP8 in HSV-1. Antisera raised against the beta-galactosidase fusion protein of recombinant phage lambda-113/2 coding for an 84 amino acid (aa) polypeptide originating from dbp BHV-2 neutralized infectivity of BHV-2 and HSV-1 in the presence of complement and precipitated [3H] glucosamine-labeled gB BHV-2 and gB-1. This antiserum also reacts with ICP8 and presumably with dbp BHV-2. Two hypotheses are discussed to explain this unexpected result: (i) epitopes in the carboxy-terminal part of gB BHV-2 and gB-1 are similar to antigenic determinants in the amino-terminal region of the gBs, thus providing cross-reacting antibody-binding sites; (iii) during gene expression a carboxy-terminal part of dbp BHV-2 and ICP8 genes might be spliced to the amino-terminal region of the glycoproteins gB BHV-2 and gB-1.

Amino Acid Sequence↗

PubMed1988

Molecular biological characterization of equine herpesvirus type 1 (EHV-1) isolates from ruminant hosts.

Isolates of equine herpesvirus type 1 (EHV-1) originating from affected antelope and cattle were compared with reference EHV-1 and EHV-4 isolates and were characterized. Based on cross-neutralization, DNA restriction profiles and blot-hybridization data these isolates could be characterized as EHV-1. One isolate (from an antelope) with a different restriction profile showed significant DNA homology with EHV-1, partial homology with EHV-4, and little or no homology with EHV-2 and HSV-1 DNAs. Blot hybridization revealed differences in DNA restriction fragments located at the termini of two isolates and size heterogeneity in the unique long/internal repeat junction fragment (UL/IR) of one isolate.

Animals↗

PubMed1988

Viral DNA from cells infected with porcine circovirus.

It has been demonstrated earlier that the genome of the porcine circovirus (PCV) isolated from virions is a circular single-stranded DNA (ssDNA). This report describes the isolation and characterization of PCV-specific DNA from infected cell cultures. Two types of double-stranded DNA (dsDNA) were observed which behaved like supercoiled and relaxed circular molecules, respectively. Both types of dsDNA displayed infectivity in transfection experiments and are regarded as replicative forms (RF) of the viral genome. In addition to complete single-stranded viral genomes and RF DNA molecules, PCV-specific subgenomic fractions of DNA molecules with a sedimentation coefficient of about 5 S were isolated from infected tissue culture cells. This 5 S DNA was also isolated from purified virions. It is single-stranded and represents a defined region of the viral genome.

Animals↗

PubMed1988

Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1.

The linear double-stranded DNA genome of herpesvirus as it is present in infectious virions needs to be circularized after infection of host cells and before DNA replication. Replicative-form genomes have to be cleaved into linear unit-length molecules during virion maturation and are most probably the substrate for inversion of the short segment relative to the long segment of the bovine herpesvirus 1 (BHV-1) genome. Those regions of the BHV-1 genome which are functionally involved in these processes have been analyzed at the molecular level by cloning and sequencing the genomic termini, the fusion of both termini from replicative-form molecules, and the junction between the short and the long genome segment. On the basis of the simple genome arrangement of BHV-1, it was inferable that the cleavage of replicative-form genomes by a hypothetical BHV-1 terminase activity may be specified by a sequence at the left end of UL (An element), which is located proximal to a reiterated beta element that makes up the cleavage site itself. The relationship of those elements in BHV-1 and the comparison to similar regions of other herpesviruses indicate consensus sequence elements which are functionally important for cleavage and isomerization of viral DNA during maturation of virions.

Animals↗

PubMed1987

Mammalian DNA polymerase alpha: a replication-competent holoenzyme form from calf thymus.

Calf thymus DNA polymerase alpha, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli, can be isolated as a distinct complex. A specific multiprotein form of the polymerase alpha, a form designated replication-competent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme alpha selectively initiates PCV DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.

Animals↗