Basic methods for the detection of PCR products.
Explore the source record for details and available documents.
Alleles↗
Biomedical subjects
F J Jenkins
Publications and source records attributed to F J Jenkins.
Retention of the herpes simplex virus type 1 (HSV-1) UL37 protein on single-stranded DNA columns requires the HSV-1 ICP8 protein.
The UL37 and ICP8 proteins present in herpes simplex virus type 1 (HSV-1)-infected-cell extracts produced at 24 h postinfection coeluted from single-stranded-DNA-cellulose columns. Experiments carried out with the UL37 protein expressed by a vaccinia virus recombinant (V37) revealed that the UL37 protein did not exhibit DNA-binding activity in the absence of other HSV proteins. Analysis of extracts derived from cells coinfected with V37 and an ICP8-expressing vaccinia virus recombinant (V8) and analysis of extracts prepared from cells infected with the HSV-1 ICP8 deletion mutants d21 and n10 revealed that the retention of the UL37 protein on single-stranded DNA columns required a DNA-binding-competent ICP8 protein.
Chromatography, Affinity↗
The herpes simplex virus UL37 protein is phosphorylated in infected cells.
The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1), nonstructural protein in infected cells. Recent studies in our laboratory have demonstrated that the UL37 protein interacts in the cytoplasm of infected cells with ICP8, the major HSV-1 DNA-binding protein. As a result of this interaction, the UL37 protein is transported to the nucleus and can be coeluted with ICP8 from single-stranded DNA columns. Pulse-labeling and pulse-chase studies of HSV-1-infected cells with [35S]methionine and 32Pi demonstrated that UL37 was a phosphoprotein which did not have a detectable rate of turnover. The protein was phosphorylated soon after translation and remained phosphorylated throughout the viral replicative cycle. UL37 protein expressed from a vaccinia virus recombinant was also phosphorylated during infection, suggesting that the UL37 protein was phosphorylated by a cellular kinase and that interaction with the ICP8 protein was not a prerequisite for UL37 phosphorylation.
Animals↗
Targets of herpes simplex virus type 1 infection in a mouse corneal model.
In animal models, spread of herpes simplex virus type 1 (HSV-1) from epithelial replication sites to the peripheral and central nervous system is known from analysis of individually dissected tissues. To examine virus spread in undissociated tissues, corneas of adult mice were inoculated with HSV-1. After 1 to 13 days groups of mice were perfused with formalin, and decalcified blocks of head and neck were embedded in paraffin. At intervals, serial sections were screened for HSV antigen. On days 1 and 2, viral antigen was restricted to cornea and conjunctiva but by days 3 and 4 was also seen in autonomic ganglia and the trigeminal system. On day 6, HSV antigen reached its maximum extent; infected sites included the trigeminal complex (ganglion, root, peripheral ophthalmic and maxillary branches and spinal nucleus and tract), ethmoid sinus and olfactory bulb, visual system, and autonomic ganglia (ciliary, pterygopalatine and superior cervical). Antigen progressively diminished on days 8 and 10, and was not detected on day 13. This method demonstrates a broader range of infected tissues and suggests a more complex pattern of HSV spread than has been previously recognized. Virus appears to reach the intracranial compartment by four different neural routes. When effects of higher and lower corneal inoculation doses were compared, a lower dose resulted in lower peak HSV titers in trigeminal ganglion and brain stem and later virus appearance in these tissues. Thus, dose may influence the kinetics of HSV spread from the peripheral inoculation site to the CNS.
Animals↗
Murine cytotoxic T lymphocytes specific for herpes simplex virus type 1 recognize the immediate early protein ICP4 but not ICP0.
