【病毒外文文獻】2015 Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Sp

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MAJOR ARTICLE Inhibition of Proprotein Convertases Abrogates Processing of the Middle Eastern Respiratory Syndrome Coronavirus Spike Protein in Infected Cells but Does Not Reduce Viral Infectivity Stefanie Gierer 1 Marcel A M ller 2 Adeline Heurich 1 Daniel Ritz 2 Benjamin L Springstein 1 Christina B Karsten 1 3 Alexander Schendzielorz 1 Kerstin Gnir 1 Christian Drosten 2 and Stefan P hlmann 1 1 Infection Biology Unit German Primate Center G ttingen 2 Institute of Virology University of Bonn Medical Center and 3 Institute for Cellular Chemistry Hannover Medical School Germany Middle East respiratorysyndrome coronavirus MERS CoV infection is associated with a high case fatality rate and the potential pandemic spread of the virus is a public health concern The spike protein of MERS CoV MERS S facilitates viral entry into host cells which depends on activation of MERS S by cellular proteases Proteolytic activation of MERS S during viral uptake into target cells has been demonstrated However it is unclear whether MERS S is also cleaved during S protein synthesis in infected cells and whether cleavage is re quired for MERS CoV infectivity Here we show that MERS S is processed by proprotein convertases in MERS S transfected and MERS CoV infected cells and that several RXXR motifs located at the border between the surface and transmembrane subunit of MERS S are required for ef cient proteolysis However blockade of pro protein convertases did not impact MERS S dependent transduction of target cells expressing high amounts of the viral receptor DPP4 and did not modulate MERS CoV infectivity These results show that MERS S is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for S pro tein activation Efforts to inhibit MERS CoV infection by targeting host cell proteases should therefore focus on enzymes that process MERS S during viral uptake into target cells Keywords MERS coronavirus protease TMPRSS2 trypsin proprotein convertase spike activation The emergence and subsequent pandemic spread of the severe acute respiratory syndrome SARS coronavirus in 2002 2003 caused almost 800 deaths and wreaked enormous economic havoc 1 2 The virus was trans mitted from bats potentially via intermediate hosts to humans demonstrating that the zoonotic transmission of novel coronaviruses from animal reservoirs to humans can pose a signi cant threat to public health 3 4 A similar outbreak scenario unfolded in 2012 when a novel coronavirus initially named human coro navirus EMC and now termed Middle East respiratory syndrome coronavirus MERS CoV was detected in a patient from Jordan hospitalized with a severe and ulti mately fatal pneumonia 5 Subsequently the virus spread within the Middle East and through travel activ ity occasionally to Europe Africa Asia and North America 6 9 The outbreak has as of 4 July 2014 en tailed 827 laboratory con rmed infections and 287 deaths 10 and adaptation of the virus to more ef cient human to human spread is a public health concern Therefore it is imperative to identify and ex plore novel targets for antiviral therapy The viral surface protein spike S a type I transmem brane protein synthesized in the constitutive secretory pathway of infected cells mediates coronavirus entry into target cells 11 12 For this the MERS CoV spike protein MERS S binds to its receptor dipeptidyl peptidase 4 DPP4 CD26 on the surface of target cells 13 anddrives Received 23 April 2014 accepted 10 July 2014 Presented in part 7th European Meeting on Viral Zoonoses St Rapha l France 24 27 May 2014 Correspondence Stefan P hlmann PhD Infection Biology Unit German Primate Center Kellnerweg 4 37077 G ttingen Germany spoehlmann dpz eu The Journal of Infectious Diseases The Author 2014 Published by Oxford University Press on behalf of the Infectious Diseases Society of America All rights reserved For Permissions please e mail journals permissions DOI 10 1093 infdis jiu407 Proprotein Convertases and MERS CoV JID 1 Journal of Infectious Diseases Advance Access published August 22 2014 at University of California San Francisco on December 23 2014 http jid oxfordjournals org Downloaded from fusion of the viral envelope with a target cell membrane which al lows delivery of viral proteins and RNA into the host cell cyto plasm the site of viral replication However MERS S and other coronavirus S proteins are synthesized as inactive precursors in in fected cells and only acquire the ability to drive membrane fusion upon processing into the surface unit S1 and the transmembrane unit S2 by host cell proteases 14 15 The activity of the respon sible proteases is essential for viral infectivity which makes these enzymes potential targets for antiviral intervention It has been demonstrated that the cysteine protease cathepsin L 14 16 17 andthetypeIItransmembraneserineprotease TMPRSS2 14 16 17 can activate MERS S during viral binding and uptake