Narrowing Down the Respective Binding Sites of Adenovirus E4orf3 and Cellular Ddx6 Proteins
Abstract
Adenovirus is often associated with localized infections in areas such as the respiratory or intestinal tract. It is a double-stranded DNA virus with a 36kb genome encoding 30-40 genes. The life cycle begins with the viral genome entering the cell and activating early gene transcription. E4orf3 is an early gene that activates the synthesis of late viral proteins and shuts down host cell protein synthesis. Ddx6 is a cellular protein present in cytoplasmic processing bodies (P-bodies), which are responsible for regulating gene expression by degrading mRNAs and repressing host cell translation. Ddx6 is known to bind to the Ad5 E4orf3 protein, but not the Ad9 E4orf3 protein. Chimeras were created using different combinations of both the Ad9 and Ad5 E4orf3 genes in order to narrow down the binding site of Ad5 E4orf3 to Ddx6. Locating this binding site will be accomplished by transfecting the viral DNA vectors into the cells and doing co-immunoprecipitations with the Ad5/Ad9 chimeric E4orf3 and Ddx6 proteins. Alternatively, co-immunoprecipitations with Ad5 E4orf3 and deletion mutants of Ddx6 will be performed to narrow down the binding site in Ddx6 for E4orf3. Together, these methods will facilitate determination of the binding sites of E4orf3 and Ddx6. Since adenovirus is a model for both virus and cellular biology, knowledge of these interactions could elucidate additional functions of the cellular P-bodies as well as be applicable to other viruses.
Session Name:
Poster Presentation Session #2 - Poster #42
Start Date
10-4-2015 12:15 PM
End Date
10-4-2015 1:00 PM
Location
HSB 3rd Floor Student Commons
Narrowing Down the Respective Binding Sites of Adenovirus E4orf3 and Cellular Ddx6 Proteins
HSB 3rd Floor Student Commons
Adenovirus is often associated with localized infections in areas such as the respiratory or intestinal tract. It is a double-stranded DNA virus with a 36kb genome encoding 30-40 genes. The life cycle begins with the viral genome entering the cell and activating early gene transcription. E4orf3 is an early gene that activates the synthesis of late viral proteins and shuts down host cell protein synthesis. Ddx6 is a cellular protein present in cytoplasmic processing bodies (P-bodies), which are responsible for regulating gene expression by degrading mRNAs and repressing host cell translation. Ddx6 is known to bind to the Ad5 E4orf3 protein, but not the Ad9 E4orf3 protein. Chimeras were created using different combinations of both the Ad9 and Ad5 E4orf3 genes in order to narrow down the binding site of Ad5 E4orf3 to Ddx6. Locating this binding site will be accomplished by transfecting the viral DNA vectors into the cells and doing co-immunoprecipitations with the Ad5/Ad9 chimeric E4orf3 and Ddx6 proteins. Alternatively, co-immunoprecipitations with Ad5 E4orf3 and deletion mutants of Ddx6 will be performed to narrow down the binding site in Ddx6 for E4orf3. Together, these methods will facilitate determination of the binding sites of E4orf3 and Ddx6. Since adenovirus is a model for both virus and cellular biology, knowledge of these interactions could elucidate additional functions of the cellular P-bodies as well as be applicable to other viruses.