A major goal of this proposal is to identify the function of the amino-terminal region of the Ad2 DNA binding protein (DBP). Mutants which enhance the ability of Ad2 to grow in African green monkey kidney cells by 1,000-fold have a single altered amino acid in their amino-terminal region. The phenotype of these host range mutants suggests that the amino-terminal domain of DBP functions in the processing of late adenovirus mRNA. Approximately 12 sites of phosphorylation occur in the amino-terminal domain near the host-range mutation site. DBP phosphorylation will be examined in productive and abortive Ad2 infections to determine if differential phosphorylation is responsible for the host range effect. Ad2 DBP mutants altered at or near these phosphorylation sites will be constructed, and these will be used to determine the function of phosphorylation at particular sites. Phosphorylation is the major postranslational mechanism for regulating protein function, but the regulatory pathways and controlling enzymes are not well understood. Inspection of the DNA sequence that encodes the amino terminus of Ad2 DBP has revealed an open reading frame (ORF) on the opposite strand that could encode a protein of 110,000 MW. Such a 110K protein would represent an extension of the late region 4 1000,000 MW nonstructural phosphoprotein (L4-100K) in the amino-terminal direction. Messenger RNA for a L4-110K protein has been detected in Ad2-infected HeLa cells. A second goal of this proposal is to determine if the L4-110K protein is essential for Ad2 reproduction. The postulated 110K protein sequence is altered in host range mutants; thus, the L4-110K gene will be examined with respect to a role in the regulation of Ad2 late gene expression and host range.
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| Lees, J A; Buchkovich, K J; Marshak, D R et al. (1991) The retinoblastoma protein is phosphorylated on multiple sites by human cdc2. EMBO J 10:4279-90 |
| Chen, Y R; Lees-Miller, S P; Tegtmeyer, P et al. (1991) The human DNA-activated protein kinase phosphorylates simian virus 40 T antigen at amino- and carboxy-terminal sites. J Virol 65:5131-40 |
| Lees-Miller, S P; Chen, Y R; Anderson, C W (1990) Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol 10:6472-81 |
