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Biomedical subjects

E K Wagner

Publications and source records attributed to E K Wagner.

At least 73 records · Page 4Linked to original sources
PubMed1986

A single regulatory region modulates both cis activation and trans activation of the herpes simplex virus VP5 promoter in transient-expression assays in vivo.

A detailed analysis of the expression of the bacterial chloramphenicol acetyltransferase gene controlled by the herpes simplex virus major capsid protein (VP5) promoter showed that this promoter can be functionally separated into an 80-base core region, which has the minimal information required to serve as a pol II promoter but which is not fully activated by viral superinfection or by cotransfections with plasmids bearing functional alpha (immediate-early) genes, and an approximately 100-base regulatory region upstream of the core, which allowed full induction of VP5 promoter-driven chloramphenicol acetyltransferase activity but which repressed the ability of the VP5 core promoter to be cis activated by the simian virus 40 enhancer. This was in distinct contrast to the situation with the alkaline exonuclease promoter (a model early promoter) and defined the regions of this promoter which can be used to study the interaction between viral promoters and putative regulatory proteins induced by viral infection.

Acetyltransferases↗
PubMed1985

An unusual spliced herpes simplex virus type 1 transcript with sequence homology to Epstein-Barr virus DNA.

High-resolution transcription mapping localized a spliced 2.7-kilobase herpes simplex virus type 1 mRNA. The 4-kilobase intron of this transcript encodes a nested set of transcripts on the opposite DNA strand. The nucleotide sequence of the DNA encoding the left-hand and right-hand exons of the spliced transcript was determined, and the salient features are presented here. Of major interest is that both exons contained regions within several hundred bases of the splice donor and acceptor sites which showed homology to two regions of the Epstein-Barr virus genome, which are themselves 3 kilobases apart. The spliced herpes simplex virus transcript encoded a translational reading frame which could encode a protein with an approximate size of 75,000 daltons. This value is in agreement with in vitro translation data. The predicted amino acid sequence of the herpes simplex virus protein had significant homology with putative amino acid sequences encoded by the homologous Epstein-Barr virus DNA sequences.

Base Sequence↗
PubMed1985

Rescue of a herpes simplex virus type 1 neurovirulence function with a cloned DNA fragment.

A herpes simplex virus type 1 (HSV-1) genetic function that is required for viral replication in the murine central nervous system was unambiguously localized. Thus, cosmid clones of either HSV-1 HindIII fragment C (0.64 to 0.87 map units) or fragment B (0.64 to 0.83 plus 0.91 to 1.0 map units) were employed to restore neurovirulence to an intertypic recombinant (RE6) that is specifically deficient in this property. The neurovirulent recombinants were generated in cell culture by cotransfecting the clone fragments and unit-length RE6 DNA and then selected in mouse brains. Either fragment efficiently conferred neurovirulence to RE6, demonstrating that no short region unique sequences are required. Analyses of the genomic structures of the neurovirulent recombinants showed that, in every case, HSV-1 information from 0.71 to 0.83 map units was incorporated into the RE6 genome. Cleavage of HindIII fragment C with EcoRI eliminated its capacity to rescue RE6. Virulence could be restored by the addition of HSV-1 BamHI fragment L (0.71 to 0.74 map units) that spans an EcoRI site at 0.72 map units. The precise location of this HSV-1 neurovirulence function is discussed.

Animals↗
PubMed1985

Virus-induced modification of the host cell is required for expression of the bacterial chloramphenicol acetyltransferase gene controlled by a late herpes simplex virus promoter (VP5).

The requirements for expression of genes under the control of early (alkaline exonuclease) and late (VP5) herpes simplex virus type 1 (HSV-1) gene promoters were examined in a transient expression assay, using the bacterial chloramphenicol acetyltransferase gene as an expression marker. Both promoters were induced, resulting in the production of high levels of the enzyme upon low-multiplicity infection by HSV-1. S1 nuclease analysis of hybrids between RNA isolated from infected cells containing HSV-1 promoter constructs and marker gene DNA demonstrated normal transcriptional initiation of the marker gene directed by the viral promoters. Viral DNA sequences no more than 125 bases 5' of the putative transcriptional cap site were sufficient for maximum activity of the late promoter. In contrast to expression controlled by the early gene, the late promoter was not active at a measurable level in uninfected cells until DNA sequences between 75 and 125 bases 5' of the transcriptional cap site were deleted. Cotransfection of cells with the expression marker controlled by HSV promoters and a cosmid containing HSV alpha (immediate-early) genes indicated that full expression of both early and late promoters requires the same virus-induced host cell modifications. Inhibition of viral DNA synthesis results in an increased rate of transient expression of marker genes under control of either early or late promoters in contrast to the situation in normal virus infection. These data provide evidence that the normal course of expression of late HSV genes involves negative modulation of potentially active promoters in the infected cell.

