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RNA 3 end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10 nsp14 exoribonuclease complex Micka l Bouvet Isabelle Imbert Lorenzo Subissi Laure Gluais Bruno Canard 1 and Etienne Decroly 1 Architecture et Fonction des Macromol cules Biologiques Unit Mixte de Recherche 7257 Ecole Sup rieure d Ing nieurs de Luminy Case 925 Centre National de la Recherche Scienti que and Aix Marseille Universit 13288 Marseille France Edited by Peter Palese Mount Sinai School of Medicine New York NY and approved April 23 2012 received for review January 22 2012 The replication transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins nsp1 16 encoded by the ORF 1a 1b This complex includes replication enzymes commonly found in positive strand RNA viruses but also a set of RNA processing activities unique to some nidoviruses The nsp14 protein carries both exoribonuclease ExoN and guanine N7 methyltransferase N7 MTase activities The nsp14 ExoN activity ensures a yet uncharacterized function in the virus life cycle and must be regulated to avoid nonspeci c RNA degradation In this work we show that the association of nsp10 with nsp14 stimulates 35 fold the ExoN activity of the latter while playing no effect on N7 MTase activity Nsp10 mutants un able to interact with nsp14 are not pro cient for ExoN activation The nsp10 nsp14 complex hydrolyzes double stranded RNA in a 3 to 5 direction as well as a single mismatched nucleotide at the 3 end mimicking an erroneous replication product In contrast di tri and longer unpaired ribonucleotide stretches as well as 3 modi ed RNAs resist nsp10 nsp14 mediated excision In addi tion to the activation of nsp16 mediated 2 O MTase activity nsp10 also activates nsp14 in an RNA processing function poten tially connected to a replicative mismatch repair mechanism RNA proofreading viral evolution protein protein interaction capping I n 2003 an outbreak of an unusually pathogenic agent spread from China to the whole world More than 8 400 people were infected with 800 case fatalities by this novel coronavirus now known as severe acute respiratory syndrome coronavirus SARS CoV Viruses from the Coronavirus and Torovirus genera con stitute the Coronaviridae virus family which together with Arteriviridae and Roniviridae belong to the Nidovirales order 1 The coronavirus genome consists of a single stranded positive sense RNA of 27 32 kb the largest size for an RNA virus ge nome The former is directly translated into two polyprotein precursors corresponding to ORF 1a called pp1a and ORF 1a elongated by ORF 1b called pp1ab following a ribosomal frameshift The ribosomal frameshift frequency allows a three to vefold excess of pp1a over pp1ab The pp1a and pp1ab poly proteins are intracellularly processed by viral proteases to yield 11 and 16 11 5 nonstructural proteins nsps respectively These nsps assemble together with cellular factors to form a huge replication transcription complex RTC associated with membrane structures derived from the endoplasmic reticulum ER 2 3 Apart from enzyme activities usually essential for RNA genome replication transcription coronaviruses also en code for a set of RNA processing activities that are either unique to genera inside Nidovirales or are found only in a few other groups of RNA viruses 4 Among these activities are two RNA nucleases an exoribonuclease nsp14 named ExoN 5 and a uridylate speci c endoribonuclease nsp15 named NendoU 6 Additionally it has recently been demonstrated that SARS CoV nsp1 in association with the 40S ribosome subunit induces an endonucleolytic degradation of host mRNAs though the enzyme responsible of the cleavage has not been identi ed 7 The presence of RNA endo and exonucleases is puzzling and therolesofnsp14andnsp15alongtheviruslifecycleareunknown Nsp14 is bifunctional with a 3 to 5 ExoN activity residing in its N terminal part 5 whereas a guanine N7 methyltransferase N7 MTase activityisembeddedintheC