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.
Highlights
RNA-dependent protein kinase (PKR) is a key interferon-stimulated enzyme involved in the innate immune response to viral infection
A recent review has highlighted the importance of virus associated RNA-I (VAI) as an essential proviral non-coding RNA [53]
The structural mechanism of inhibition of RNA-activated Protein Kinase (PKR) by VAI RNA is not fully understood, previous in vitro studies have highlighted the importance of the central stem-loop (CS) of VAI in the inhibition of PKR self-association [10,11,12,13,14, 19]
Summary
RNA-dependent protein kinase (PKR) is a key interferon-stimulated enzyme involved in the innate immune response to viral infection. PKR is a Ser/Thr kinase that consists of tandem copies of a conserved double-stranded RNA binding motif (dsRBMs, residues 1–169) at the Nterminal domain, and a C-terminal kinase domain [1]. Upon viral infection and subsequent production of viral dsRNAs, PKR binds viral dsRNA, which enables self-association and a conformational change resulting in auto-phosphorylation on two threonine residues (Thr446 and Thr451) that overhang the enzyme’s active site [2]. Phosphorylated PKR in turn phosphorylates its target substrate eukaryotic initiation factor 2α (eIF2α) at Ser, which slows the translation of viral proteins, helping the host cell’s response [3,4,5]. Activation of PKR follows a bimolecular reaction mechanism [8, 9]
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