Acetyltransferases↗
PubMed1984

Regulation of HSV transcription.

Herpes simplex virus (HSV) transcripts share many features with both cellular mRNA and the mRNAs expressed by other nuclear-replicating DNA viruses: they are capped, polyadenylated, and generally have an approximately 150 base leader between the cap and translation initiation codon. Further, certain sequence features around individual transcription units are shared: HSV promoters contain TATA and often CATC boxes, and the sequence signal indicating a polyadenylation site (AATAAAA) is standard. In spite of these similarities, there is one very distinct difference between the structure of HSV transcription units and those of other nuclear replicating DNA viruses. Each HSV transcript appears to be controlled by its own promoter and encodes a specific polypeptide. Further, the high degree of splicing seen with most eukaryotic and viral mRNAs is not seen in HSV. Certainly, HSV and other herpesviruses do express some spliced transcripts, but these are in the minority (with HSV at least). Thus a whole hierarchy of potential control points utilized in eukaryotic gene expression is missing or rarely utilized in HSV. Despite this, the high density of gene packaging and relatively complex arrangement of partially overlapping transcripts is seen in HSV transcription as it is in other, smaller DNA viruses.

Chromosome Mapping↗
PubMed1984

Molecular basis of the glycoprotein-C-negative phenotype of herpes simplex virus type 1 macroplaque strain.

The basis for the inability of the macroplaque (MP) strain of herpes simplex virus type 1 to express mature glycoprotein C (gC) was examined. RNA transfer (Northern) blot analysis with hybridization probes from the region of the herpes simplex virus type 1 DNA known to encode the gC gene indicated that gC mRNA was produced in MP-infected HeLa cells at levels relative to other mRNAs comparable with that seen in KOS-infected cells. Comparative nucleotide sequence analysis of the gC gene from the MP and KOS strains, coupled with the results of recently reported marker rescue experiments, indicates that the inability of MP to produce gC is due to a frameshift mutation in the gC-coding sequence. Because two different (out-of-phase) open reading frames overlap the gC-coding sequence in the region of the mutation, MP mRNA can encode two gC-related polypeptides. Two polypeptides of the predicted size and precipitable by anti-gC antibodies were produced by in vitro translation of MP mRNA. These polypeptides have not been detected in extracts from infected cells with the same antibodies. Comparative nucleotide sequence analyses led to several corrections in the published sequence for the gC gene and the 17,800-molecular-weight polypeptide gene just to the right in KOS DNA. These relatively minor effects on the predicted amino code sequence of gC are tabulated.

Amino Acid Sequence↗
PubMed1984

Herpes simplex virus types 1 and 2 homology in the region between 0.58 and 0.68 map units.

The homology between herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively) DNA between 0.58 and 0.674 map units was compared by Southern and dot blot analysis with DNA of one type of virus as a hybridization probe against the other type. Regions of high homology were interspersed with regions of detectably lower homology. However, only one region (between 0.647 and 0.653 map units) contained few or no homologous sequences. In situ RNA blot hybridization demonstrated that the mRNA species transcribed in the right-hand portion of the region are homologous between HSV-1 and HSV-2, as was previously found for the left-hand portion. A 2.7-kilobase HSV-2 transcript in the right-hand portion of the studied region was clearly that encoding HSV-2 glycoprotein C. Comparative nucleotide sequence analysis of specific regions demonstrated that homologous translational reading frames could be identified in the virus types. This analysis also demonstrated that homology could be abruptly lost outside such reading frames. Comparison of regions of homology with published HSV-1 transcription maps suggests that there can also be large divergence within translational reading frames. Some, but not complete, sequence homology was seen in the putative promoter sequence for the 730-base HSV-1 mRNA mapping to the right of glycoprotein C and the corresponding HSV-2 DNA. This suggests that the rather strict conservation of promoter sequences between homologous HSV-1 and HSV-2 transcripts seen in other regions of the genome may not be a necessary feature between these virus types.

