Biomedical subjects

J R Smiley

Publications and source records attributed to J R Smiley.

At least 55 records · Page 3Linked to original sources

PubMed1988

Regulated expression of stably transfected herpes simplex virus thymidine kinase genes in continuous cell lines expressing a temperature-sensitive mutant form of the immediate-early protein ICP4.

We stably transfected the herpes simplex virus type 1 thymidine kinase gene into a continuous cell line expressing a temperature-sensitive form of the viral immediate-early protein ICP4. In these cells, expression of the thymidine kinase gene was regulated in a temperature-sensitive manner, partially reproducing the controls that operate during a viral infection.

Gene Expression Regulation↗

PubMed1988

Abundant expression of herpes simplex virus glycoprotein gB using an adenovirus vector.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major component of infected cell membranes and virion envelopes. Glycoprotein B is known to be essential for entry of viruses into cells and may play important roles in virus-induced cell fusion and other alterations in cell morphology. In order to study the biochemical and immunological properties of gB in isolation from other HSV-1 polypeptides we have constructed human adenovirus vectors capable of expressing high levels of gB. The gB gene was coupled to the SV40 early promoter and inserted into the E3 region of two adenovirus vectors, one in which the E1 region was deleted (AdgB-1) and another which contained E1 sequences (AdgB-2). In AdgB-1 the orientation of the chimeric gB-SV40 gene was right to left, i.e., opposite to the direction of late and E3 mRNA transcription, whereas in AdgB-2 the orientation was left to right. Human 293 cells which express E1 functions supported replication of AdgB-1 and gB was expressed in these cells but not in mouse cells and only at very low levels in human cells other than 293. Replication of AdgB-2 was not limited to 293 cells and the virus was able to induce synthesis of gB at levels equal to or higher than those expressed in HSV-1-infected human or mouse cells. Microscopic examination of AdgB-2-infected cells revealed extensive vacuolization in a manner completely uncharacteristic of adenovirus-infected cells, and fluorescent antibody staining indicated that gB was not only present at the cell surface but also concentrated in the cytoplasmic vacuoles.

Adenoviridae↗

PubMed1987

Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts.

We constructed nondefective herpes simplex virus type 1 recombinants bearing the intact rabbit beta-globin gene inserted into the viral gene for thymidine kinase to study the expression of a cellular gene when it is present in the viral genome during lytic viral infections. The globin promoter was activated to high levels during productive infection of Vero cells, giving rise to properly spliced and processed cytoplasmic globin transcripts. Expression of globin RNA occurred with early kinetics, was not affected by blocking viral DNA replication, and was strongly inhibited by preventing viral immediate-early protein synthesis with cycloheximide. These results support the hypothesis that temporal control of herpes simplex virus early gene expression is accomplished by mechanisms that are not restricted to viral promoters. In addition, these data show that a cellular transcript can be correctly processed and can accumulate to high levels during viral infection; this indicates that the mechanisms of virally induced shutoff of host RNA accumulation and degradation of host mRNAs do not depend on sequence-specific differentiation between host and viral RNAs. These findings also suggest that herpesviruses have considerable potential as high-capacity gene transfer vectors for a variety of applications.

Animals↗

PubMed1987

Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes.

To determine which viral molecule(s) is recognized by herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL), target cells were constructed which express individual HSV glycoproteins. A mouse L cell line, Z4/6, which constitutively expressed high levels of HSV type 2 (HSV-2) gD (gD-2) was isolated and characterized previously (D. C. Johnson and J. R. Smiley, J. Virol. 54:682-689, 1985). Despite the expression of gD on the surface of Z4/6 cells, these cells were not killed by anti-HSV-2 CTL generated following intravaginal infection of syngeneic mice. In contrast, parental Z4 or Z4/6 cells infected with HSV-2 were lysed. Furthermore, unlabeled Z4/6 cells were unable to block the lysis of HSV-2-infected labeled target cells. Cells which express HSV-1 gB (gB-1) were isolated by transfecting L cells with the recombinant plasmid pSV2gBneo, which contains the HSV-1 gB structural sequences and the neomycin resistance gene coupled to the simian virus 40 early promoter and selecting G418-resistant cell lines. One such cell line, Lta/gB15, expressed gB which was detected by immunoprecipitation and at the cell surface by immunofluorescence. Additionally, cells expressing HSV-1 gC (gC-1) or gE (gE-1) were isolated by transfecting Z4 cells, which are L cells expressing ICP4 and ICP47, with either the recombinant plasmid pGE15neo, which contains the gE structural sequences and the neomycin resistance gene, or pDC17, which contains the gC structural gene coupled to the gD-1 promoter. A number of G418-resistant cell lines were isolated which expressed gC-1 or gE-1 at the cell surface. Anti-HSV-1 CTL generated following footpad infection of syngeneic mice were unable to lyse target cells expressing gB-1 or gE-1. In contrast, target cells expressing very low levels of gC-1 were killed as well as HSV-1-infected target cells. Furthermore, infection of gC-1-transformed target cells with wild-type HSV-1 or a strain of HSV-1 that does not express gC did not result in a marked increase in susceptibility to lysis. These results suggest that murine class I major histocompatibility complex-restricted anti-HSV CTL recognize gC-1 but do not recognize gB, gD, or gE as these molecules are expressed in transfected syngeneic target cells. The results are discussed in terms of recent evidence concerning the specificity of antiviral CTL.

