Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro.

Horwitz, Marshall S.

doi:10.1073/pnas.75.9.4291

Cited by 71 publications

(28 citation statements)

References 34 publications

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“…Several mutants with temperature sensitive (tsj alterations in the DBP have been indispensable in defining several of the functions of this protein. The ts alterations at the nonpermissive temperature diminish the single-strand, DNA-binding activity of the protein in vitro (52) and inhibit DNA replication in vivo (13,47,53) and in vitro (20). Normal turn-off of viral early genes during the late phase of the lytic cycle is also disrupted at the nonpermissive temperature in cells infected with the ts mutant (6,9,10,37,38).…”

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confidence: 99%

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Klessig¹,

Brough²,

Cleghon³

1984

Mol. Cell. Biol.

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The DNA-bindihg protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central iole in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by IBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DB3P is totic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50-to 200-fold, and within 8 h its synthesis fromb the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.A number of multifunctional proteins in euearyotes have been defined by,a combination of genetic and biochemical studies. One of the best characterized of these is the simian virus 40 (SV40) large T antigen, which exhibits separate functional domains as discerned by mapping of sets of mutations which alter one but not another of the various protein functions.The human adenovirus 72-kilodalton DNA-binding protein (DBP) also plays several independent roles during the infectious cycle of this virus. Several mutants with temperature sensitive (tsj alterations in the DBP have been indispensable in defining several of the functions of this protein. The ts alterations at the nonpermissive temperature diminish the single-strand, DNA-binding activity of the protein in vitro (52) and inhibit DNA replication in vivo (13, 47, 53) and in vitro (20). Normal turn-off of viral early genes during the late phase of the lytic cycle is also disrupted at the nonpermissive temperature in cells infected with the ts mutant (6,9,10,37,38). The DBP directly affects early gene turn-off as well as DNA replication, since inhibition of viral DNA synthesis with either drugs or ts lesions in other viral early genes does not affect early gene expression (9, 10). In addition, at the nonpermissive temperatui*, the ts mutants transform rat cells in culture with an enhanced frequency compared with wild-type (wt) virus (16,54).A second class of mutants which greatly enhance the ability of this human virus to grow in monkey cells defines yet a second set of functions of DBP (1,26,29). In monkey cells infected with wt human adenovirus, the viral early genes are properly expressed (3), and viral DNA replication occurs no...

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Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Klessig¹,

Brough²,

Cleghon³

1984

Mol. Cell. Biol.

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“…The growth of HeLa S3 cells in suspension cultures, infection with Ad type 2 (Ad2) at an input multiplicity of 4,000 virions per cell, and preparation of deproteinized Ad2 DNA have been previously described (12,28).…”

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“…The standard Ad2 DNA synthesis reaction previously described (13) 12,000 x g for S min, were washed with ether and suspended in electrophoresis buffer containing 2% sodium dodecyl sulfate (SDS) and 1% mercaptoethanol. After the samples were boiled for 5 min and then cooled to room temperature, they were electrophoresed on a 12.5% polyacrylamide gel with 0.1% SDS until the dye front reached the bottom of the 10-cm gel (26).…”

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confidence: 99%

Inhibition of Adenovirus DNA Synthesis In Vitro by Sera from Patients with Systemic Lupus Erythematosus

Horwitz¹,

Friefeld²,

Keiser³

1982

Mol. Cell. Biol.

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Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60°C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%6. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-doublestranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases a, (, y and had no antibody to the 72,000-dalton DNAbinding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.Sera from patients with systemic lupus erythematosus (SLE) and other connective tissue diseases contain antibodies to a wide variety of nuclear and cytoplasmic macromolecules, including single-stranded (ss) and double-stranded (ds) DNA, RNA, nucleohistones, and ribonucleoprotein (RNP) particles (29). These antibodies may distinguish between different disease entities or prognostic subclasses and, in the case of antibodies to dsDNA, provide a means for monitoring the level of SLE disease activity (23,29,35). Although the antigens recognized by these antibodies play critical roles in intracellular processes, the pathogenicity of antibodies to these macromolecules is unknown.To evaluate the possibility that the various antinuclear antibodies may alter cellular functions, we studied the effects of such antibodies on the replication of adenovirus (Ad) DNA in vitro by subcellular soluble fractions. In this model system, Ad DNA synthesis is initiated on template dsDNA which contains a 55,000-dalton

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“…This protein is known to be coded for by sequences in a limited region (0.62-0.68 map unit) of the genome [2-41. It is required both for chain initiation [ S ] and elongation [6,7] during adenovirus D N A replication. In addition, it has been identified as a component of DNA replication complexes [8-121. A temperature-sensitive mutant of Ads, ts125, harbors a mutation in region Ez of the genome [13], and is deficient in viral D N A replication at elevated tempcratures.…”

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Protein Kinases Associated with the Adenovirus Single‐Stranded DNA‐Binding Protein

Cajean-Feroldi

Loeb

Meguenni

et al. 1981

European Journal of Biochemistry

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Protein kinase activities copurifying with the 7200‐MrDNA‐binding protein of adenovirus on DNA‐cellulose chromatography and gel filtration in acrylamide/agarose have been partially characterized and purified. One of these kinases was found to phosphorylate efficiently the viral DNA‐binding protein in vitro and to be stimulated severalfold by the addition of histones, protamine, or polyamines. The kinase does not, however, phosphorylate histones, protamine, casein, or phosvitin. A second protein kinase was also recovered from single‐stranded DNA‐cellulose which is able to phosphorylate the 72000‐Mr DNA‐binding protein, but which is inhibited by the addition of histones. Phosphorylation in vitro of the 72000‐Mr DNA‐binding protein from the ts125 mutants of adenovirus by the histone‐stimulated protein kinase was found to be thermosensitive.

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Temperature-sensitive replication of H5ts125 adenovirus DNA in vitro.

Cited by 71 publications

References 34 publications

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Inhibition of Adenovirus DNA Synthesis In Vitro by Sera from Patients with Systemic Lupus Erythematosus

Protein Kinases Associated with the Adenovirus Single‐Stranded DNA‐Binding Protein

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