Base Sequence↗
PubMed1983

Physical location of a herpes simplex virus type-1 gene function(s) specifically associated with a 10 million-fold increase in HSV neurovirulence.

In this paper we present a technique developed to physically locate the HSV-1 gene(s) which restore neurovirulence to a non-neurovirulent HSV intertypic recombinant described in the preceding report. In brief, tissue culture cells are co-transfected with unit length RE6 DNA and restriction endonuclease fragmented HSV-1 (strain 17 Syn+) DNA. In this way, random recombinations between RE6 and 17 Syn+ are produced. An in vivo enrichment in mouse brains is then employed to select recombinants which have incorporated the HSV-1 gene(s) associated with neurovirulence. In each of five cases where neurovirulent recombinants were isolated by this procedure, restriction enzyme and Southern DNA transfer analysis revealed that HSV-1 information from 0.71 to 0.83 map units had been incorporated into the RE6 genome. Confirmation of the role of this portion of the genome for HSV neurovirulence was obtained by similar cotransfection and in vivo rescue experiments performed with an electrophoretically purified HSV-1 DNA fragment which encompasses this region. Subsequent genome structure analysis of neurovirulent recombinants generated by this procedure revealed that only type-1 information from 0.71 to 0.83 map units had been incorporated into RE6. Thus an HSV-1 gene function(s) which resides in this region of the viral DNA is associated with a 10 million-fold increase in the neurovirulence of the virus. Potential applications of this in vivo selection technique are discussed.

Animals↗
PubMed1983

Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.

Base Sequence↗
PubMed1983

High-resolution characterization of herpes simplex virus type 1 transcripts encoding alkaline exonuclease and a 50,000-dalton protein tentatively identified as a capsid protein.

Four partially overlapping mRNAs (1.9, 2.3, 3.9, and 4.5 kilobases [kb]) were located between 0.16 and 0.19 map units on the herpes simplex virus type 1 genome. Their direction of transcription was found to be from right to left. The 2.3-kb mRNA was found to be early (beta), whereas the others were late (beta gamma). Partial sequence analysis of the DNA encoding these genes indicated that the promoter for the 2.3-kb mRNA shares structural features with other early (beta) promoters. In vitro translation of hybrid-selected mRNA indicated that among the proteins these mRNAs encode are an 82,000-dalton (d) polypeptide reactive with a monoclonal antibody against herpes simplex virus type 2 alkaline exonuclease and a 50,000-d polypeptide weakly reactive with a polyclonal antibody made against the capsid protein VP19C. Further experiments suggested that the 2.3-kb mRNA encodes the 82,000-d polypeptide, whereas one (or both) of the larger mRNAs encodes the 50,000-d protein. A novel finding was that the 1.9-kb mRNA appears to share part of the translational reading frame for alkaline exonuclease, but any polypeptide it encodes does not react with the monoclonal antibody to this enzyme.

Base Sequence↗
PubMed1982

Herpes simplex virus mRNA species mapping in EcoRI fragment I.

We described the detailed characterization and high-resolution mapping of nine herpes simplex virus type 1 mRNAs encoded in EcoRI fragment I. Four of these mRNAs are partially colinear and encode the same sized polypeptide in vitro. Nucleotide sequence analysis of the DNA around the 5' ends of these mRNAs suggested that the larger may encode a small (ca. 100-dalton) polypeptide not resolvable by in vitro translation.

Base Sequence↗
PubMed1982

Detailed characterization of an apparently unspliced beta herpes simplex virus type 1 gene mapping in the interior of another.

We precisely localized the coding region and determined the nucleotide sequence of a 1.2-kilobase beta herpes simplex virus type 1 mRNA which underlies the 3' region of the 5.2-kilobase beta mRNA mapping in HindIII fragment K. This mRNA, which lacks readily detectable splices, has its own promoter by the criteria of identification of putative herpes simplex virus type 1 control sequences and in vitro transcription by a Manley polymerase system.

