Solution conformation of adenovirus virus associated RNA-I and its interaction with PKR

Abstract

Adenovirus virus-associated RNA (VAI) provides protection against the host antiviral response in part by inhibiting the interferon-induced double stranded RNA-activated protein kinase (PKR). VAI consists of three base-paired regions; the apical stem responsible for the interaction with double-stranded RNA binding motifs (dsRBMs) of PKR, the central stem required for inhibition, and the terminal stem. The solution conformation of VAI and VAI lacking the terminal stem were determined using SAXS that suggested extended conformations that are in agreement with their secondary structures. Solution conformations of VAI lacking the terminal stem in complex with the dsRBMs of PKR indicated that the apical stem interacts with both dsRNA-binding motifs whereas the central stem does not. Hydrodynamic properties calculated from ab initio models were compared to experimentally determined parameters for model validation. Furthermore, SAXS envelopes were used as a constraint for the in silico modeling of tertiary structure for RNA and RNA-protein complex. Finally, full-length PKR was also studied, but concentration-dependent changes in hydrodynamic parameters prevented ab initio shape determination. Taken together, results provide an improved structural framework that further our understanding of the role VAI plays in evading host innate immune responses.

Keywords: AS; AUC; Adenovirus; CS; D(max); DLS; EM; NSD; PKR; Protein–RNA interactions; RNA-activated protein kinase; SAXS; SEC; Small angle X-ray scattering; TS; VA(I); VA(I) RNA; VA(I) lacking the terminal stem; VA(I)AS; VA(IΔ)TS; adenovirus virus-associated RNA; analytical ultracentrifugation; apical stem-loop; apical stem-loop of VA(I); central stem-loop; double-stranded RNA binding domains; dsRBMs; dsRNA; dynamic light scattering; eIF2; electron microscopy; electron pair-distance distribution function; eukaryotic initiation factor 2; hydrodynamic radius; in silico structure determination; maximum particle dimension; normalized spatial discrepancy; p(r) function; r(G); r(H); radius of gyration; s(20)(w); sedimentation co-efficient of water at 20°C; size exclusion chromatography; small angle X-ray scattering; terminal stem-loop.