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. 2016 Mar 29;7(2):e00258.
doi: 10.1128/mBio.00258-16.

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

Joshua M Thornbrough  1 , Babal K Jha  2 , Boyd Yount  3 , Stephen A Goldstein  1 , Yize Li  1 , Ruth Elliott  1 , Amy C Sims  3 , Ralph S Baric  4 , Robert H Silverman  2 , Susan R Weiss  5
Affiliations

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

Joshua M Thornbrough et al. mBio. .

Abstract

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 CBetacoronavirus, 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 ABetacoronavirusPDEs 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.

Importance: Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV). MERS-CoV, like other coronaviruses, carries genes that encode accessory proteins that antagonize the host antiviral response, often the type I interferon response, and contribute to virulence. We found that MERS-CoV NS4b and homologs from related lineage C bat betacoronaviruses BtCoV-SC2013 (SC2013) and BtCoV-HKU5 (HKU5) are members of the 2H-phosphoesterase (2H-PE) enzyme family with phosphodiesterase (PDE) activity. Like murine coronavirus NS2, a previously characterized PDE, MERS NS4b, can antagonize activation of the OAS-RNase L pathway, an interferon-induced potent antiviral activity. Furthermore, MERS-CoV mutants with deletion of genes encoding accessory proteins NS3 to NS5 or NS4b alone or inactivation of the PDE can activate RNase L during infection of Calu-3 cells. Our report may offer a potential target for therapeutic intervention if NS4b proves to be critical to pathogenesis inin vivomodels of MERS-CoV infection.

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Figures

FIG 1
FIG 1
Predicted structure of MERS-CoV NS4b phosphodiesterase. (A) Structure of Rattus norvegicus AKAP7γ/δ (PDB: 2VFK) (37). (B) Tertiary structural homology model of MERS-CoV NS4b. (C) Structure of NS2 (PDB: 4Z5V) (36). The structures were visualized and analyzed in UCSF Chimera 1.8.
FIG 2
FIG 2
Cellular and viral 2′,5′-phosphodiesterase (PDE) domains. (A) Alignment of known and predicted cellular and viral 2′,5′-PDE sequences. Catalytic motifs [H-Φ-(S/T)-Φ] are indicated by the red and blue boxes. Rat (Rattus norvegicus [Rn]) and mouse (Mus musculus [Mm]) AKAP sequences and human (H) and bat (Bt) coronavirus sequences are shown. (B) Comparison of known features of full-length M. musculus AKAP7γ, MHV NS2, RVA VP3 CTD and lineage C NS4b proteins including nuclear localization sequence (NLS) and PDE domains. PKA-RII-α-BD, a binding domain for regulatory subunit (RII) of cAMP-dependent protein kinase A (37), guanylyltransferase (Gtase), and methyltransferase (Mtase) domains are also indicated (32).
FIG 3
FIG 3
Lineage C NS4b proteins are catalytically active 2′,5′-phosphodiesterases. (A) Abrogation of catalytic activity by mutation of the second catalytic His of NS4b proteins and comparison with MHV NS2 and RVA VP3 CTD. The kinetic data were obtained using 10 µM 2-5A and 1 µM enzyme concentration with the exception of MBP, which was used at 10 µM. Data shown are from one representative experiment of three carried out with separate enzyme preparations. (B to D) Steady-state enzyme kinetic curves of velocity versus concentration of 2-5A for MHV NS2 (B), MERS-CoV (C), BtCov-SC2013 (D), and BtCoV-HKU5 (E) NS4b proteins. Kinetic reactions were carried out in triplicate and expressed as means ± standard errors of the means (SEM) (error bars). Substrate-dependent velocity measurements are shown from one representative experiment carried out twice in triplicate with separate enzyme preparations.
FIG 4
FIG 4
Construction of NS4b-expressing recombinant MHV. (A) Diagrammatic representation of MHVMut genome (expressing catalytically inactive NS2H126R). The letters designate genes encoding structural proteins, and the numbers designate ORFs encoding nonstructural proteins. ORFs 1a and 1b together comprise 20 kb and are not to scale. (B) Wild-type or catalytic mutant NS4b genes are lined up with the MHV ORF4a or ORF4b insertion site to construct chimeric viruses. Nuclear localization signals (NLSs) and phosphodiesterase (PDE) domains are indicated.
FIG 5
FIG 5
Expression and localization of MERS-CoV NS4b and BtCoV homologs expressed by chimeric MHVs. (A) Lysates of chimeric-virus-infected RNase L−/− BMM were analyzed on denaturing polyacrylamide gels and analyzed by immunoblotting with antibody against Flag (α-Flag) to detect NS4b, antibody against MHV nucleocapsid (N) protein, and antibody against GAPDH. Data are representative of three experiments carried out twice in 17Cl-1 cells and once each in B6 and RNase L−/− BMM with similar results. (B) Murine L2 cells infected with chimeric MHVs were fixed and stained with DAPI as well as antibodies against Flag to detect NS4b and MHV N. These data are from one representative experiment of three.
FIG 6
FIG 6
Lineage C PDE NS4b proteins functionally replace MHV NS2 in primary BMM. (A to D) Representative one-step growth curve of WT and mutant (Mut) MHV (A) and chimeric viruses expressing WT and mutant NS4b proteins (diagrammed in Fig. 4B), WT and mutant MHV-MERS (B), WT and mutant MHV-MERSΔ1-52 (C), and WT and mutant MHV-SC2013 (D) in B6 and RNase L−/− BMM (MOI of 1 PFU; n = 3). Statistical significance was determined by two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons and is indicated as follows: *, P value of <0.05; **, P < 0.01; ***, P < 0.001. Values that are not significantly different (ns) are indicated. Values are means ± SEM (error bars). These data are from one representative experiment of three experiments. (E) rRNA degradation pattern 15 h postinfection in B6 BMM. Data are from one representative experiment of three (MOI of 1 PFU; n = 3). The positions of 28S and 18S rRNAs are shown to the right of the gel.
FIG 7
FIG 7
Catalytically active MERS-CoV NS4b rescues replication of MHVMut in the livers of B6 mice. Liver titers (n = 19 or 20) day 5 postinfection (MOI of 2,000 PFU) of MHVWT and MHVMut (A) or MHV-MERSWT and MHV-MERSMut (B). Statistical significance was determined by χ2 test with Yates’ correction and is indicated as follows: *, P  value of <0.05; ***, P < 0.001. Values are means ± SEM (error bars). Values that are not significantly different (ns) are indicated. These data are pooled from four experiments
FIG 8
FIG 8
MERS-CoV inhibits rRNA degradation in human airway cells. (A to C) rRNA degradation pattern from cells 36 h postinfection (A), quantification by RNA integrity number (RIN) (B), and replication kinetics (C) of mock-infected and MERS-CoV-, MERS-ΔNS4b-, and MERS-ΔNS3-5-infected Calu-3 cells (MOI of 1 PFU; n = 3). Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons and indicated as follows: ***, P value of <0.001; ns, not significant. Values are means ± SEM (error bars). RNA from Sindbis virus-infected human A549 cells (run separately on the Agilent Bioanalyzer) is shown in panel A as a marker for the RNase L-induced pattern of degradation of human rRNA. (D) rRNA degradation pattern from cells 24 and 48 h after MERS-NS4bH182R infection of Calu-3 cells. The positions of 28S and 18S rRNAs are indicated in panels A and D. Data shown are from one representative experiment of two.
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