terminalpart 8 9 The N7 MTase domain is not functionally separable from the ExoN domain because N terminal deletions of the protein impair nsp14 N7 MTase activity 8 Nsp14 contains essential ExoN exo ribo nucleases containing a conserved Asp Glu Asp Asp motif DEDD ExoN superfamily motifs and has been shown to hy drolyze single and double stranded RNAs ssRNA and dsRNA to nal products of 8 12 nt and 5 7 nt respectively This enzyme hasbeenproposedtobeinvolvedinreplicationandrecombination during minus strand discontinuous transcription 5 10 11 However murine hepatitis virus MHV and SARS CoV viruses ExoN mutants exhibit growth defects but are competent for replication in cell culture 12 13 Notably nsp14 mutant viruses exhibit a mutator phenotype with an overall 12 to 20 fold increase in mutation frequency and up to 14 fold increase in mutation rate compared with WT Nsp14 was therefore pro posed to be involved in proofreading repair and or re combination mechanisms essential to maintain the integrity of the astonishingly long CoV s RNA genome for a review see ref 14 However the mechanisms by which nsp14 ExoN safeguards replication delity remain to be discovered Nsp14 is known to interact with nsp10 15 16 whose crystal structure is known 17 18 Nsp10 also interacts with nsp16 in vitro forms a complex whose structure has been recently solved and switches on an RNA cap 2 O MTase activity carried by nsp16 9 19 21 In this work we demonstrate a second regulatory role for SARS CoV nsp10 Nsp14 is converted by nsp10 into a 35 fold more potent exoribonuclease Because substrate requirements of the nsp10 nsp14 complex include dsRNAs having a mismatched or Watson Crick base paired 3 end our results suggest that nsp14 is a proofreading enzyme in agreement with the mutator phenotype observed for coronavirus nsp14 mutants 12 14 Results SARS CoV nsp10 Protein Interacts with the nsp14 Protein The nsp10 and nsp14 proteins were previously shown to interact using yeast and mammalian two hybrid systems 15 16 We sought to demonstrate this interaction using an in vitro system For this purpose SARS CoV Strep nsp10 and nsp14HN proteins were coexpressed in Escherichia coli and the bacterial cell lysate was incubated with Strep Tactin beads to bind the Strep tagged Author contributions M B B C and E D designed research M B I I L S L G and E D performed research M B B C and E D analyzed data and M B B C and E D wrote the paper The authors declare no con ict of interest This article is a PNAS Direct Submission 1 To whom correspondence may be addressed E mail etienne decroly afmb univ mrs fr or bruno canard afmb univ mrs fr This article contains supporting information online at www pnas org lookup suppl doi 10 1073 pnas 1201130109 DCSupplemental 9372 9377 PNAS June 12 2012 vol 109 no 24 www pnas org cgi doi 10 1073 pnas 1201130109 nsp10 protein As shown in Fig 1A nsp14 remains associated with nsp10 when both proteins are coexpressed whereas nsp14 alone is unable to bind to the beads We thus con rm that nsp14 stably interacts with nsp10 Nsp10 Protein Activates nsp14 ExoN Activity To discover a potential function for this interaction we produced both proteins in dividually in E coli and puri ed them separately using af nity followed by size exclusion chromatography Nsp10 was re covered with an N terminal hexahistidine tag His 6 and nsp14 was recovered as an untagged protein Materials and Methods We rst incubated a 5 end 32 P labeled ssRNA substrate named p H4 Table S1 with nsp14 in the presence or absence of nsp10 Reaction products were separated by denaturing Urea PAGE and revealed using autoradiography Nsp14 alone exhibits nuclease activity albeit weak with this RNA substrate Fig 1B lane 2 major degradation products are indicated by in agreement with others 5 11 The nsp10 protein does not carry any nuclease activity under these conditions Fig 1B lane 3 but incubation of both proteins results in a strong nuclease activity lane 4 major degradation products