Adenovirus 5 early region 1A host range mutants hr3, hr4, and hr5 contain point mutations which generate single amino acid substitutions

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Abstract
The early region 1A (E1A) gene of adenovirus 5 encodes two proteins, 289AA and 243AA, which are translated from mRNAs of 13S and 12S, respectively. These two E1A proteins are identical except for an internal stretch of 46 amino acids unique to the larger protein. The 289AA protein activates transcription from promoters of other early adenoviral genes. The adenovirus type 5 host range mutants hr3, hr4, and hr5 are unable to activate transcription of these early viral genes. We show here that hr3, hr4, and hr5 each contain a distinct missense mutation in the E1A gene. We first localized the mutations in a series of constructed wild-type-hr hybrid E1A genes by using a biological assay which can discriminate between functional and nonfunctional E1A proteins. We then identified the mutations by DNA sequencing. In hr3 lysine replaced methionine at position 176, and in hr4 phenylalanine replaced leucine at position 173; both substitutions occurred in the region unique to the 289AA protein. In hr5, due to the splicing patterns of the two mRNAs, asparagine replaced serine as the last amino acid in the unique region of the 289AA protein at position 185, while aspartic acid replaced glycine at position 139 in the 243AA protein, which is the last amino acid common to both proteins before the unique region. These results substantiate the role of the 289AA protein in transcriptional activation and underscore the importance of the unique region as the basis of the functional difference between the two E1A proteins. Implications as to how these mutations affect the structure and function of the E1A proteins in transcriptional activation and transformation are discussed.