Vaccinia virus recombinants expressing the herpes simplex virus type 1 (HSV-1) genes encoding ICP0 or ICP4 were used to identify the precise target antigen(s) of murine anti-viral cytotoxic T lymphocytes (CTL) specific for the non-structural immediate early proteins. These studies revealed that HSV-1-specific CTL, restricted to class I major histocompatibility complex genes of the H-2k haplotype but not the H-2d or H-2b haplotypes, would lyse autologous cells expressing ICP4. HSV-1-specific CTL derived from various mice strains failed to lyse target cells expressing ICP0. Calculation of the frequencies of H-2k-restricted virus-specific CTL demonstrated that approximately a third of the total HSV-1-specific response was directed against ICP4. Immunization of mice with either recombinant vaccinia virus or transfected L cells expressing ICP4 induced HSV-1-specific lymphoproliferation and delayed hypersensitivity but CTLs were not induced. More importantly, such immunized animals were unable to resist or control a subsequent challenge with virulent HSV-1.
Animals↗
Identification and characterization of the herpes simplex virus type 1 protein encoded by the UL37 open reading frame.
The UL37 open reading frame of the herpes simplex virus type 1 (HSV-1) DNA genome is located between map units 0.527 and 0.552. We have identified and characterized the UL37 protein product in HSV-1-infected cells. The presence of the UL37 protein was detected by using a polyclonal rabbit antiserum directed against an in vitro-translated product derived from an in vitro-transcribed UL37 mRNA. The UL37 open reading frame encodes for a protein with an apparent molecular mass of 120 kDa in HSV-1-infected cells; the protein's mass was assigned on the basis of its migration in sodium dodecyl sulfate-polyacrylamide gels. The UL37 protein is not present at detectable levels in purified HSV-1 virions, suggesting that it is not a structural protein. Analysis of time course experiments and experiments using DNA synthesis inhibitors demonstrated that the UL37 protein is expressed prior to the onset of viral DNA synthesis, reaching maximum levels late in infection, classifying it as a gamma 1 gene. Elution of HSV-1-infected cell proteins from single-stranded DNA agarose columns by using a linear KCl gradient demonstrated that the UL37 protein elutes from this matrix at a salt concentration similar to that observed for ICP8, the major HSV-1 DNA-binding protein. In addition, computer-assisted analysis revealed a potential ATP-binding domain in the predicted UL37 amino acid sequence. On the basis of the kinetics of appearance and DNA-binding properties, we hypothesize that UL37 represents a newly recognized HSV-1 DNA-binding protein that may be involved in late events in viral replication.
Animals↗
Role of the herpes simplex virus 1 internal repeat sequences in pathogenicity.
Three independently isolated herpes simplex virus type 1 recombinant viruses containing a deletion of approximately 14 kilobase pairs, representing greater than 95% of the internal repeat DNA sequences, were analyzed for their pathogenicity in mice. The recombinant viruses were found to be avirulent, exhibiting drastically increased LD50 values over wild-type herpes simplex virus 1(F) by intracerebral injection, nonneuroinvasive, unable to spread from the cornea to sensory ganglion, and unable to establish a reactivable latent infection in trigeminal ganglion following either intracerebral inoculation or inoculation of scarified corneas. The potential role of diploid genes in herpes simplex virus pathogenesis in the mouse is discussed.
Animals↗
Characterization and nucleotide sequence of two herpes simplex virus 1 genes whose products modulate alpha-trans-inducing factor-dependent activation of alpha genes.
