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Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis Vineet D Menachery a b Lisa E Gralinski b Hugh D Mitchell d Kenneth H Dinnon III b Sarah R Leist b Boyd L Yount Jr b Rachel L Graham b Eileen T McAnarney a b Kelly G Stratton d Adam S Cockrell b Kari Debbink e Amy C Sims b Katrina M Waters d Ralph S Baric b c Department of Microbiology and Immunology University of Texas Medical Branch Galveston Texas USA a Department of Epidemiology University of North Carolina at Chapel Hill Chapel Hill North Carolina USA b Department of Microbiology and Immunology University of North Carolina at Chapel Hill Chapel Hill North Carolina USA c Pacific Northwest National Laboratory Richland Washington USA d Department of Natural Sciences Bowie State University Bowie Maryland USA e ABSTRACT Coronaviruses CoVs encode a mixture of highly conserved and novel genes as well as genetic elements necessary for infection and pathogenesis raising the possibility of common targets for attenuation and therapeutic design In this study we focused on highly conserved nonstructural protein 16 NSP16 a viral 2 O methyltransferase 2 O MTase that encodes critical functions in immune modulation and infection Using reverse genetics we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV MERS CoV NSP16 D130A and evaluated the effect on viral infection and pathogenesis While the absence of 2 O MTase activity had only a marginal impact on propagation and replication in Vero cells dNSP16 mutant MERS CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures and in vivo Further examination indicated that dNSP16 mutant MERS CoV had a type I interferon IFN based attenu ation and was partially restored in the absence of molecules of IFN induced proteins with tetratricopeptide repeats Importantly the robust attenuation permitted the use of dNSP16 mutant MERS CoV as a live attenuated vaccine platform protecting from a challenge with a mouse adapted MERS CoV strain These studies demonstrate the importance of the conserved 2 O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting IMPORTANCE Coronavirus CoV emergence in both humans and livestock repre sents a significant threat to global public health as evidenced by the sudden emer gence of severe acute respiratory syndrome CoV SARS CoV MERS CoV porcine epi demic diarrhea virus and swine delta CoV in the 21st century These studies describe an approach that effectively targets the highly conserved 2 O MTase activ ity of CoVs for attenuation With clear understanding of the IFN IFIT IFN induced proteins with tetratricopeptide repeats based mechanism NSP16 mutants provide a suitable target for a live attenuated vaccine platform as well as therapeutic develop ment for both current and future emergent CoV strains Importantly other ap proaches targeting other conserved pan CoV functions have not yet proven effective against MERS CoV illustrating the broad applicability of targeting viral 2 O MTase function across CoVs KEYWORDS IFIT MERS CoV SARS CoV coronavirus emerging virus live attenuated methyltransferase vaccine Received 8 August 2017 Accepted 26 October 2017 Published 15 November 2017 Citation Menachery VD Gralinski LE Mitchell HD Dinnon KH III Leist SR Yount BL Jr Graham RL McAnarney ET Stratton KG Cockrell AS Debbink K Sims AC Waters KM Baric RS 2017 Middle East respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis mSphere 2 e00346 17 https doi org 10 1128 mSphere 00346 17 Editor Ana Fernandez Sesma Icahn School of Medicine at Mount Sinai Copyright 2017 Menachery et al This is an open access article distributed under the terms of the Creative Commons Attribution 4 0 International license Address correspondence to Ralph S Baric Rbaric email unc edu Targeting 2 O methyltransferase offers universal approach for live attenuated CoV vaccine generation in outbreak RESEARCH ARTICLE Host Microbe Biology crossm November December 2017 Volume 2 Issue 6 e00346 17 msphere asm org 1 on December 16 2017 by guest http msphere asm org Downloaded from T he emergence of Middle East respiratory syndrome coronavirus MERS CoV in 2012 represents the second severe CoV to strike the human population since the beginning of the 21st century 1 Similar to its predecessor severe acute respiratory syndrome CoV SARS CoV MERS CoV is characterized by severe lung infection and high mortality rates 2 Associated with elderly patients and nosocomial spread MERS CoV is likely harbored in camel populations with periodic reintroductions into humans followed by periodic nosocomial outbreaks in hospital settings 3 Impor tantly with the continued rate of globalization MERS CoV remains an ongoing threat for future outbreaks both in and outside the Middle East as evidenced by the large outbreak in South Korea 4 Together these factors highlight the importance of examining CoV pathogenesis and developing conserved therapeutic