41 - Finding The Binding Site of Adenovirus E4 11K and P-body Component DDX6

Abstract

Adenovirus (Ad) is a non-enveloped virus with an icosahedral capsid and a linear double-stranded DNA genome. Adenovirus typically infects vertebrates and is best known for causing the common cold. The adenovirus early region 4 (E4) 11k protein, product of open reading frame 3 (ORF3), acts to inhibit host cell protein synthesis while promoting viral protein synthesis. During infection, the E4 11k protein of the Ad5 serotype, binds to the cellular RNA helicase enzyme, and cellular processing body (P-body) component, Ddx6. Adenovirus 5 E4 11k re-localizes Ddx6 and co-localizes with it in aggresomes, whereas Ad9 E4 11k does not. Aggresomes are aggregates of misfolded proteins marked with γ-tubulin that form near a cell’s nucleus. The overall goal is to determine the binding site between Ddx6 and Ad5 E4 ORF3 in order to determine the importance of their interaction during viral infection. The current objective is to optimize the transfection method in order to find the protocol that yields the greatest transfection efficiency. HeLa cells will be transfected with RFP-tagged Ddx6 and HA-tagged E4 ORF3. The transfection reagent used will be polyethylenimine (PEI). Concentrations and ratios of DNA and the transfection medium, either NaCl or OPTI-MEM, will be the variables manipulated to optimize transfection. Proteins will be isolated from transfected cells and analyzed in a Western blot to determine the optimal transfection conditions needed to visualize the Ddx6 and Ad E4 11k interactions. Once this is determined, Ad5/9 chimeras of E4 ORF3 will be used to narrow down the Ad5 E4 ORF3/Ddx6 binding site. Finding the binding site will contribute to the knowledge of the role of P-bodies in gene expression during viral infections.