DETERMINGING THE BINDING SITE OF ADENOVIRUS E4 11K TO CELLULAR DDX6**
Adenovirus is a linear double-stranded DNA virus known to cause illnesses such as conjunctivitis and the common cold. The virus uses the host cell’s machinery as a means of its own genome replication and protein synthesis. Early genes often modulate this disruption of the host cell. Our protein of interest is the early viral protein E4 11k and is encoded in early region 4 open reading frame 3 (E4 ORF3) of the genome. E4 11k is known to play key roles in the inhibition of host cell protein synthesis, while facilitating the stimulation of viral protein synthesis. In serotype 5 of adenovirus (Ad5), E4 11k has been observed to localize with cellular processing bodies (P-bodies) and binds to the P-body protein, Ddx6. This interaction is thought to play a role in the reorganization of P-bodies to aggregates of misfolded proteins, termed aggresomes. Our goal is to determine the binding site between these two proteins and to observe the consequences of disrupting that binding on the viral lifecycle. To do this, Ad5 and Ad9 hybrids of the viral E4 ORF3 gene will be transfected into A549 cells, a human lung carcinoma cell line. Our current objective is to optimize our transfection methods. We have used a number of different transfection reagents and methods such as the use of lipofectamine, and various protocols using polyethylenimine (PEI) and calcium phosphate. Following the success of our transfection, we will perform a co-immunoprecipitation assay to narrow down the region of binding between the two proteins. Alanine amino acid substitutions will be used to further narrow the binding site. Once the binding site has been determined, the interaction between E4 11k and Ddx6 can be disrupted and the role E4 11k has on the re-organization of P-bodies and host cell protein shut off can be determined.
McGraw*, Kathryn E.; Moon*, Courtney F.; and Karen, Kasey A.
"DETERMINGING THE BINDING SITE OF ADENOVIRUS E4 11K TO CELLULAR DDX6**,"
Georgia Journal of Science, Vol. 76, No. 1, Article 56.
Available at: https://digitalcommons.gaacademy.org/gjs/vol76/iss1/56