into target cells However activation of viral glyco proteins including activation of the S protein of certain strains of the coronavirus infectious bronchitis virus IBV 18 may also proceed in the constitutive secretory pathway of infected cells and is often accomplished by furin and other proprotein convertases 19 22 Whether MERS S is also cleaved during the passage of the secretory pathway and whether these cleav ageeventscontributetoSproteinactivationisatpresent unknown Here we show that proprotein convertases process MERS S in transfected and infected cells and we demonstrate that the integrity of several RXXR motifs located at the border of the S1 and S2 subunit is required for S protein processing Treat ment of MERS CoV infected cells with a proprotein convertase inhibitor PCI abrogated S protein cleavage but did not alter viral infectivity indicating that S protein processing in infected cells is dispensable for MERS S activation MATERIALS AND METHODS Plasmids Expression plasmids encoding MERS S 14 vesicular stomatitis virus glycoprotein VSV G 23 Zaire ebolavirus glycoprotein EBOV GP 23 Lassa virus glycoprotein LASV GPC 23 DPP4 13 and TMPRSS2 24 have been described previously Plasmids pGAL4 VP16 25 pGAL5 luc 25 pNL4 3 Luc R E 26 and p96ZM651gag opt 27 as well as plasmids encoding MERS S 14 and EBOV GP 28 with a C terminal V5 tag have also been described A Constructs expressing LASV GPC and MERS S potential cleavage site mutants PCM 1 626 AXXA 629 PCM2 691 AXXA 694 PCM3 748 AXXA 751 and PCM4 884 AXXA 887 with a C terminal V5 tag were generated by polymerase chain reaction PCR based mutagenesis The integrity of all PCR ampli ed sequenc es was con rmed by automated sequence analysis Cell Culture 293T cells were propagated in Dulbecco s modi ed Eagle s me dium DMEM Life Technologies supplemented with 10 fetal bovine serum FBS PAN Biotech penicillin and streptomycin Caco 2 cells were cultured in DMEM GlutaMAX medium In vitrogen supplemented with 10 FBS penicillin and strepto mycin Vero B4 cells were cultured in DMEM supplemented with 5 FBS 1 L glutamine 1 sodium pyruvate 1 nones sential amino acids all from Life Technologies and antibiotics as stated above All cell lines were grown in humidi ed atmo sphere at 37 C and 5 CO 2 Analysis of MERS S Expression and Cleavage For the detection of MERS S expression in transfected cells 293T cells underwent calcium phosphate transfection with the respective plasmids encoding S proteins with a C terminal V5 antigenic tag For immunoblotting the lysates were separated by sodium dodecyl sulfate gel electrophoresis and transferred onto nitrocellulose membranes Hartenstein MERS S expres sion was detected using a monoclonal antibody directed against the V5 tag Invitrogen or a polyclonal antibody directed against the S2 subunit of the MERS S protein Sino Biological For detection of MERS S protein in infected cells Caco 2 and Vero B4 cells were infected with MERS CoV human betacor onavirus 2c EMC 2012 at a multiplicity of infection MOI of 0 01 and 5 respectively At 24 hours after infection the cells were harvested and treated with NuPAGE LDS sample buffer Invitrogen boiled for 20 minutes at 95 C and analyzed by Western blot as describedabove As a loading control the mem branes were incubated with anti actin antibody Sigma Inhibition of Proprotein Convertases To assess the role of proprotein convertase activity in MERS S processing in transfected 293T cells the cells were transfected with plasmids encoding MERS S EBOV GP and LASV GPC or with empty plasmid The medium was changed 6 hours after transfection and PCI Merck was added to the fresh medi um at the indicated concentrations Medium was replaced again 32 hours after transfection and inhibitor was replenished Cells were lysed 48 hours after transfection and cleavage of viral glyco proteins was detected by Western blot using a monoclonal anti body recognizing the V5tag As a loading control an anti actin antibody was used To assess whether proprotein convertase activity is required for MERS S driven cell cell fusion a previously described cell cell fusion assay was used 24 which is based on mixing effec tors cells expressing the trans activator VP16 with target cells expressing luciferase under the control of a VP16 responsive promoter Speci cally 293T target cells that expressed MERS S or no glycoprotein and effector cells that were transfected to express DPP4 and or TMPRSS2 were incubated with 1 M PCI One dayafter transfection the cells were mixed to allow cell cell fusion and medium was supplemented with PCI at 1 M nal concentration Cell cell fusion was quanti ed by determination of luciferase activity in cell lysates at 48 hours after cocultiva tion using a commercially available kit PJK 2 JID Gierer et al at University of California San Francisco on December 23 2014 http jid oxfordjournals org Downloaded from Foranalysis of the importance ofproproteinconvertase activity for MERS S driven virus cell fusion a previously established