Determination of the Binding Site of Adenovirus E4ORF3 and Ddx6
Abstract
Adenovirus is a double stranded DNA virus known to cause illnesses such as respiratory infections and conjunctivitis. Like many other viruses, adenovirus hijacks the host cell’s machinery in order to synthesize viral proteins. Our gene of interest, E4ORF3, codes for an early viral protein known to disrupt host cell protein synthesis. E4ORF3 has the capability of binding and disrupting the function of a cellular processing body (P-body) protein, Ddx6. The binding of these two proteins can only be seen in serotype Ad5 in which cellular P-body proteins are relocalized to aggresomes. A similar serotype, Ad9, does not show this binding capability although the P-bodies are disrupted. In order to determine the significance of the role E4ORF3 plays in Ad5 viral infections, the binding site of these two proteins must be determined and then disrupted. To do this, various hybrids of Ad5 and Ad9 were created by using modern gene synthesis technology. After successfully transfecting these hybrids into HeLa cells, a co-immunoprecipitation assay will be used to confirm which hybrid successfully binds to Ddx6. These binding mutants will eventually help us understand how the interaction of E4ORF3 and Ddx6 contributes to late viral protein synthesis and P-body reorganization.
Determination of the Binding Site of Adenovirus E4ORF3 and Ddx6
Adenovirus is a double stranded DNA virus known to cause illnesses such as respiratory infections and conjunctivitis. Like many other viruses, adenovirus hijacks the host cell’s machinery in order to synthesize viral proteins. Our gene of interest, E4ORF3, codes for an early viral protein known to disrupt host cell protein synthesis. E4ORF3 has the capability of binding and disrupting the function of a cellular processing body (P-body) protein, Ddx6. The binding of these two proteins can only be seen in serotype Ad5 in which cellular P-body proteins are relocalized to aggresomes. A similar serotype, Ad9, does not show this binding capability although the P-bodies are disrupted. In order to determine the significance of the role E4ORF3 plays in Ad5 viral infections, the binding site of these two proteins must be determined and then disrupted. To do this, various hybrids of Ad5 and Ad9 were created by using modern gene synthesis technology. After successfully transfecting these hybrids into HeLa cells, a co-immunoprecipitation assay will be used to confirm which hybrid successfully binds to Ddx6. These binding mutants will eventually help us understand how the interaction of E4ORF3 and Ddx6 contributes to late viral protein synthesis and P-body reorganization.