Dissection of the Adenoviral VA RNAI Central Domain Structure Reveals Minimum Requirements for RNA-mediated Inhibition of PKR

Jo L. Wilson,Virginia K. Vachon,S. Sunita,Samantha L. Schwartz,Graeme L. Conn

doi:10.1074/jbc.m114.550046

Abstract

Virus-associated RNA I (VA RNAI) is a short (∼160-nucleotide) non-coding RNA transcript employed by adenoviruses to subvert the innate immune system protein double-stranded RNA-activated protein kinase (PKR). The central domain of VA RNAI is proposed to contain a complex tertiary structure that contributes to its optimal inhibitory activity against PKR. Here we use a combination of VA RNAI mutagenesis, structural analyses, as well as PKR activity and binding assays to dissect this tertiary structure and assess its functional role. Our results support the existence of a pH- and Mg(2+)-dependent tertiary structure involving pseudoknot formation within the central domain. Unexpectedly, this structure appears to play no direct role in PKR inhibition. Deletion of central domain sequences within a minimal but fully active construct lacking the tertiary structure reveals a crucial role in PKR binding and inhibition for nucleotides in the 5' half of the central domain. Deletion of the central domain 3' half also significantly impacts activity but appears to arise indirectly by reducing its capacity to assist in optimally presenting the 5' half sequence. Collectively, our results identify regions of VA RNAI critical for PKR inhibition and reveal that the requirements for an effective RNA inhibitor of PKR are simpler than appreciated previously.

Highlights

Adenoviruses use the short non-coding transcript Virus-associated RNA I (VA RNAI) to inhibit host antiviral kinase PKR
What is the nature of its tertiary structure, and what role does it play in PKR inhibition? The analyses presented here reveal the defining features of the central domain tertiary structure but demonstrate that it is not required for activity against PKR
A Putative pH-dependent VA RNAI Central Domain Tertiary Structure Is Stabilized by Mg2ϩ and PKR—A new VA RNAI variant was created (Fig. 1A) that combined two previously characterized mutations: a 21 base pair truncation of the terminal stem (TS⌬21) that mirrors that produced by Dicer processing in vivo [8] and a six-nucleotide deletion in the apical stem (A2dl2) [31]

Summary

Background

Adenoviruses use the short non-coding transcript VA RNAI to inhibit host antiviral kinase PKR. Our results support the existence of a pH- and Mg2؉-dependent tertiary structure involving pseudoknot formation within the central domain This structure appears to play no direct role in PKR inhibition. A single VA RNAI transcript possesses multiple activities It can inhibit two antiviral proteins (PKR and OAS1), potentially regulate gene expression via the cellular RNAi system, and saturate the RNA-processing enzyme Dicer. The requirements for optimal inhibitory activity against PKR are far simpler than appreciated previously, offering a satisfying explanation for the consistently observed inhibition of PKR by VA RNAI molecules from different adenovirus serotypes despite their significant differences at the sequence and secondary structural levels

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

CONCLUSIONS