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PLoS One. 2017 Oct 20;12(10):e0186849. doi: 10.1371/journal.pone.0186849. eCollection 2017.

Impact of the structural integrity of the three-way junction of adenovirus VAI RNA on PKR inhibition.

Author information

1
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
2
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4,Warsaw, Poland.
3
Manitoba Institute for Materials, University of Manitoba, Winnipeg, Manitoba, Canada.
4
Laboratory of Bioinformatics and Protein Engineering, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, ul. Umultowska 89, Poznan, Poland.
5
Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada.
6
DiscoveryLab, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada.
7
Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, University Dr. NW Calgary, Alberta, Canada.
8
Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada.

Abstract

Highly structured RNA derived from viral genomes is a key cellular indicator of viral infection. In response, cells produce the interferon inducible RNA-dependent protein kinase (PKR) that, when bound to viral dsRNA, phosphorylates eukaryotic initiation factor 2α and attenuates viral protein translation. Adenovirus can evade this line of defence through transcription of a non-coding RNA, VAI, an inhibitor of PKR. VAI consists of three base-paired regions that meet at a three-way junction; an apical stem responsible for the interaction with PKR, a central stem required for inhibition, and a terminal stem. Recent studies have highlighted the potential importance of the tertiary structure of the three-way junction to PKR inhibition by enabling interaction between regions of the central and terminal stems. To further investigate the role of the three-way junction, we characterized the binding affinity and inhibitory potential of central stem mutants designed to introduce subtle alterations. These results were then correlated with small-angle X-ray scattering solution studies and computational tertiary structural models. Our results demonstrate that while mutations to the central stem have no observable effect on binding affinity to PKR, mutations that appear to disrupt the structure of the three-way junction prevent inhibition of PKR. Therefore, we propose that instead of simply sequestering PKR, a specific structural conformation of the PKR-VAI complex may be required for inhibition.

PMID:
29053745
PMCID:
PMC5650172
DOI:
10.1371/journal.pone.0186849
[Indexed for MEDLINE]
Free PMC Article

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