Investigating the Interaction between Adenoviral Protein E4 11k and Cellular Processing Body Protein Ddx6**


Adenovirus is a non-enveloped virus with an icosahedral capsid encompassing a double-stranded DNA genome. In immunocompetent individuals, adenovirus most commonly causes infections that are localized to the upper respiratory and gastrointestinal tracts. However, immunocompromised people experience more serious, generalized infections. Our lab focuses on the adenovirus early region 4 11 k protein (E4 11k), which has been shown to inhibit host cell protein synthesis as well as stimulate viral late protein synthesis. During an adenovirus serotype 5 infection (Ad5), E4 11k has been shown to bind to the cellular processing body (p-body) protein, Ddx6. The two proteins then co-localize in aggresomes, which are juxtanuclear aggregates of misfolded proteins marked by �-tubulin. Our lab aims to determine the binding location between the two proteins. In order to determine this site, we had Ad5 and Ad9 chimeras of the E4 ORF3 gene, which encodes the E4 11k protein, synthesized. The E4 11k protein encoded by Ad9 does not bind to Ddx6, only E4 11 k from Ad5 has been shown to bind Ddx6. Therefore, the chimeras will allow us to narrow down the binding site to a particular region on the E4 11k protein. We have begun using the Ad5/Ad9 E4 ORF3 chimeras in transfections to express them in the human cell line, HeLa, to observe cellular responses. We plan to use confocal microscopy to visualize whether aggresomes are able to form using the various chimeras. Once we find the binding region that allows for E4 11k to reorganize Ddx6 to aggresomes, we will perform alanine substitutions to disrupt the binding to observe any changes that result during an infection. These results will contribute to our knowledge of the roles that p-bodies play during viral infections, which may also help to understand their basic role in cell biology.


Young Harris Undergraduate Research Initiative

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