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

Sui, Baokun; Huang, Junhua; Jha, Babal K.; Yin, Ping; Zhou, Ming; Fu, Zhen F.; Silverman, Robert H.; Weiss, Susan R.; Peng, Guiqing; Zhao, Ling

doi:10.1099/jgv.0.000395

Cited by 7 publications

(6 citation statements)

References 28 publications

(29 reference statements)

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“…For comparison, the recently solved structure of lineage A Betacoronavirus mouse hepatitis virus (MHV) NS2, a 2H-PE with 2′,5′-PDE activity, is also shown ( Fig. 1C ) ( 36 ).…”

Section: Resultsmentioning

confidence: 99%

Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

Thornbrough

Jha

Yount

et al. 2016

mBio

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Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage C Betacoronavirus, and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2′,5′-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage A Betacoronavirus PDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention.

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“…For comparison, the recently solved structure of lineage A Betacoronavirus mouse hepatitis virus (MHV) NS2, a 2H-PE with 2′,5′-PDE activity, is also shown ( Fig. 1C ) ( 36 ).…”

Section: Resultsmentioning

confidence: 99%

Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

Thornbrough

Jha

Yount

et al. 2016

mBio

Self Cite

139

182

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“…The portion of ns2 deleted in MHV-ExoN(-) P250 lies outside the PDE catalytic domain, in a region of unknown function. C-terminally truncated ns2 retains enzymatic activity ( 55 ), but whether these specific deletions and fusions disrupt PDE activity remains to be tested. Nevertheless, ns2 is dispensable for MHV replication in immortalized cells ( 56 , 57 ).…”

Section: Resultsmentioning

confidence: 99%

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

Graepel

Case

et al. 2017

mBio

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The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication.

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“…2H phophodiesterase enzymes are highly divergent in sequence outside of the active site H-X-S/T motif 28 , yet structurally homologous. A DALI search 39 (Supplementary Table 2 ) reveals that the closest structural homologs of yUsb1 (after hUsb1) include putative 2′–5′ ligases 40 , 41 , 2′,3′-cyclic nucleotide 3′-phosphodiesterases (such as the well-characterized enzyme ThpR) 42 , 43 , 2′–5′ phosphodiesterases 44 , 45 and several proteins of unknown activity 46 , 47 . Thus, the fold of Usb1 is more reminiscent of a 2′,3′-cyclic phosphodiesterase than an exoribonuclease, and the closest homologs tend to open cyclic phosphates to produce a 2′ phosphate instead of a 3′ phosphate.…”

Section: Discussionmentioning

confidence: 99%

Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

et al. 2017

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U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3′ phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2–8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.

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

Cited by 7 publications

References 28 publications

Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

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