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Updated: Jun 21, 2026

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells
Published on: November 12, 2015
The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures
Ahmed M Wahid1, Veronica K Coventry, Graeme L Conn
1Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, M1 7DN.
Abstract:
VA RNA(I) is a non-coding adenoviral transcript that counteracts the host cell anti-viral defenses such as immune responses mediated via PKR. We investigated potential alternate secondary structure conformations within the PKR-binding domain of VA RNA(I) using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNA(I) apical stem can adopt two different conformations and that it exists as a mixed population of these two structures. In contrast, in two sequence variants we designed to eliminate one of the possible structures, while leaving the other intact, each formed a unique secondary structure. This clarification of the apical stem pairing also suggests a small alteration to the apical stem-loop secondary structure. The relative ability of the two apical stem conformations to bind PKR and inhibit kinase activity was measured by isothermal titration calorimetry and PKR autophosphorylation inhibition assay. We found that the two sequence variants displayed markedly different activities, with one being a significantly poorer binder and inhibitor of PKR. Whether the presence of the VA RNA(I) conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation.
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