Herpes simplex viruses encode a structural protein which induces, in trans, expression of alpha genes, the first set of genes to be expressed after infection of permissive cells. This protein, designated as the alpha-trans-inducing factor (alpha-TIF), maps within the BamHI F fragment, and its gene has been sequenced. In the course of mapping the domain of the alpha-TIF gene, it was noted that the intact BamHI fragment was consistently more effective than the complete domain of the alpha-TIF gene in inducing expression of alpha genes. Cotransfections of DNA fragments containing an alpha indicator gene and the alpha-TIF gene with various regions of the BamHI F DNA fragment revealed that the sequences located 3' to the alpha-TIF gene raised the activity of the alpha-TIF gene to nearly the same level as that of the intact BamHI F fragment. The nucleotide sequence and S1 nuclease mapping analyses revealed the presence of two transcribed open reading frames capable of encoding polypeptides with translated molecular weights of 77,357 and 70,527. To determine whether the effect of these sequences in trans on alpha-TIF-mediated induction of alpha genes was due to expression of these genes or competition for transcriptional factors, we constructed plasmids that contained both genes. Into each or both of these genes we inserted, near the translation initiation sites, 14-base-pair linkers carrying translational stop codons (TAG) in all three reading frames. Analyses of these plasmids indicated that the gene encoding the 70,527-molecular-weight polypeptide reduced alpha-TIF-dependent induction of alpha genes, whereas the gene encoding the 77,357-molecular-weight polypeptide increased this activity. Insertion of the stop codons abolished these activities.
Amino Acid Sequence↗
Site-specific mutagenesis of large DNA viral genomes.
Explore the source record for details and available documents.
DNA, Viral↗
Herpes simplex virus 1 recombinants with noninverting genomes frozen in different isomeric arrangements are capable of independent replication.
Herpes simplex virus 1 genome consists of two covalently linked components, L and S, that invert relative to each other to yield four equimolar isomeric populations designated P (prototype), Is (inversion of S component), Il (inversion of L component), and Ils (inversion of L and S components) differing in the orientation of the two components. Previous studies have yielded recombinant genomes frozen in the P isomeric arrangement, reinforcing suggestions that the four isomers may not be functionally equivalent. We report the isolation of recombinants produced by insertional mutagenesis with alpha TK mini-Mu that are frozen in Is and Ils arrangements. Thus, all isomeric forms of herpes simplex virus DNA appear to be capable of independent replication and must be considered as functionally equivalent.
Base Sequence↗
Transcription initiation sites and nucleotide sequence of a herpes simplex virus 1 gene conserved in the Epstein-Barr virus genome and reported to affect the transport of viral glycoproteins.
Earlier reports have localized mutations which affect the processing and transport of herpes simplex virus 1 glycoproteins to a region located between the genes specifying glycoprotein B and the major viral DNA-binding protein (beta 8). The nucleotide sequence of this region contains a single long open reading frame encoding a 780-amino-acid protein with a predicted molecular weight of 83,845. To confirm the existence of this protein, rabbit polyclonal antibody was made against a synthetic peptide made according to the predicted sequence of a hydrophilic domain near the carboxy terminal of the protein. This antibody reacted with an infected cell protein of an apparent molecular weight of 95,500. We designated this protein infected cell protein 18.5 (ICP18.5). S1 nuclease analysis suggested that the 5.6-kilobase mRNA encoding ICP18.5 is initiated predominantly from one site, but three weaker initiation sites also seemed to occur within a 74-base-pair stretch of DNA. This gene appears to be conserved in the Epstein-Barr virus (EBV) genome, inasmuch as 174 of the 780 amino acids of ICP18.5 align with corresponding amino acids predicted by the EBV open reading frame BALF3. The EBV gene is located adjacent to the gene specifying a homolog of the herpes simplex virus 1 glycoprotein B.
Amino Acid Sequence↗
Genetic engineering of novel genomes of large DNA viruses.
Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.
Base Sequence↗
Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome.
