Narrowing Down the Binding Site of Adenovirus E4 11k on the Cellular Protein Ddx6

Adenovirus is a double-stranded DNA virus that can cause self-limiting localized infections. Upon entry into a cell, a virus expresses proteins that interact with various cellular pathways allowing the virus to control the cell. This results in the inactivation of many pathways, including repair and mRNA regulation mechanisms. The viral gene, E4ORF3, functions in turning on late viral expression and turning off host cell synthesis. The protein encoded by E4ORF3 is E4 11k, which interacts with the P-body protein, Ddx6. We hypothesize that the binding of E4 11k to Ddx6 plays a critical role in the disruption of late gene expression. We aim to identify the binding site on Ddx6 for the E4 11k protein. Ddx6 deletion mutants will be used in order to remove the possible binding sites of E4 11k. Then a co-immunoprecipitation will be performed in order to determine the specific binding site of E4 11k on Ddx6. The E4 11k protein will only be co-immunoprecipitated when the Ddx6 mutant contains the binding site. Ddx6 mutants with alanine substitutions in the region containing the binding site will then be created and additional co-immunoprecipitation assays will be performed. Once the binding site is narrowed down to a single amino acid or sequence of amino acids, the function of E4 11k regarding late gene expression during adenovirus infection can be identified in human cells. Additionally, P-bodies are not fully understood, but several viruses have been shown to disrupt them, including HIV and HCV. These studies could contribute to a better understanding of their role in normal cells as well as during viral infections