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Crystal Structure of an Adenovirus Virus-Associated RNA

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Crystal Structure of an Adenovirus Virus-Associated RNA ARTICLE https://doi.org/10.1038/s41467-019-10752-6 OPEN Crystal structure of an adenovirus virus-associated RNA Iris V. Hood1, Jackson M. Gordon 1, Charles Bou-Nader1, Frances E. Henderson1, Soheila Bahmanjah1 & Jinwei Zhang 1 Adenovirus Virus-Associated (VA) RNAs are the first discovered viral noncoding RNAs. By mimicking double-stranded RNAs (dsRNAs), the exceptionally abundant, multifunctional VA 1234567890():,; RNAs sabotage host machineries that sense, transport, process, or edit dsRNAs. How VA-I suppresses PKR activation despite its strong dsRNA character, and inhibits the crucial anti- viral kinase to promote viral translation, remains largely unknown. Here, we report a 2.7 Å crystal structure of VA-I RNA. The acutely bent VA-I features an unusually structured apical loop, a wobble-enriched, coaxially stacked apical and tetra-stems necessary and sufficient for PKR inhibition, and a central domain pseudoknot that resembles codon-anticodon interac- tions and prevents PKR activation by VA-I. These global and local structural features col- lectively define VA-I as an archetypal PKR inhibitor made of RNA. The study provides molecular insights into how viruses circumnavigate cellular rules of self vs non-self RNAs to not only escape, but further compromise host innate immunity. 1 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, 50 South Drive, Room 4503, Bethesda, MD 20892, USA. Correspondence and requests for materials should be addressed to J.Z. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:2871 | https://doi.org/10.1038/s41467-019-10752-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10752-6 denoviruses infect the respiratory system and the gastro- a intestinal and urinary tracts and can be life-threatening PKR 118 94 A Basic 115 for immunocompromised patients. Late in infection, L8 Kinase 7– 8 Stem 7 adenoviruses produce two extraordinarily abundant (10 10 dsRBM2 S7 97 L9 copies per cell), highly structured noncoding RNAs termed virus- 3′ 110 L6 S5 S4 S3 S1 associated RNAs (VA-I and VA-II; ~160 nucleotides (nts)), 5′ L10 93 100 fi 1,2 dsRBM1 L2 leading to their discovery as the rst viral noncoding RNAs .At b 70 8090 92 119120 least one VA RNA exists in all known adenovirus serotypes and 107 123 most (~80%) contain both3. The multifunctional VA RNAs 106 66 55 50 45 40 interfere with essentially all host systems that interface with 125 65 60 Apical stem Tetrastem double-stranded RNAs (dsRNAs), from their sensing by protein 36 1.2 L8 kinase R (PKR), export by Exportin-5, processing by Dicer, c WT VA-I 127 1.0 ΔTS (crystal) L10 editing by ADAR, to activation of oligoadenylate synthetases 130 (OAS), etc4. Dicer-processed terminal strands of VA are further 0.8 0.6 Stem 3 30 assembled into functional RISC complexes that may target PKR 0.4 additional host systems5,6. Collectively, VA RNAs contribute ~60 phosphorylation 0.2 fold to viral titers and confer adenoviruses general resistance to 0.1 ′ ′ interferon-mediated antiviral defense7. 0 10 100 1000 5000 5 3 VA-I RNA (nM) PKR is a central component of the interferon response and the best-characterized target of VA RNAs. PKR recognizes dsRNAs d produced during viral replication or bidirectional transcription, and exerts antiviral and antiproliferative effects through signaling pathways including NF-kB, TNF, STATs, p53, etc8. Beyond its immune function, PKR plays additional roles in neurodegenera- tive diseases9, cognition and memory10, malignant transforma- tion11, etc. Dimerization of PKR on sufficiently long dsRNA e L8 (>30–33 base pairs) induces PKR autophosphorylation and acti- L10 vation of its latent kinase activity12. Activated PKR phosphor- ylates the alpha subunit of eukaryotic translation initiation factor 80° ′ 2 (eIF2α) to block cap-dependent translation initiation and pro- 5 duction of new viral particles13. Indeed, PKR-deficient mice are exceptionally susceptible to intranasal infection by vesicular sto- fl 14 matitis and in uenza viruses . To counter the debilitating 3′ restriction by PKR, nearly all known viruses have evolved protein or RNA antagonists to evade or inactivate PKR. The constant tug- Fig. 1 Overall structure of Adenovirus 2 VA-I RNA. a Cartoon model of VA-I of-war with viral antagonists has driven intense positive selection and PKR interactions. S: stem; L: loop. S1 and L2 were absent from the of PKR in vertebrates15. Besides manipulation by viral elements, crystal construct. b Secondary structure of crystallized VA-I RNA. PKR activity is subject to tight control by host protein factors Tetrastem: green, Loop 8: violet, Loop 10: orange, engineered sequences: including NF9016, TRBP17, ADAR118, etc. Furthermore, endo- gray. Three single-stranded, helix-capping purines, A36, G106, and