pseudotyping strategy was used 14 pNL4 3 Luc R E 26 vec tors bearing MERS S LASV GPC EBOV GP or VSV G or no glycoprotein were generated in the presence or absence of 1 M PCI As target cells 293T cells transfected with DPP4 encoding plasmid or empty plasmid were seeded into 96 well plates and pretreated with 0 5 M PCI for 60 minutes at 37 C The cells were then incubated with pseudotypes for 8 hours followed by replacement of infection medium with culture medium contain ing inhibitor Transduction ef ciency was measured after 72 hours by determining luciferase activities in cell lysates To determine the role of proprotein convertase activity in in fection withauthenticMERS CoV Caco 2 cells seededin24 well plates were incubated with dimethyl sulfoxide or rising concen trations of PCI for 1 hour at 37 C and were then inoculated with MERS CoV MOI 0 01 and 0 001 in quadruplicates in the pres enceofinhibitor Afterincubationfor 30minutes at 4 C the cells werewashed and again incubated in culture medium with the in hibitor At 24 hours after infection the cells were washed and harvested and the pelletwas lysed withRIPA lysis buffer supple mented with 4xNuPAGE Invitrogen and boiled for 20 minutes at 95 C S protein expression in lysates was detected by Western blot using a polyclonal antibody directed against the S2 subunit of MERS S Sino Biological In parallel for quanti cation of viral RNA 50 L of the cell supernatant was dissolved in RAV1 buffer Macherey Nagel for RNA extraction followed by quantitative reverse transcription PCR analysis using the upE assay as previously described 29 Quanti cation of infec tious particles was done by plaque assay using Vero B4 cells as published elsewhere 6 Brie y 10 fold dilutions of supernatants were tested in duplicates using cell monolayers of Vero B4 cells After 1 hourof virus adsorption cells werewashed and overlayed with a 1 2 avicel resin After 3 days the plates were xed with 7 paraformaldehyde and stained with crystal violet solution To investigatethe in uenceofPCI on the formationofMERS CoV induced cytopathogenic effects Vero B4 cells were infected at an MOI of 0 1 and xed with 7 paraformaldehyde 42 hours after infection MERS CoV antigen detection was performed by incu bation with a serum specimen from a patient with MERS as de scribed elsewhere 30 Bound antibodies were detected with a cyanine 2 labeled goat anti human immunoglobulin G second ary antibody Dianova Nuclei were stained by mounting the slides with DAPI containing ProLong Gold antifade mounting medium Life technologies RESULTS MERS S Is Proteolytically Processed in Transfected and Infected Cells To investigate MERS S cleavage in virus producing cells we de termined whether the S protein is cleaved in transfected and infected cells Western blot analysis of S protein transfected 293T cells and MERS CoV infected Vero B4 cells with an an tibody speci c to the S2 subunit of MERS S revealed 2 promi nent S protein bands with molecular weights of 170 kDa and 90 kDa Figure 1 in keeping with our previous results 14 The 170k Da band corresponds to uncleaved MERS S while the presence of the 90 kDa band indicates ef cient processing of MERS S into an N terminal S1 subunit not detected and a C terminal S2 subunit Figure 1 Several RXXR Motifs Located at the Border of the S1 and S2 Subunits Are Required for Processing of MERS S Inspection of the sequences located at the border of the S1 and S2 subunits of MERS S revealed the presence of 4 RXXR se quences Figure 2A which might represent recognition sites for proprotein convertases 22 To determine the importance of these motifs for MERS S cleavage we changed the arginine residues in 626 RXXR 629 691 RXXR 694 748 RXXR 751 and 884 RXXR 887 to alanine residues creating the potential cleavage site mutants PCM1 PCM2 PCM3 and PCM4 Fig ure 2A All S protein mutants were expressed with the same ef ciency as wild type MERS S in transfected cells Figure 2B The only exception was PCM2 for which consistently no ex pression was detected not shown Importantly mutation of potential cleavage sites 3 and 4 alone impacted S protein pro cessing the presence of the 90 kDa band was reduced in cells expressing PCM3 and a band with a molecular weight slightly Figure 1 The Middle East respiratory syndrome coronavirus MERS CoV spike protein MERS S is cleaved in transfected and infected cells 293T cells were transfected with a plasmid encoding the MERS S protein or with empty plasmid pcDNA Vero B4 cells were either infected with MERS CoV at a multiplicity of infection of 5 or mock infected Subse quently the cells were lysed and analyzed by Western blot using a poly clonal antibody directed against the S2 subunit of MERS S A actin antibody served as a loading control Similar results were obtained in 2 separate experiments Proprotein Convertases and MERS CoV JID 3 at University of California San Francisco on December 23 2014 http jid oxfordjournals org Downloaded from higher than 90 kDa was observed upon expression of PCM4 Figure 