are indicated by As the RNA was labeled at its 5 end the laddering degradation pattern is suggestive of a 3 to 5 directionality We determined optimal reaction conditions as well as metallic ion requirements of the nsp10 nsp14 ExoN activity Interestingly we found that the latter critically depends both on metallic ions such as Mg 2 and on the presence of the Zn 2 ions of nsp10 Fig S1 and SI Text We also determined nsp10 nsp14 concen tration ratio yielding the maximal ExoN activity As shown in Fig 1C the ExoN activity is stimulated by nsp10 in a dose dependent manner until a 35 fold stimulation reached with a fourfold excess of nsp10 over nsp14 At equimolar ratio as used in Fig 1B the ExoN activity is stimulated around 20 fold compared with the ExoN activity exhibited by nsp14 alone We infer that besides the recently identi ed stimulation effect of nsp10 onto nsp16 MTase activity 9 nsp10 also stimulates the ExoN activity of nsp14 In contrast the presence of nsp10 has no effect on nsp14 mediated N7 MTase activity as reported before 9 ExoN Activity Exhibited by nsp10 nsp14 Involves the DEDDh Catalytic Residues of nsp14 To formally demonstrate that nsp10 does not have any nuclease activity switched on by the presence of nsp14 we mutated the nsp14 conserved catalytic residues D 90 XE 92 D 243 H268 and D273 of the DEDDh ExoN motifs to alanine residues We also substituted residue D 331 from the DxG motif implicated in S adenosyl methionine SAM binding 8 and used it as a negative control As shown in Fig 2A mutations of residues belonging to motif Exo I II or III completely abrogate ExoN activity Nsp14 mutants migrate at their expected molec ular weight after puri cation and are known to keep their native folding because they exhibit N7 MTase activity in vitro 9 We also observe that the SAM binding site mutation within the N7 MTase domain D 331 A dampens ExoN activity However a vefold excess of nsp14 D 331 A allows one to observe an ExoN activity comparable to that of WT These results demonstrate that nsp10 nsp14 ExoN activity involves the nsp14 DEDDh cat alytic residues and that nsp10 acts as an activation cofactor de void of nuclease activity per se Moreover the SAM binding mutant D 331 A reveals a cross talk between nsp14 N7 MTase and ExoN catalytic sites a result in agreement with the obser vation made by others that deletions in the ExoN domain of nsp14 lead to inactivation of the N7 MTase activity 8 ActivationofSARS CoVnsp14ExoNActivityRequiresDirectInteraction with nsp10 We selected residues N40 G69 H80 D82 localized onthesurfaceofthensp10proteintoperformalaninesubstitutions Fig 3A Strep tagged nsp10 mutants were coexpressed with nsp14 and puri ed using Strep Tactin resin Proteins eluted from Strep Tactin were separated by LabChip Caliper and peaks corresponding to nsp10 and nsp14 were quanti ed Fig 3B pres ents the percentage of interaction between nsp14 and nsp10 mu tants relative to nsp10 WT black bars G69A and H80A mutants completely lose their ability to interact with nsp14 whereas N40A and toalesserextent D82Akeeptheirinteractionproperties We next analyzed the consequence of these nsp10 mutations on the nsp10 nsp14 ExoN activity p H4 RNA was incubated with nsp14 in the presence of nsp10 WT or nsp10 mutants RNA hydrolysis was quanti ed upon denaturing Urea PAGE Fig 3B presents the percentage of nsp14 ExoN activity obtained with each nsp10 mu tant relative to nsp10 WT gray bars The results show that in teraction of nsp10 and nsp14 is required for the stimulation of ExoN activity Moreover we note that residues annihilating both nsp10 nsp14interactionandExoNactivity e g G69andH80 are localized within a surface area involved in the nsp10 nsp16 in teraction Fig 3A 19 20 Because these data suggested a com mon interaction surface for both nsp14 and nsp16 with nsp10 we performed competition experiments to evaluate complex stabili ties AsshowninFig S2A a16 foldmolarexcessofnsp16isunable to alter nsp10 nsp14 mediated ExoN