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Nucleic Acids Research Advance Access originally published online on July 27, 2009
Nucleic Acids Research 2009 37(17):5830-5837; doi:10.1093/nar/gkp595
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Nucleic Acids Research, 2009, Vol. 37, No. 17 5830-5837
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures

Ahmed M. Wahid1,2, Veronica K. Coventry2 and Graeme L. Conn1,3,*

1Manchester Interdisciplinary Biocentre, 2Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, M1 7DN and 3Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA

*To whom correspondence should be addressed. Tel: +1 404 727 5965; Fax: +1 404 727 2738; Email: graeme.l.conn{at}emory.edu

Received December 15, 2008. Revised June 26, 2009. Accepted June 29, 2009.

VA RNAI 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 RNAI using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNAI 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 RNAI conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation.

Present address: Graeme L. Conn, Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA.


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