Genetic analysis of the adenovirus DNA binding protein

The 72 kilodalton DNA protein (DBP) of human adenovirus is a multifunctional polypeptide involved in several aspects of vital replication. In order to better understand DBP, adenovirus mutants containing alterations in DBP were isolated and analyzed. Ad2ta400 is an adenovirus type 2 mutant containing three DBP mutations. Two of the mutations map to the amino-terminal domain of DBP and confer a cold-resistant host range phenotype which allows the virus to grow efficiently in monkey cells at 32.5?C. The third mutation maps in the carboxyl-terminal domain of DBP and causes a temperature-sensitive, DNA replication negative phenotype at 39.5?C in both human and monkey cells. At 39.5?C, Ad2ts400 is capable of enhancing viral growth in monkey cells, indicating that this activity of DBP is independent of the role of the protein in viral DNA replication. A genetic system was developed which allows the isolation of DBP mutants of any desired genotype. The mutations are first engineered into a cloned DBP gene using techniques of in vitro, site-directed mutagenesis. The mutated genes are then introduced into the viral chromosome by homologous recombination in vivo. Mutant virus are propagated by growth in human cell lines which express DBP and thus complement DBP-defective virus. The systems was used to construct five adenovirus type 5 DBP deletion mutants designated Ad5d1801 through Ad5d1805. All five mutants fare completely defective of growth in normal human cell lines. The mutant Ad5d1804 encodes a truncated protein which contains the entire amino-terminal domain of DBP including the histidine-to-tyrosine alteration at amino acid 130 which normally enhances viral growth in monkey cells. The truncated protein, however, failed to enhance late viral gene expression in monkey cells. The mutant Ad5d1802, which encodes no detectable DBP or DBP fragment, was used to study the role of DPB in early gene regulation of human cells. Contrary to previously published results, the absence of DBP function had no apparent effect on either process.