targets for the treatment of current and future emergent strains Like all members of the CoV family MERS CoV maintains a balance of conserved and novel viral proteins within its genome 5 It is a member of the group 2C CoV family and a wealth of distinct accessory open reading frames and nonstructural proteins NSPs have already been established to have important roles in modulation of the host immune response 6 Similarly a number of viral proteins highly conserved in structure replication and fidelity are also maintained in the CoV backbone 7 Among these MERS CoV NSP16 provides a potent target for therapeutic development A 2 O methyltransferase 2 O MTase CoV NSP16 has been implicated in the capping of viral RNA and prevention of its recognition by the intracellular sensor MDA5 and antiviral effectors including members of the IFIT interferon IFN induced proteins with tetratri copeptide repeats family 8 Generation of mutants with changes in the NSP16 KDKE active site resulted in IFN mediated in vitro and in vivo attenuation of both mouse hepatitis virus MHV and SARS CoV 9 10 Therefore an approach targeting MERS CoV NSP16 might be anticipated to result in attenuation and potentially provide a universal platform for CoV vaccines against future emergent strains Using reverse genetics to target residues in the highly conserved active site we evaluated MERS CoV infection outcomes in the context of inactive NSP16 dNSP16 Consistent with previous studies of SARS CoV 10 the dNSP16 MERS CoV mutant maintained no significant attenuation in terms of replication or the initial host immune response However both primary human airway epithelial HAE cells and in vivo studies in a MERS CoV mouse model demonstrated robust attenuation of dNSP16 mutant growth and pathogenesis Notably attenuation was both IFN and IFIT1 dependent providing a clear mechanism for attenuation Importantly the dNSP16 mutant also provided robust protection against a lethal MERS CoV challenge and maintained attenuation in the mouse adapted backbone Together the results illustrate the broad conservation and necessity of NSP16 in CoV pathogenesis and highlight the targeting of this protein as a rapid response platform for future emergent CoV strains RESULTS A combination of structural and biochemical approaches has established a critical role for CoV NSP16 in 2 O MTase activity Fig 1A Stabilized by interactions with NSP10 orange NSP16 has been identified as a structurally conserved AdoMet dependent methyltransferase 11 despite divergence in protein sequence across organisms an invariant KDKE motif red within the methyltransferase core is required to mediate its activity 12 This KDKE motif is highly conserved within all of the NSP16 sequences examined in the CoV family Fig 1B Importantly mutation of any of the KDKE residues has been shown to ablate 2 O MTase activity 11 In addition previous alteration of this motif in both group 2b SARS CoV 10 and group 2a MHV 9 disrupted 2 O MTase activity and attenuated various aspects of infection On the basis of high conservation in the CoV family we hypothesized that disruption of the KDKE motif would also attenuate other emerging CoV families including group 2c MERS CoV Utilizing a MERS CoV reversed genetic system 13 we disrupted the KDKE motif by mutating two nucleotides to produce a D130A change Fig 1A The resulting disrupted NSP16 dNSP16 mutant had no significant defect noted in stock titer generation not shown Menachery et al November December 2017 Volume 2 Issue 6 e00346 17 msphere asm org 2 on December 16 2017 by guest http msphere asm org Downloaded from similarly infection of both Vero cells and Calu 3 2B4 cells a respiratory epithelial cell line at a low multiplicity of infection MOI demonstrated only modest attenuation at late time points Fig 1C and D Together these results indicate that NSP16 activity is not required for replication Similar in vitro host responses of SARS CoV and MERS CoV dNSP16 mutants Having established replication competence in both Vero and Calu 3 2B4 cells we next evaluated the induction of host pathways following infection Calu 3 2B4 cells infected at an MOI of 5 demonstrated no differences in replication not shown and only modest differences in host induction zero genes with a log 2 change in expression ofH110221 5 fold similar to observations with NSP16 mutant SARS CoV compared with wild type WT SARS CoV 10 However unlike in studies with SARS CoV a rapid cytopathic effect CPE by 24 h limited the analysis to early time points with WT and dNSP16 mutant FIG 1 NSP16 is highly conserved in the CoV family A MERS CoV NSP16 NSP10 complex Shown is NSP16 gray highlighting the conserved KDKE motif red required for 2 O MTase activity Also shown is the NSP10 scaffold required for MERS CoV orange The inset displays conserved KDKE left and the D130A mutation right that disrupts function Homology models were created with Modeler