Base Sequence↗
PubMed1981

Uninfected cell polymerase efficiently transcribes early but not late herpes simplex virus type 1 mRNA.

The sequences of the DNAs encoding the 5' ends of one early and one late herpes simplex virus type 1 mRNA were analyzed, and the 5' ends of these mRNA species were precisely located. Neither mRNA species is spliced and the noncoding strand of the DNA contains recognizable T-A-T-A and C-A-T boxes upstream from their respective 5' ends. The early mRNA was efficiently transcribed by a commercially available uninfected cell lysate system, but the late mRNA was not. This difference between early and late mRNAs appears to be general in this virus.

Base Sequence↗
PubMed1981

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.

Base Sequence↗
PubMed1981

Characterization of a major late herpes simplex virus type 1 mRNA.

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

Base Sequence↗
PubMed1981

Herpes simplex virus type 1 HindIII fragment L encodes spliced and complementary mRNA species.

We have used DNA bound to cellulose to isolate and translate in vitro herpes simplex virus type 1 (HSV-1) mRNA's encoded by HindIII fragment L (mapping between 0.592 and 0.647), and 8.450-base-pair (8.45-kb) portion of the long unique region of the viral genome. Readily detectable, late mRNA's 2.7 and 1.9 kb in size encoding 69,000- and 58,000-dalton polypeptides, respectively, were isolated. A very minor late mRNA family composed of two colinear forms, one 2.6 kb and one 2.8 kb, was isolated and found to encode only an 85,000-dalton polypeptide. A major early mRNA, 1.8 kb in size encoding a 64,000-dalton polypeptide, was also isolated. High-resolution mapping of these mRNA's by using S1 nuclease and exonuclease VII digestion of hybrids between them and 5' and 3' end-labeled DNA fragments from the region indicated that the major early mRNA contained no detectable splices, and about half of its 3' end was complementary to the 3' region of the very minor 2.6- to 2.8-kb mRNA's encoded on the opposite strand. These mRNA's also contained no detectable splices. The major late 2.7-kb mRNA was found to be a family made up of members with no detectable splices and members with variable-length (100 to 300 bases) segments spliced out very near (ca. 50 to 100 bases) the 5' end.

Base Sequence↗
PubMed1980

Isolation and translation of mRNA encoded by a specific region of the herpes simplex virus type 1 genome.

We have examined in detail the major mRNA species encoded by the region of the herpes simplex virus type 1 genome encoded by HindIII fragment K (0.53-0.59 from the left end of the prototype arrangement of the genome) by using this restriction fragment bound to cellulose as a reagent for isolation of this mRNA. Before viral DNA replication in infected cells (early), a major species of viral mRNA 5.2 kilobases (kb) in length is abundant. After the onset of viral DNA replication (late), four mRNA species are abundant: 7, 5.2, 3.8, and 1.8 kb in size. We have used reverse transcriptase from avian myeloblastosis virus to make DNA complementary to these RNA species and their 3' ends. We have shown by hybridization of this complementary DNA to Southern blots of herpes simplex virus type 1 DNA that the 7-, 5.2-, and 1.8-kb mRNA species have their 3' ends to the right of 0.59 and are at least partially colinear. The 3.8-kb mRNA has a 3' end mapping to the left of the 3' ends of these other species. In vitro translation of HindIII fragment K-specific mRNA in a reticulocyte lysate system yielded three major polypeptide products: 140,000, 122,000, and 54,000 daltons (d). Less prominent species of 86,000 and 65,000 d also were produced. Translation of size-fractionated HindIII fragment K-specific mRNA showed that the 7-, 5.2-, and 3.8-kb mRNA's encoded the 54,000-, 140,000-, and 122,000-d polypeptides, respectively. The 140,000-d polypeptide was the major polypeptide translated using early HindIII fragment K-specific mRNA as a template. The 3.8-kb mRNA also encoded the 86,000-d polypeptide, whereas the 1.8-kb mRNA encoded a polypeptide that was indistinguishable from the 54,000-d polypeptide encoded by the 7-kb mRNA, in addition to the 65,000-d polypeptide. The implications of the data are discussed.

DNA Restriction Enzymes↗