Animals↗

PubMed1985

Activation and inhibition of expression of the 72,000-Da early protein of adenovirus type 5 in mouse cells constitutively expressing an immediate early protein of herpes simplex virus type 1.

It has been previously reported that immediate early proteins of pseudorabies and cytomegalo viruses can substitute for the products of the human adenovirus type 5 (Ad5) E1A gene in the activation of early Ad5 transcription. In the present report the effect of one of the herpes simplex virus type 1 (HSV-1) immediate early genes, ICP4, on Ad5 early gene expression has been examined using mouse cell lines that constitutively express ICP4. These lines as well as nonproducers were infected with wild-type (wt) Ad5 or with various Ad5 E1A mutants and the levels of expression of the Ad5 E2A 72K DNA binding protein were measured by immunoprecipitation with a monoclonal antibody specific for 72K. With dl 312, which lacks E1A, some 72K expression was seen in nonproducer lines but levels were considerably higher in the producer lines. A similar result was also obtained using dl 312-infected nonproducer cells that were superinfected with HSV-1 virions. These data suggest that HSV-1 ICP4 can substitute for E1A in the activation of expression of early Ad5 proteins. With wt Ad5, 72K was also expressed at high levels in nonproducer mouse cells, however, in the ICP4 producer cell lines, a marked inhibition of 72K expression was observed and this inhibition correlated with the amount of ICP4 present. Using the E1A mutants pm 975 and hr 1, this inhibition was found to be specific for the products of the 1.1-kb E1A mRNA. These data suggest that ICP4 and E1A proteins either directly inhibit each other, or more likely, operate independently and competitively on factors required for viral gene activation.

Adenovirus Early Proteins↗

PubMed1985

Signals for site-specific cleavage of HSV DNA: maturation involves two separate cleavage events at sites distal to the recognition sequences.

Mature Herpes Simplex Virus (HSV) genomes are cleaved from concatemeric precursors by a site-specific mechanism. These cleavage events are probably coupled to the encapsidation process. Sequences within the terminal repeat of HSV DNA are necessary for the cleavage and packaging reactions, and are also thought to be responsible for high frequency genome isomerization events. Here we present evidence to show that two viral DNA cleavage and packaging signals reside within a 250 bp subfragment of the terminal repeat, that the termini of mature viral DNA are generated by a process involving two separate DNA cleavages at sites distal to the cleavage signals, and that the sequences between these two cleavage sites are duplicated by the DNA maturation system.

Base Sequence↗

PubMed1985

Cells that constitutively express the herpes simplex virus immediate-early protein ICP4 allow efficient activation of viral delayed-early genes in trans.

To study the role of herpes simplex virus type 1 immediate-early proteins in the transcriptional activation of herpes simplex virus genes, we isolated stably transformed cells expressing herpes simplex virus type 1 ICP4, an immediate-early protein known from previous studies to be necessary for delayed-early and late transcription. These cells efficiently expressed six delayed-early herpes simplex virus genes introduced by viral superinfection, in the absence of de novo viral protein synthesis. In contrast, the delayed-early gene encoding alkaline exonuclease and the late gene encoding the capsid protein VP5 were expressed at much lower levels. Expression of a second late gene, that for glycoprotein C, was undetectable under the same experimental conditions. These results suggest that many, but not all, delayed-early genes are efficiently activated by ICP4; in addition, they demonstrate that although the late gene for VP5 is detectably activated by ICP4, its full expression requires additional factors.

Animals↗

PubMed1985

Intracellular transport of herpes simplex virus gD occurs more rapidly in uninfected cells than in infected cells.

