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

Abstract

Background: Adenoviruses use the short non-coding transcript VA RNA I to inhibit host antiviral kinase PKR. Results: VA RNA I contains a pH-and Mg 2ϩ -sensitive tertiary structure that, unexpectedly, is not required for PKR inhibition. Conclusion: Structural requirements for an RNA inhibitor of PKR are simpler than appreciated previously. Significance: These findings explain how non-coding RNAs of varied sequence and structure can efficiently inhibit PKR.

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs

“…We found that the addition of an N-terminal SUMO (smt3) tag allowed soluble expression of both shorter constructs. Importantly, the addition of the SUMO tag had no discernible effect on hPKR activation or inhibition by poly(rI:rC) dsRNA and Ad2 VA RNA I (30,32), respectively (Fig. 2, A and B).…”

“…RNA in Vitro Transcription and Purification-A truncated form of human adenovirus type 2 (Ad2) VA RNA I (94 nucleotides), which retains full wild-type activity but lacks the entire terminal stem ("TS⌬21") and a short sequence in the apical stem ("A2dl2") was in vitro transcribed using T7 RNA polymerase under previously established optimal conditions (31,32). HIV-1 TAR RNA (59 nucleotides) was also prepared by T7 RNA polymerase run-off transcription using established conditions (33,34).…”

“…In the latter case quenched samples were applied promptly to a BioDot SF (Bio-Rad) microfiltration system, and proteins were bound to a nitrocellulose membrane under vacuum. The membrane was washed to remove unreacted [␥- 32 P]ATP and then air-dried. For both membranes and dried SDS-PAGE gels, the extent of PKR and eIF2␣ phosphorylation was determined by exposure to a phosphor storage screen and analysis using a Typhoon FLA 7000 PhosphorImager and ImageQuant software (GE Healthcare).…”

“…The activation of PKR follows a bell-shaped curve where the extent of activation initially increases with the concentration of dsRNA but then declines at the highest activator concentrations as PKR binds in a monomeric form to different dsRNA molecules (37,38). Assays of PKR autophosphorylation in the presence of [␥- 32 P]ATP are established as a direct readout of PKR activation (32) and were used to assess the activation and inhibition of each IDL deletion mutant by poly(rI:rC) dsRNA and VA RNA I , respectively. In both cases the regulation of PKR by RNA was essentially unaffected by any of the three IDL deletions; PKR activation followed a bell-shaped curve with approximately the same maximum poly(rI:rC) dsRNA concentration, and inhibition by VA RNA I was accomplished with similar IC 50 values (Fig.…”

Viral noncoding RNAs: more surprises

“…PKR is activated by binding to dsRNA, originating from viral transcription, whereupon it phosphorylates the translation initiation factor eIF2 to inhibit protein synthesis in virus-infected cells (de Haro et al 1996). The cytoplasmic VA RNA I competes with dsRNA for binding to PKR but, instead of stimulating, inhibits activation of PKR, thereby enabling viral protein synthesis (Mathews and Shenk 1991;Wilson et al 2014). VA RNAs can also act as substrates for the RNase III enzyme Dicer, with the products being incorporated into functional Argonaute (AGO)-containing RNA-induced silencing complexes (RISCs) (Aparicio et al 2006(Aparicio et al , 2010.…”