2B Processing of PCM1 was comparable to that seen for MERS S wild type However combined mutation of poten tial cleavage sites 1 and 3 PCM1 PCM3 abrogated S protein processing Figure 2B suggesting that also potential cleavage site 1 contributes to S protein cleavage in this experimental set ting Similar effects were seen when potential cleavage sites 3and4weresimultaneouslyaltered PCM3 PCM4 or when all 3 potential cleavage sites were mutated in parallel Figure 2 RXXR motifs located at the border between S1 and S2 are re quired for ef cient processing of the Middle East respiratory syndrome coro navirus spike protein MERS S A The domain organization of the MERS S protein is schematically depicted The MERS S sequence at the border be tween the S1 and S2 subunits is shown RXXR motifs which constitute po tential cleavage sites are highlighted and the predicted start of the S2 subunit is underlined The mutations introduced into the potential cleavage sitesinMERS Sareshown B 293T cells were transfected with expression plasmids coding for MERS S wild type and the indicated MERS S mutants equipped with a C terminal V5 tag Transfection of empty plasmid pcDNA served as negative control Expression of S proteins in cell lysates was deter mined by Western blot using a V5 tag speci c monoclonal antibody Expres sion of actin in cell lysates was assessed as a loading control The results shown are representative for at least 3 independent experiments Abbrevia tions CT cytoplasmic tail PCM potential cleavage site mutant RBD receptor binding domain SP signal peptide TM transmembrane domain Figure 3 The Middle East respiratory syndrome coronavirus spike pro tein MERS S is cleaved by proprotein convertases 293T cells were trans fected with expression plasmids encoding MERS S Zaire ebolavirus glycoprotein EBOV GP or Lassa virus glycoprotein LASV GPC all equipped with a C terminal V5 tag Cells transfected with empty plasmid pcDNA served as negative control Subsequently cells were incubated with the indicated concentrations of the proprotein convertase inhibitor PCI At 48 hours after transfection glycoprotein expression was analyzed by Western blot using a V5 tag speci c monoclonal antibody Detection of actin served as loading control The results shown are representative of three independent experiments 4 JID Gierer et al at University of California San Francisco on December 23 2014 http jid oxfordjournals org Downloaded from PCM1 PCM3 PCM4 Collectively these results indicate that MERS S cleavage is reduced or altered upon mutation of potential cleavage sites 1 3 and 4 MERS S Is Processed by Proprotein Convertases We next sought to identify the proteases responsible for MERS S cleavage For this we investigated whether S protein cleavage can be blocked by a cell permeable tripeptide derivative con taining an Arg X Arg motive which is known to inhibit several proprotein convertases including furin 31 Treatment with this PCI reduced the production of the 90 kDa band in cells transfected to express MERS S Figure 3 Moreover the pres ence of PCI diminished processing of the Zaire ebolavirus gly coprotein EBOV GP into GP1 and GP2 Figure 3 which depends on the activity of the proprotein convertase furin 32 In contrast processing of the glycoprotein of Lassa virus LASV GPC which is mediated by the proprotein convertase SKI 1 S1P 33 was not suppressed Figure 3 This nding is in keeping with the known differences in substrate speci city and thus inhibitor sensitivity of SKI I SIP compared with propro tein convertases with speci city for basic amino acids like furin 34 Collectively these results show that the activity of propro tein convertases is essential for MERS S processing in transfect ed cells Proprotein Convertase Activity Is Not Essential for Ef cient MERS S Driven Cell Cell and Virus Cell Fusion We next assessed whether proprotein convertase activity is re quired for S protein driven cell cell fusion For this we used a previously described assay 24 which is based on directed ex pression of S protein and receptor protease in effector and tar get 293T cells respectively Expression of MERS S in effector cells allowed inef cient fusion with control transfected 293T target cells which are known to express low amounts of endog enous DPP4 35 and the ef ciency of cell cell fusion was markedly increased when target cells were transfected with DPP4 and or TMPRSS2 encoding plasmid Figure 4A Howev er the continuous presence of PCI in target and effector cell cultures before during and after mixing had no appreciable ef fect on fusion ef ciency Figure 4A suggesting that proprotein convertase activity is dispensable for MERS S driven cell cell fusion Figure 4 Proprotein convertase activity is dispensable for Middle East respiratory syndrome coronavirus spike protein MERS S driven cell cell and virus cell fusion A Fusion of 293T effector cells transfected to express MERS S with target cells transfected to express DPP4 and or TMPRSS2 or control transfected with empty