A mouse L cell line which expresses the herpex simplex virus type 1 immediate-early polypeptides ICP4 and ICP47 was cotransfected with a cloned copy of the BglII L fragment of herpes simplex virus type 2, which includes the gene for gD, and the plasmid pSV2neo, which contains the aminoglycosyl 3'-phosphotransferase (agpt) gene conferring resistance to the antibiotic G418. A G418-resistant transformed cell line was isolated which expressed herpes simplex virus type 2 gD at higher levels than were found in infected cells. The intracellular transport and processing of gD was compared in transformed and infected cells. In the transformed Z4/6 cells gD was rapidly processed and transported to the cell surface; in contrast, the processing and cell surface appearance of gD in infected parental Z4 cells occurred at a much slower rate, and gD accumulated in nuclear membrane to a greater extent. Thus, the movement of HSV-2 gD to the cell surface in infected cells is retarded as viral glycoproteins accumulate in the nuclear envelope, probably because they interact with other viral structural components.

Animals↗

PubMed1985

Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

Cell Line↗

PubMed1984

Unstable heterozygosity in a diploid region of herpes simplex virus DNA.

We have examined the behavior of a herpes simplex virus strain KOS isolate in which the two inverted repeats flanking the short segment of viral DNA differ in length by approximately 60 base pairs. We find that individual viral DNA molecules exist which contain the two distinguishable repeats, demonstrating that heterology between the repeats is tolerated. However, viruses heterozygous for the two different repeats are unstable, segregating both classes of homozygotes at a high frequency. We propose that this segregation is a consequence of the high-frequency recombination events which also result in genome segment inversion.

Animals↗

PubMed1984

Effects of deletions on expression of the herpes simplex virus thymidine kinase gene from the intact viral genome: the amino terminus of the enzyme is dispensable for catalytic activity.

We have transferred two deletions affecting the 5' end of the herpes simplex virus thymidine kinase (TK) gene into the intact viral genome. One, extending from -12 to +189, had no effect on TK mRNA synthesis and only a small effect on TK activity, although the first 27 codons of the TK polypeptide were deleted. The other, extending from -85 to +85, severely impaired TK mRNA synthesis. We conclude that the amino terminus of the TK polypeptide is dispensable for catalytic activity, and that expression of TK in viral infections requires some of the same promoter elements used in uninfected cells.

Animals↗

PubMed1984

Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2.

Herpes simplex virus type 2 is a common human venereal pathogen which causes lethal neurological illness after intravaginal inoculation into BALB/cJ mice. To investigate whether an attenuated, nonlethal strain of this virus would confer immunity after inoculation of mice, we constructed a strain containing a partial deletion of the thymidine kinase gene, which is necessary for viral replication and spread in sensory ganglia. Unlike its wild-type counterpart, this deletion-containing strain of herpes simplex virus type 2 caused mild clinical disease and was not lethal when studied in an age-dependent murine model of intravaginal infection. Furthermore, after intravaginal infection, the deletion-containing strain could not be isolated from sensory ganglia at a time when wild-type virus was abundant. Of greater significance, intravaginal inoculation with the deletion-containing strain rendered mice completely resistant to rechallenge with a 10-fold 50% lethal dose of wild-type virus. These results suggest that a strain of herpes simplex virus type 2 containing a deletion of the thymidine kinase gene will be useful in studying the cellular basis of mucosal immunity in the genital tract.

Animals↗

PubMed1984

Transactivation of a late herpes simplex virus promoter.

We have asked whether the promoter for the gene encoding the major capsid protein (VP5) of herpes simplex virus functions in uninfected mouse cells. Our experimental strategy was to first fuse the VP5 promoter to the herpes simplex virus thymidine kinase (TK) structural sequence and then to use the resulting hybrid gene to transform TK- cells to TK+. The recombinant gene transferred TK at an extremely low frequency by comparison with the wild-type TK gene, and the TK transcripts present within the resulting rare transformants initiated within the TK structural gene, rather than in the vicinity of the VP5 promoter. However, after infection with herpes simplex virus, large amounts of RNA driven from the VP5 promoter accumulated. We conclude that the VP5 promoter does not function in uninfected cells but is efficiently activated by virally coded factors, most likely one or more immediate-early proteins.

Animals↗

PubMed1983

Positive control of the herpes simplex virus thymidine kinase gene requires upstream DNA sequences.

We examined the ability of deleted versions of the herpes simplex virus type 1 thymidine kinase gene, present in biochemically transformed mouse cells, to be induced in trans to a higher level of expression by superinfecting herpes simplex virus immediate early gene products. The results demonstrate that sequences mapping between -200 and -80 and between -70 and -12 are required for induction. As these regions are largely coincident with the previously identified thymidine kinase gene promoter, the results suggest that herpes simplex virus immediate early gene products or their metabolic product activate thymidine kinase expression by acting at the promoter region to increase the rate of transcription.

Base Sequence↗

PubMed1982