activity We then tried to de stabilizensp10 nsp16interactionusingnsp14 AsshowninFig S2B the presence of nsp14 increases the 2 O MTase activity of nsp10 nsp16 and the effect was even higher in the presence of the nsp10 Fig 1 Nsp10interactswithnsp14 andnsp10 nsp14 showsenhancedExoNactivity A SARS CoVnsp14HNandStrep nsp10 proteinscoexpressedorexpressed alone were incubated with Strep Tactin Sepharose Strep Tactin bound proteins were eluted with D desthiobiotin and analyzed by SDS PAGE and Coomassie blue staining Lane1 corresponds tothe molecular size markers lane 2 to Strep nsp10 expressed alone lane 3 tonsp14HNexpressed alone and lane 4to Strep nsp10coexpressedwithnsp14HN B AutoradiogramofRNAcleavageproducts Synthetic p H4RNAwasradiolabeledatits5 endusingPNKinthepresenceof 32 P ATP theasteriskindicatesthe 32 P labelingposition p H4RNAwasincubatedat37 CinTris HClbuffer40mM pH8 DTT5mMwithnoprotein lane1 0 7 Mofnsp14 lane2 nsp10 lane3 orbothproteins lane4 duringa90 minperiod Thereactionproductswerethenseparatedona20 wt vol denaturing Urea PAGE and revealed using photostimulated plates and a FujiImager Fuji C p H4 RNA was hydrolyzed with xed nsp14 concentration 50 nM in the presence of increasing concentration of nsp10 ranging from 0 to 1 600 nM nsp10 nsp14 is indicated below the bar graph ExoN activity was quanti ed using denaturing Urea PAGE followed by measuring the hydrolysis of p H4 corresponding band using a FujiImager and Image Gauge software analysis Bouvet et al PNAS June 12 2012 vol 109 no 24 9373 BIOCHE MISTRY nsp14complex Weconcludethatonceformed bothcomplexesare stable Nsp10 nsp14 might stimulate further nsp10 nsp16 2 O MTase activity suggesting a cross talk between nsp14 and nsp16 MTase activities both of which are involved in RNA capping Substrate Requirements of the SARS CoV nsp10 nsp14 ExoN To de termine the nsp10 nsp14 ExoN activity substrate requirements we used a set of 5 end radiolabeled RNAs as substrates their secondary structures were predicted using the Mfold RNA modeling server and are presented in Fig S3 ExoN assays in dicate that nsp10 nsp14 ExoN activity requires an RNA 3 end engaged in a duplex structure SI Text and Fig S4 This nding is also illustrated in Fig 4 where a ssRNA oligonucleotide RNA11 incubated with the enzyme complex nsp10 nsp14 is barely degraded major degradation products are indicated by Upon annealing of its complementary strand carrying a biotin RNA11revbiot at its 3 end the resulting dsRNA heteroduplex is rapidly hydrolyzed by nsp10 nsp14 into shorter products of 3 4 nt indicated by in Fig 4 Together these results show that nsp10 nsp14 ExoN shows no obvious sequence preference but requires the 3 end to be engaged in a stable RNA duplex Nsp14 ExoN Activity Requires a Free 3 Hydroxyl End and Proceeds 3 to 5 Wenextinvestigatedthe effectof a modi cation of the RNA 3 OH end on nsp10 nsp14 ExoN activity Fig 5A shows that dsRNA oligonucleotides carrying either a 3 terminal puromycin p H4 puro or phosphate H4 pCp vs H4 pC OH resist nsp10 nsp14 mediated hydrolysis We also analyzed the hydrolysis of 3 end labeled H4 pC OH its hydrolysis leads to the immediate recovery of a unique product comigrating with a pC OH control demonstrating that nsp10 nsp14 proceeds 3 to 5 Because the nsp10 nsp14 ExoN activity was proposed to be functionally linked to the nsp15 NendoU activity e g in a proofreading mechanism 14 we assayed nsp15 NendoU products as substrates for the ExoN activity of nsp10 nsp14 For this purpose RNAs containing an internal CUU or GUU se quences H2 CUU N 10 and H5 GUU N 10 were incubated with nsp15 to generate a terminal 2 3 cyclic phosphate RNAs H2 CU U P and H5 GU U P 22 23 As shown in Fig 5B in contrast to unmodi ed RNAs used as controls RNAs carrying a2 3 cyclic phosphate end resist nsp10 nsp14 mediated hydro lysis We conclude that irrespective of a putative role of nsp14 in mismatch repair nsp15 mediated RNA endonucleolytic cleavage is unlikely to serve as an entry point for