An earlier technique for insertional mutagenesis of large viral genomes involved the insertion of the thymidine kinase (TK) gene at a specific target site, cotransfection of the fragment carrying the insertion with the intact viral genome, and selection of the progeny for viral recombinants expressing the TK gene. The inserted TK gene could then be replaced by cotransfection of the recombinant DNA with fragments carrying a foreign sequence or a deletion in the target sequence. To enable the probing of larger target domains and facilitate insertional mutagenesis, we extended this technique by insertion of a 2.2-kilobase-pair (kbp) herpes simplex virus 1 (HSV-1) chimeric alpha TK gene into the 7.5-kbp mini-Mu-phage (alpha TK-mini-Mu) and lysogenized Escherichia coli with the helper Mu phage and the alpha TK-mini-Mu. Induction of phage multiplication of the lysogenized E. coli after transformation with plasmids carrying HSV-1 DNA and subsequent infection of E. coli RecA+ lysogenized with Mu phage yielded plasmid populations carrying randomly inserted alpha TK-mini-Mu DNA. Application of this procedure for insertional mutagenesis of the BamHI B fragment, which spans the junction between the unique and reiterated sequences of the L component of viral DNA, yielded two types of recombinants. Viral recombinant designated RBMu1 contained the intact alpha TK-mini-Mu inserted into unique sequences of BamHI B fragment. In recombinant RBMu2, the alpha TK-mini-Mu was inserted at or in the repeated sequences, but approximately equal to 14 kbp comprising most of the internal reiterations of the viral genome in the prototype arrangement were deleted.
Animals↗
Nucleotide sequence and predicted amino acid sequence of a protein encoded in a small herpes simplex virus DNA fragment capable of trans-inducing alpha genes.
The five alpha genes of herpes simplex virus 1 are the first set of genes to be expressed after infection. Previous studies have shown that alpha genes resident in eukaryotic cells are induced by infection with herpes simplex virus 1 or 2 but not by other herpesviruses and indicate that the alpha trans-inducing factor was a structural component of the virion. This factor induces genes linked to a bona fide promoter and containing at the 5' end a small sequence derived from the promoter-regulatory domains of alpha genes. We report the sequence of a small DNA fragment shown previously to be capable of expressing the alpha trans-inducing factor in transient expression systems. The only gene encoded in its entirety in this fragment is predicted to specify a 479 amino acid protein with a Mr of 53,053. The precise termini of the 1.74-kilobase mRNA specifying this protein were determined in our 5' and 3' S1 nuclease protection studies.
Amino Acid Sequence↗
Characterization of mRNAs that map in the BglII N fragment of the herpes simplex virus type 2 genome.
The BglII N DNA fragment (0.580 to 0.620 map units) of herpes simplex virus type 2 strain (333) is of interest because of its transforming potential. This fragment contains either partial or the complete coding sequences for nine mRNA transcripts that can be detected during a lytic infection. Subclones of the BglII N DNA fragment were generated in plasmid vectors, and the approximate locations of the mRNA transcripts were mapped by RNA blot hybridization technology. Precise 5' or 3' ends (or both) of these mRNA species were determined by S1 nuclease mapping, using the BglII N subclones as DNA probes. At least four mRNA transcripts are fully encoded in the BglII N fragment. The coding regions for all of the mRNA transcripts are densely packaged along the BglII N fragment with less than 150 base pairs between neighboring mRNA ends. Analysis of both neutral and alkaline gels failed to reveal the presence of any detectable introns. This manuscript reports a detailed transcription map for this region.
Animals↗
Simian virus 40 promoters direct expression of the tetracycline gene in plasmid pACYC184.
Insertion of HindIII DNA fragments into the HindIII site of plasmid pACYC184 destroys the promoter of the plasmid tetracycline resistance gene and causes Escherichia coli cells harboring recombinant plasmids to be tetracycline sensitive and chloramphenicol resistant. The HindIII-C DNA fragment of simian virus 40 contains the two virus promoters and the virus origin of replication. We report the isolation of recombinant plasmids that contained the simian virus 40 HindIII-C DNA fragment at the HindIII site but were capable of conferring tetracycline resistance to E. coli cells. The viral promoter sequences contained in the HindIII-C fragment presumably replaced the inactivated tetracycline resistance gene promoter sequences and enabled transcription of the tetracycline resistance gene.
DNA Restriction Enzymes↗
Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA.
RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.
Animals↗
Genital cancer and viruses.
Explore the source record for details and available documents.
Adolescent↗