【病毒外文文獻】2008 Fusion core structure of the severe acute respiratory syndrome coronavirus (SARS-CoV)_ In search of potent SARS-CoV

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Journal of Cellular Biochemistry 104 2335 2347 2008 Fusion Core Structure of the Severe Acute Respiratory Syndrome Coronavirus SARS CoV In Search of Potent SARS CoV Entry Inhibitors Ling Hon Matthew Chu 1 Siu Hong Chan 2 Sau Na Tsai 2 Yi Wang 3 Christopher Hon Ki Cheng 4 Kam Bo Wong 4 Mary Miu Yee Waye 1 4 5 and Sai Ming Ngai 1 2 1 Molecular Biotechnology Program The Chinese University of Hong Kong Hong Kong China 2 Department of Biology The Chinese University of Hong Kong Hong Kong China 3 Department of Cell Biology and Genetics College of Life Sciences Peking University Beijing China 4 Department of Biochemistry The Chinese University of Hong Kong Hong Kong China 5 Department of Biochemistry Croucher Laboratory for Human Genomics The Chinese University of Hong Kong Hong Kong China Abstract Severe acute respiratory coronavirus SARS CoV spike S glycoprotein fusion core consists of a six helix bundle with the three C terminal heptad repeat HR2 helices packed against a central coiled coil of the other three N terminal heptad repeat HR1 helices Each of the three peripheral HR2 helices shows prominent contacts with the hydrophobic surface of the central HR1 coiled coil The concerted protein protein interactions among the HR helices are responsible for the fusion event that leads to the release of the SARS CoV nucleocapsid into the target host cell In this investigation we applied recombinant protein and synthetic peptide based biophysical assays to characterize the biological activities of the HR helices In a parallel experiment we employed a HIV luc SARS pseudotyped virus entry inhibition assay to screen for potent inhibitory activities on HR peptides derived from the SARS CoV S protein HR regions and a series of other small molecule drugs Three HR peptides and five small molecule drugs were identified as potential inhibitors ADS J1 which has been used to interfere with the fusogenesis of HIV 1 onto CD4 cells demonstrated the highest HIV luc SARS pseudotyped virus entry inhibition activity among the other small molecule drugs Molecular modeling analysis suggested that ADS J1 may bind to the deep pocket of the hydrophobic groove on the surface of the central coiled coil of SARS CoV S HR protein and prevent the entrance of the SARS CoV into the host cells J Cell Biochem 104 2335 2347 2008 C223 2008 Wiley Liss Inc Key words SARS CoV fusion core peptide Severe acute respiratory syndrome corona virus SARS CoV is the causative agent respon sible for the worldwide epidemic of respiratory disease and the death of over 800 patients in 2003 Chim et al 2003 Ksiazek et al 2003 Lee et al 2003 Intensive researches are underway to search for viral proteins to be used for drug target for anti SARS therapies in preparation of future recurrences of SARS outbreaks Amongst different SARS CoV proteins the spike S protein is of major focus in SARS research for its vital role in target cell recogni tion and virus entry Choy et al 2004 Hofmann and Pohlmann 2004 As a type I membrane glycoprotein S protein is the major component of the characteristic spikes on the surface of SARS CoV It contains a total of 1 255 amino acid residues and is the largest structural protein amongst other C223 2008 Wiley Liss Inc Abbreviations used CD circular dichroism HIV 1 human immunodeficiency virus type 1 HR heptad repeat HR1 heptad repeat 1 HR2 heptad repeat 2 HR1 DP deep pocket region of heptad repeat 1 LS light scattering MALDI TOF Matrix assisted laser desorption ionization time of flight MHV murine hepatitis virus MW molecular weight Native PAGE native polyacrylamide gel electrophoresis S protein spike glycoprotein SARS severe acute respiratory syndrome SARS CoV severe acute respiratory syndrome coronavirus SEC size exclusion chromatography Tm transition midpoint temperature Grant sponsor SARS Special RGC Grant number CUHK4536 03M Grant sponsor Croucher Foundation Grant sponsor Special SARS funding from GreaterChina Technology Group Ltd Correspondence to Sai Ming Ngai Department of Bio logy The Chinese University of Hong Kong Shatin Hong Kong China E mail smngai cuhk edu hk Received 17 December 2007 Accepted 24 March 2008 DOI 10 1002 jcb 21790 SARS CoV proteins S protein consists of S1 residues 12 680 and S2 residues 681 1 255 subunits respectively Spiga et al 2003 Lio and Goldman 2004 The S1 subunit of the S protein is responsible for host cell receptor binding and the S2 subunit is responsible for the membrane fusion process The