in the Max Planck Institute Bioinformatics Toolkit The known crystal structure of the NSP16 NSP10 complex 3R24 in the RCSB Protein Data Bank was used as the template structure 38 Homology models were then manipulated with MacPyMol B Heat maps were constructed from a set of representative CoVs from all four genogroups by using alignment data paired with neighbor joining phylogenetic trees built in Geneious v 9 1 5 and visualized in EvolView http evolgenius info Trees show the degree of genetic similarity of NSP16 across CoV families C and D Viral replication of dNSP16 mutant MERS CoV red relative to WT MERS CoV black in Vero C and Calu 3 2B4 D cells following infection at an MOI of 0 01 P H11021 0 001 Student t test MERS CoV NSP16 Is Necessary for Pathogenesis November December 2017 Volume 2 Issue 6 e00346 17 msphere asm org 3 on December 16 2017 by guest http msphere asm org Downloaded from MERS CoV Further David based analysis compared the network host responses to the MERS CoV and SARS CoV dNSP16 mutants Fig 2 Over the first 24 h of infection both MERS CoV and SARS CoV dNSP16 mutant infections showed no significant func tional enrichment of any categories relative to corresponding WT infections consistent with the lack of replication attenuation However at late times H1102224 h postinfection SARS CoV produced robust changes in several host pathways including cytokine responses inflammation and extracellular activity Similarly changes in apoptosis transcription repression and regulation of phosphorylation indicated a host response more hostile to viral infection While a more rapid CPE following both WT and dNSP16 mutant MERS CoV infections precluded an equivalent finding at late time points the SARS CoV results suggest that the absence of NSP16 activity eventually initiates host response changes that contribute to attenuation at late time points MERS CoV dNSP16 mutant attenuated in primary and in vivo models To further examine the replicative capacity of the dNSP16 mutant we infected both HAE cells and mice expressing human dipeptidyl peptidase 4 the receptor for MERS CoV Primary HAE cell cultures were challenged with WT and dNSP16 MERS CoV at a low MOI Fig 3A While robust replication was observed following WT infection dNSP16 MERS CoV had significant attenuation that corresponded well to previous results obtained with dNSP16 mutant SARS CoV 10 We next examined dNSP16 mutant MERS CoV replication phenotypes in the context of in vivo infection by using an adenovirus BALB c mouse transduction model 14 While neither infection produced weight loss not shown WT MERS CoV replicated efficiently at both days 2 and 4 postinfection Fig 3B in contrast no detectable replication was seen following infec tion with dNSP16 mutant MERS CoV The lack of replication may be due to residual IFN FIG 2 dNSP16 mutant MERS CoV infection produces minimal changes in early host responses Changes in functional host gene clusters on the basis of RNA expression following infection at an MOI of 5 of Calu 3 2B4 cells with dNSP16 mutant MERS CoV left or SARS CoV right relative to the WT control virus The heat map plots significant enrichment of clustered functional categories as determined by David analysis for each mutant over time Only marginal changes were noted during the first 24 h for both SARS CoV and MERS CoV dNSP16 mutants After 24 h right significant changes due to SARS CoV were noted MERS CoV had a significant CPE after 24 h postinfection precluding analysis Menachery et al November December 2017 Volume 2 Issue 6 e00346 17 msphere asm org 4 on December 16 2017 by guest http msphere asm org Downloaded from responses associated with initial adenovirus infection For greater clarity we next infected CRISPR Cas9 targeted mice that include mutations in Dpp4 at positions 288 and 330 288 330 H11001 H11001 conferring efficient WT MERS CoV infection and growth in mice but no clinical disease 15 Following infection no changes were observed in weight loss in either group of mice consistent with previous findings data not shown However absence of NSP16 activity severely attenuated dNSP16 mutant virus replica tion at both days 2 and 4 postinfection Fig 3C Coupled with data from HAE cell cultures and the adenovirus model the results demonstrate clear attenuation of dNSP16 mutant MERS CoV relative to the control dNSP16 mutant MERS CoV attenuation is mediated by IFN and IFIT1 Having established a deficit in dNSP16 mutant MERS CoV replication in relevant in vitro and in vivo models we next sought to evaluate the mechanism of attenuation Previous work by our lab and others had established increased susceptibility of dNSP16 mutants to type I IFN 9 10 however dNSP16 mutant SARS CoV had not shown augmented type I IFN stimulation following infection 10 Consistent with this finding infection with dNSP16 mutant MERS CoV produced stimulation of type I IFN transcript similar to that seen