Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.
The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed.
Morin
N
N
Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855-0759.
Delsert
C
C
Klessig
D F
DF
D000260:Adenoviruses, Human / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D010948:Viral Plaque Assay; D015678:Viral Structural Proteins / Q000235:genetics
D004268:DNA-Binding Proteins; D015678:Viral Structural Proteins