Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

Abstract

Synthesis of 2=-5=-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined H⌽(S/T)⌽ (where ⌽ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave ␤-sheet that contains the catalytic cleft, but it lacks two ␣-helical regions and two ␤-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A. IMPORTANCEThe C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2=-5=-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two ␣-helical regions and ␤-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A. O ne of the earliest steps in the battle between a virus and its host is the detection of pathogen-associated molecular patterns by cellular sensors. For RNA viruses, these patterns may be found in RNA products synthesized during viral transcription and replication. A number of cytoplasmic cellular molecules have been described that sense foreign RNA and resp...

Crystal structure of the mouse hepatitis virus ns2 phosphodiesterase domain that antagonizes RNase L activation

“…The arginine that is important for stabilizing 2-5A has previously been reported to be present in the R-loop region and is also strictly conserved in those three proteins ( Fig. 2a, d) (Ogden et al, 2015). All of the aforementioned characteristics demonstrate the structural conservation of the PDE catalytic domain of these three 2H phosphoesterases, despite lacking significant sequence homology ( Fig.…”

“…2a). The structures of AKAP7 CD (PDB ID 2VFY) and VP3-CTD (PDB ID 5AF2), which exhibit substantial structural homology, have previously been determined (Brandmann & Jinek, 2015;Ogden et al, 2015). The structural alignment of ns2 7-167 , AKAP7 CD and VP3-CTD indicated that all of these structures form similar positive electrostatic concave regions in the 29,59-PDE activity domain, which is putatively involved in the binding and cleavage of 2-5A ( Fig.…”

Inhibition of the OAS/RNase L pathway by viruses

“…In recombinant MHV viruses, rotavirus VP3 can substitute for ns2 and rescue replication in bone marrow-derived macrophages and in mouse liver. Sequence alignments suggest that a similar PDE occurs in all group A and likely group B and C rotavirus strains [26,27].…”

New advances in our understanding of the “unique” RNase L in host pathogen interaction and immune signaling

“…In contrast, in RNase L knockout B6 mice, both viruses replicated similarly, suggesting that ns2 directly acts through OAS-RNase L [56]. Similarly, rotavirus A (RVA) encodes for the viral protein 3 (VP3), which is structurally homologous in its C-terminal domain to MHV-ns2 [54,57,58]. Although from evolutionarily non-related viruses, VP3 degraded 2-5A in similar manner as ns2 in vitro and in intact cells, whereas mutation of the two conserved histidines in the PDE catalytic site of VP3 prevented 2-5A degradation.…”

“…The 2H central domain of the murine AKAP7 restored replication of ns2 mutant MHV chimeric virus whereas MHV chimeras with a histidine-to-arginine mutation in the His-X-Thr motif of AKAP7 central domain failed to replicate in bone marrow macrophages of B6 mice [61]. Due to structural homology, it was proposed that AKAP7 could be the evolutionary precursor of viral 2 0 ,5 0 PDE members [58,61]. In addition, phosphodiesterase 12 (PDE12) is a member of the exonuclease-endonuclease phosphatase family that localizes in mitochondria and regulates mRNA turnover by removing the polyA tails [62,63].…”