A123, genous, highly structured RNAs also modulate PKR activity, such are in red italics. Leontis–Westhof symbols74 denote non-canonical base as snoRNAs19, the IFN-γ and TNF-α pre-mRNAs20,21, nc886 pairs. Arrowheads denote chain connectivity. Dotted red lines indicate RNA22, etc. Recently, additional endogenous RNAs have been hydrogen bonds. G60 is disordered. c Inhibitory activities of wild-type and shown to directly associate with PKR, including nuclear trans- ΔTS (deletion of terminal stem; crystallization construct) VA-I. Several ΔTS posable elements and mitochondrial dsRNAs proposed to med- RNAs showed an enhanced inhibitory activity than WT, as previously iate mitochondria–cytosol communication, especially during reported26. Error bars here and thereafter represent standard deviations stress23. (s.d.) from three independent replicates. d, e Two views of the overall Among the expanding collection of PKR-regulatory RNAs, structure of VA-I, colored as in b VA-I is the best-characterized RNA antagonist24. It is comprised of an elongated apical stem loop, a central domain proposed to extraordinarily stable and yet fluid enough to diverge into two contain a pseudoknot, and a terminal stem thought to be dis- distinct conformations. The structurally complex central domain pensable for PKR binding and inhibition (Fig. 1a, b; Supple- has been long proposed to contain a 3-bp pseudoknot between mentary Fig. 1)3,25,26. Although the importance of higher-order Loop 8 and Loop 10, based on covariation of the loop sequen- RNA structure to PKR modulation is well recognized27, no high- ces25. Yet its functional importance for PKR regulation has resolution structure of VA, or of any other complex RNA mod- remained unclear. UV-melting analysis of the central domain ulator of PKR is known. Nonetheless, extensive phylogenetic, revealed an unusual sensitivity to solution pH, the structural basis biochemical, and small-angle X-ray scattering (SAXS) analyses of of which remains unknown30. At the junction between the apical VA-I uncovered a number of unusual features of this RNA. The and central domains is an essentially invariant tetrastem structure fi apical domain is exceptionally thermostable (Tm~83 and ~95 °C, (Fig. 1b). The speci c sequence of the tetrastem, in addition to its in the absence and presence of 1 mM Mg2+, respectively26,28), secondary structure, is required for full inhibitory activity against causing it to migrate much slower on denaturing Urea-PAGE and PKR31. What drives the near-universal sequence conservation of to resist denaturation by up to 6 M urea. Yet, structural-probing this functionally crucial element? analyses revealed sensitivity of this region to both ssRNA-specific To address these open questions and understand how viral and dsRNA-specific RNases (RNase A and V1, respectively)25,29. RNAs employ unique tertiary structures to confound and defeat This led to the proposal that the VA-I apical region exists as two host immune proteins, we determined a 2.7 Å crystal structure of structurally different, functionally distinct conformers25,29. the apical and central domains of VA-I RNA of adenovirus ser- However, it is perplexing how a single RNA region is both otype 2 (Fig. 1b–e; Table 1; Supplementary Figs. 2 and 3). This 2 NATURE COMMUNICATIONS | (2019) 10:2871 | https://doi.org/10.1038/s41467-019-10752-6 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10752-6 ARTICLE Table 1 Summary of crystallographic statistics of a native from the colinear, or cis global dsRNA arrangements that gen- 34,35 dataset erally activate PKR . It was shown that bending a 51-bp dsRNA by inserting adenosine bulges of increasing sizes at its center progressively reduced its potential to activate PKR, by as Native much as 10-fold when a ~93° bend is produced with a 6- Data collection adenosine bulge35. Further, when two bends were introduced in Space group P4122 Cell dimensions the same dsRNA, a more bent cis-bulged global shape activated a, b, c (Å) 100.5, 100.5, 130.7 PKR far less than a more linear trans-bulged global shape. α, β, γ (°) 90, 90, 90 Contrary to the acutely bent VA-I that functions as a PKR Resolution (Å)a 100.0–2.74 (2.81–2.74) inhibitor, the structural model of the PKR-activating TNF-α pre- a Rmerge (%) 15.2 (364) mRNA 3' UTR features two parallel helices that drive exceptional <I>/<σ(I)>a 12.2 (0.82) PKR activation21. Similar near-parallel, adjacent helices in Completeness (%)a 99.9 (100.0) riboswitches, and ribozymes also drive PKR activation36. Thus, Redundancya 8.0 (8.2) the sharply bent global shape of VA-I appears to have evolved to CC 1/2 0.998 (0.340) avoid near-linear, extended configurations that are hallmarks of CC* 1.000 (0.712) Refinement PKR-activating RNAs. Resolution (Å)a 37.02–2.74 (2.84–2.74) No. of reflectionsb 18,173 (1771) An apical structure with both ssRNA and dsRNA character- R R a work/ free (%) 22.0 (43.0)/23.6 (42.5) istics.
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