following in vivo infection of Dpp4 mutant 288 330 H11001 H11001 CRISPR mice with the WT virus Fig 4A In contrast while both the WT and mutant viruses were sensitive to IFN treatment dNSP16 mutant MERS CoV had a significant reduction in replication relative to the control virus Fig 4B These attenuation results are consistent with reports of NSP16 mutants of other CoVs and are in contrast to the equivalent replication observed without pretreatment Fig 1C 8 Extending this analysis further we examined the role of IFIT1 and IFIT2 gene expression in this attenuation phenotype in previously con FIG 3 dNSP16 mutant MERS CoV is attenuated in primary cultures and in vivo A Primary HAE cells infected with WT black or dNSP16 mutant red MERS CoV at an MOI of 0 01 and monitored over a time course B Day 2 and 4 lung titers of adenovirus transduced mice expressing human Dpp4 infected with WT black or dNSP16 mutant red MERS CoV C Day 2 and 4 lung titers of 288 330 H11001 H11001 CRISPR Cas9 targeted mice infected with WT black or dNSP16 mutant red MERS CoV P H11021 0 05 P H11021 0 01 P H11021 0 001 Student t test n H11005 H113504 for each experimental group at each time point over multiple experiments MERS CoV NSP16 Is Necessary for Pathogenesis November December 2017 Volume 2 Issue 6 e00346 17 msphere asm org 5 on December 16 2017 by guest http msphere asm org Downloaded from structed stable short hairpin RNA shRNA knockdown cell lines 10 Similar to SARS CoV knockdown of IFIT1 augmented replication of dNSP16 mutant MERS CoV in the context of type I IFN pretreatment Fig 4C In addition knockdown augmented WT MERS CoV infection suggesting sensitivity to IFIT1 activity despite the presence of NSP16 Notably IFIT2 knockdown had only a modest nonsignificant impact on repli cation contrasting with results obtained with SARS CoV Overall the data indicate that dNSP16 mutant MERS CoV attenuation is driven by sensitivity to type I IFN mediated by the activity of IFIT1 rather than augmented IFN responses NSP16 mutant vaccination protects from a lethal MERS CoV challenge On the basis of IFN and IFIT1 attenuation phenotypes targeting of NSP16 offers a potential platform strategy for live attenuated vaccine generation While previous work by our group had shown that the dNSP16 mutant SARS CoV conferred protection from a lethal challenge similar phenotypes in other more distant CoVs are essential for establishing universal principles of attenuation across a virus family To test this hypothesis Dpp4 288 330 H11001 H11001 mutant mice were vaccinated with dNSP16 mutant MERS CoV and subse quently challenged with a mouse adapted MERS CoV strain Fig 5 15 Following the challenge dNSP16 mutant vaccinated mice showed only modest weight loss in sig nificant contrast to the severe disease seen in the control group Fig 5A In addition both viral replication and lung hemorrhage were significantly reduced in the context of the dNSP16 mutant vaccine Fig 5B and C Importantly serum analysis revealed robust virus neutralization with values similar to those seen in serum from WT virus infected mice Fig 5D Together the results indicate that dNSP16 mutant MERS CoV can function as a vaccine platform that not only induces high levels of neutralizing antibodies but provides compete protection from a lethal MERS CoV challenge NSP16 mutation attenuates mouse adapted MERS CoV Despite conferring pro tection in the WT MERS CoV backbone it was unclear if the NSP16 mutant would be sufficiently attenuated in a virulent MERS CoV backbone To address this question we inserted the dNSP16 mutation D130A into the mouse adapted MERS CoV backbone 15 Following infection mouse adapted MERS CoV produced rapid weight loss and death Fig 6A In contrast the mouse adapted dNSP16 mutant produced only modest weight loss and 100 survival following infection In addition the replication of the dNSP16 mutant was significantly attenuated relative to that of the WT at days 2 and 4 postinfection Fig 6B Finally hemorrhage scoring of the lung revealed minimal disease in dNSP16 mutant immunized mice relative to control mice at day 4 postin fection Fig 6C Overall the results demonstrate robust attenuation of MERS CoV pathogenesis in the context an NSP16 mutation FIG 4 dNSP16 mutant MERS CoV attenuated by type I IFN treatment via IFIT1 A IFN H9252 IFN H92514 and IFN H92517 RNA expression in the lungs of 288 330 H11001 H11001 CRISPR Cas9 targeted mice at 2 days postinfection with WT black or dNSP16 mutant red MERS CoV Values are representative of log 2 fold expression relative to mock infected lungs as measured by real time reverse transcription PCR B Vero cells were treated with type I IFN 1 000 U 16 h prior to infection with either WT black or dNSP16 mutant red MERS CoV C Vero cells expressing shRNA targeting IFIT1 blue or IFIT2 green or a no shRNA control cells black were pretreated with IFN H9252 PBL Laboratories and infected with