Construction, characterization, and utilization of cell lines which inducibly express the adenovirus DNA-binding protein.
To further our understanding of structure-function relationships within the multifunctional adenovirus DNA binding protein (DBP) a more diverse collection of mutants is necessary. DBP-expressing cell lines (gmDBP) were previously constructed that complemented DBP-negative mutants for viral growth. However, they did not allow severely defective viruses to form plaques. Since efficient mutant construction is reliant on plaque isolation of the desired mutant virus as a final step, additional gmDBP cell lines were constructed which allow all DBP-negative mutants to form plaques. Here we describe the construction and characterization of 12 new gmDBP cell lines. The utility of these lines was demonstrated by the efficient construction of a new defective mutant, H5in804, using a combination of DBP-expressing lines. The H5in804 mutation adds 22 amino acids at the carboxyl end of an otherwise wild type protein. Characterization of H5in804 revealed that it was altered in its ability to replicate viral DNA. The depression of DNA synthesis most probably results from a reduced ability of H5in804 DBP to bind ssDNA.
Brough
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Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855.
Cleghon
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Klessig
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D017502:Adenovirus E2 Proteins; D003001:Cloning, Molecular; D004261:DNA Replication / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics*
D017502:Adenovirus E2 Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003907:Dexamethasone