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A protein adopting a unique membrane conformation allows for a new viral immunoevasion strategy : a study of the function, targeting and membrane topology of adenovirus E3/6.7K protein

University of British Columbia

Adenovirus persistently infects the eye, ear and the respiratory and digestive tracts in humans. Its persistence is due to in part to immunosubversive proteins coded by the E3 region of the viral genome. This thesis project started from the enticing prospect that E3/6.7K, one of the two remaining uncharacterized proteins coded by the E3 region, also has a role in virus-host cell interaction. Previously, E3/6.7K was shown to be an endoplasmic reticulum (ER) localized membrane glycoprotein. Our first functional observation was that in the presence of E3/6.7K there was a reduced inflammatory response in the lungs of virally infected mice. Consequently, cells that express E3/6.7K were protected against apoptosis induced by TNF as well as other Death Inducers, such as Fas Ligand or TRAIL. The release of inflammatory mediators by cells stimulated with TNF was also significantly reduced in the presence of E3/6.7K. More importantly, we observed that E3/6.7K prevents the efflux of calcium ions from the ER. The release of calcium ions from the ER is known to be important for the generation of mediators of inflammation and apoptosis. The effect of E3/6.7K on calcium ion homeostasis can explain the protein's protective effects against inflammatory cytokines. In an effort to explore the E3/6.7K protein in further detail, we examined its structure and membrane targeting. In spite of being an ER-localized membrane protein, E3/6.7K lacks the targeting signals that are necessary for a newly synthesized protein to be translocated across the lumen of the ER. We found that E3/6.7K translocated across the ER membrane following complete synthesis in contrast to the signal sequence-dependent mode of targeting characteristic for most other proteins secreted by higher eukaryotes. Analysis of the orientation of E3/6.7K indicated that it can assume two distinct orientations including the first encountered example of a 'hairpin' topology with both its N and C termini in the lumen of the ER. Ultimately, these studies challenged the current model of membrane protein structure and uncovered a new viral immunoevasion mechanism.

Thesis/Dissertation

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2009-09-23

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http://hdl.handle.net/2429/13132

Zoology

University of British Columbia

UBCV

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