nsp14 ExoN SARS CoV nsp14 ExoN Activity Is Able to Excise a 3 Mismatched Nucleotide We assayed the ability of nsp14 to excise 3 end mis matched nucleotides on a dsRNA mimicking an erroneous rep lication product Fig S5 For this purpose a 40 mer RNA LS1 carrying a 3 biotin group was annealed to a radiolabeled reverse complement RNA carrying no mismatch LS2 or adding one two three or four mismatched nucleotides to its 3 end LS3 LS6 Fig 6A shows that nsp10 nsp14 is able to excise a single 3 terminal mismatch but excision capability strongly decreases when a longer mismatch is introduced at the 3 end The time Fig 2 Mutagenesis analysis of nsp14 ExoN activity Residues from the nsp14 ExoN catalytic site and from the SAM binding site of the nsp14 MTase domain weremutatedintoalanine Equalamountsofeachnsp14mutantwereincubated with nsp10 and p H4 RNA for 0 2 and 30 min In lane 5XD331A the concen tration of the mutant was vefold higher The panel shows the time dependent hydrolysis of p H4 RNA after Urea PAGE separation and autoradiography Fig 3 Nsp10 nsp14 interaction is required for nsp14 ExoN stimulation A Selection of nsp10 surface mutants and their position in the nsp10 nsp16 dimer The nsp10 nsp16 complex image Upper was represented using PyMOL and an enlargement of nsp10 is shown Lower with the position of mutated amino acids highlighted in blue nsp16 is represented in green nsp10 in gold and nsp10 zinc structural ions as gray spheres B Quanti cation of nsp10 nsp14 interaction and corresponding ExoN activity Nsp10 WT or mutants proteins carrying a Strep TagII were coexpressed with nsp14HN After puri cation on Strep Tactin beads eluted proteins were separated using LabChip Caliper and the intensities of peaks corresponding to nsp10 and nsp14 proteins were quanti ed Molecular ratio obtained for nsp10 WT nsp14 complex was taken as 100 Results are presented in percentage of interaction compared with the nsp10 WT nsp14 complex black bars For ExoN quanti cation equal amounts of nsp10 mutants were incubated with nsp14 and p H4 RNA After 30 min of incubation reaction products were separated using Urea PAGE revealed using a FujiImager and quanti ed using Image Gauge software Results are pre sented in percentage of ExoN activity nsp10 WT nsp14 was taken as 100 of activity gray bars 9374 www pnas org cgi doi 10 1073 pnas 1201130109 Bouvet et al course of RNA degradation shown in Fig 6B indicates that a mismatched nucleotide is excised at the same rate as that of aregularWatson Crick base pair whereas the RNA excision rate decreases as a function of the extension of unpaired sequences To determine the substrate preference between a Watson Crick and a mismatched 3 end base pair we performed competition experiments using a radiolabeled substrate carrying either a U AoraU C3 end base pair Cold substrates were used as challengers competing for nsp10 nsp14 binding and degrada tion As shown in Fig S6 A and B a substrate carrying a 3 end mismatch competes out more ef ciently the perfectly matched 3 end substrate than the other way around This result indi cates that a mismatched 3 end base pair might actually be the preferred substrate for nsp10 nsp14 ExoN activity Finally we compared the excision ef ciency using different mismatched base pairs Single mismatches of any type A G A A A C U G U C U U were removed ef ciently initial velocity measure ments of nsp14 mismatch excision indicate that the ability of the ExoN to degrade these substrates does not depend on the nature of the nucleotide misincorporated at the 3 end Fig S6C Because the main RNA dependent RNA polymerase RdRp nsp12 is known to interact with nsp14 15 we conclude that nsp10 nsp14 is able to ef ciently remove a 3 terminal mismatch present on an RNA supposedly being synthesized by nsp12 Longer mismatched structures unlikely to be synthesized by the viral polymerase do not act as nsp10 nsp14 ExoN substrates Discussion SARS CoV encodes several RNA processing activities including RNA