Determination of the Binding Site of Adenovirus E4 ORF3 and Ddx6
Abstract
Adenovirus is a double-stranded DNA virus that causes illnesses such as colds, respiratory infections, and pink eye. Its genome is approximately 36kb in size and contains around 30-40 genes. The gene of interest in this study is E4 ORF3, which is an early region protein necessary for viral protein synthesis and inhibition of host cell protein synthesis. E4 ORF3 binds with cellular protein Ddx6 in adenovirus serotype 5. Ddx6 is located in P-bodies, which regulate mRNA expression through degradation and inhibit translation of host cell proteins. E4 ORF3 redistributes Ddx6 from the P-body. Adenovirus serotype 9 does not bind to Ddx6, so to determine the binding site of Ad5 E4 ORF3 and Ddx6, Ad5 and Ad9 chimeras were made through overlap PCR. Hybrids were inserted into a bacterial expression plasmid and transformed into E. coli cells. After unsuccessful attempts to produce hybrids in the mammalian expression vector pcDNA3.1, Ad9/5 and Ad9/5 jr. were ordered from GenScript, which utilizes modern gene synthesis technology. We are currently trying to transfect the hybrids into HeLa cells in order to conduct co-immunoprecipitations with Ddx6 to determine the binding site for Ddx6 on E4 ORF3. Determining the binding site allows further study of the interaction of E4 ORF3 and Ddx6 and the process of P-body reorganization and viral infection. This information is relevant to other viruses and the functions of P-bodies.
Determination of the Binding Site of Adenovirus E4 ORF3 and Ddx6
Adenovirus is a double-stranded DNA virus that causes illnesses such as colds, respiratory infections, and pink eye. Its genome is approximately 36kb in size and contains around 30-40 genes. The gene of interest in this study is E4 ORF3, which is an early region protein necessary for viral protein synthesis and inhibition of host cell protein synthesis. E4 ORF3 binds with cellular protein Ddx6 in adenovirus serotype 5. Ddx6 is located in P-bodies, which regulate mRNA expression through degradation and inhibit translation of host cell proteins. E4 ORF3 redistributes Ddx6 from the P-body. Adenovirus serotype 9 does not bind to Ddx6, so to determine the binding site of Ad5 E4 ORF3 and Ddx6, Ad5 and Ad9 chimeras were made through overlap PCR. Hybrids were inserted into a bacterial expression plasmid and transformed into E. coli cells. After unsuccessful attempts to produce hybrids in the mammalian expression vector pcDNA3.1, Ad9/5 and Ad9/5 jr. were ordered from GenScript, which utilizes modern gene synthesis technology. We are currently trying to transfect the hybrids into HeLa cells in order to conduct co-immunoprecipitations with Ddx6 to determine the binding site for Ddx6 on E4 ORF3. Determining the binding site allows further study of the interaction of E4 ORF3 and Ddx6 and the process of P-body reorganization and viral infection. This information is relevant to other viruses and the functions of P-bodies.