functional ele ments of S2 subunit that are involved in SARS CoV membrane fusion are a putative fusion peptide FP plus two highly conserved 4 3 hydrophobic repeat domains heptad repeat 1 HR1 and heptad repeat 2 HR2 The function of these three sequences had been elucidated in the context of prototypic type I fusion proteins such as human immunodeficiency virus 1 HIV 1 gp160 Eckert and Kim 2001b This suggests a similar membrane fusion mechanism for the SARS CoV S protein In HIV 1 binding of S1 subunit to target host cell surface receptor induces global changes in the conformation of S protein converting the S2 subunit from a native pre fusion state to a intermediate pre hairpin state This receptor induced conforma tional change is expected to facilitate the release of the fusion peptide FP and position it to interact with the target host cell membrane The intermediate pre hairpin then resolves itself into a final fusogenic state Eckert and Kim 2001a Biophysical studies on SARS CoV S protein suggests a similar conformational rearrangement which leads to the association of the two coiled coil regions within S2 subunit where three N terminal HR1 helices interact with three peripheral C terminal HR2 helices in an anti parallel manner to form a stable six helix coiled coil bundle structure which is known as SARS CoV S protein fusion core Liu et al 2004 Tripet et al 2004 Zhu et al 2004 Xu et al 2004b Such fusion core structure facilitates the juxtaposition of the SARS CoV onto target cell membranes destabilizes the membrane lipid bilayers and ultimately causes membrane fusion and the release of SARS CoV nucleocapsid into the host cell The high resolution structures from X ray crystallographic analysis of SARS CoV S pro tein Supekar et al 2004 Xu et al 2004a Duquerroy et al 2005 indicated the presence of the coiled coil structure in the heptad repeat regions of the S protein fusion core and was confirmed by other biophysical investigations Liu et al 2004 Tripet et al 2004 Zhu et al 2004 Xu et al 2004b In this study we have further characterized and confirmed the bio logically important interaction of the HR1 and HR2 in the fusion core The results can facilitate us to investigate the entry inhibitory activities of the HR peptides derived from HR regions and a series of small molecule drugs that may attenuate the SARS CoV infection in a pseudo typed virus system In conclusion three HR peptides and five small molecule drugs were identified as potential inhibitors RESULTS Design of Heptad Repeats By applying the Multicoil program Wolf et al 1997 on the S1 polypeptide sequence we have predicted that the HR1 region of S1 covers 112 amino acid residues residues 899 1 010 However it is unlikely that the whole region of HR1 would interact with HR2 which only covers 48 amino acid residues residues 1 145 1 192 Two different sites of interactions have been proposed by two individual research teams 1 Residues 916 950 of HR1 and residues 1 151 1 185 of HR2 Tripet et al 2004 and 2 Residues 902 952 of HR1 and residues 1 145 1 184 Xu et al 2004b In order to clarify the detailed site of interactions between the two heptad repeats and acquire accurate information for the SARS CoV S protein fusion core we have designed syn thetic peptides and recombinant proteins that cover both of the HR1 and HR2 regions to study the corresponding protein protein interac tions Three synthetic peptides were synthe sized HR1 a residues 899 926 HR1 b residues 916 950 and HR2 residues 1 151 1 185 based on the interacting site of HR1 and HR2 suggested by Tripet et al 2004 Fig 1 Three recombinant proteins were designed according to Xu et al 2004b His 6 HR1 residues 899 958 GST HR2 or GST removed HR2 residues 1 145 1 192 and HR 2 Helix HR 2 Helix is an artificial mini coiled coil where HR1 899 958 and HR2 1 145 1 192 are connected by a 10 amino acid linker GGSGEFGGSG HR1 Interacts With HR2 to Form a Six Helix Bundle of HR1 3 HR2 3 Native polyacrylamide gel electrophoresis PAGE was used to study the interactions between HR2 and HR1 a HR1 b peptides HR2 is negatively charged pH 6 8 and it enters 2336 Chu et al the gel to give a single band Fig 2 lane 1 In contrast HR1 a carries zero charge and HR1 b is positively charged at pH 6 8 Both HR1 a and HR1 b did not enter the gel and could not be visualized upon native gel electrophoresis lanes 2 5 When HR1 b was pre incubated with HR2 an extra band of lower mobility was observed The relative intensity of the resulting protein bands correlated stochiometrically to the molar ratio of HR1 b HR2 sample mixtures lanes 6 and 7 No mobility shift of the HR2 band was observed when HR2 was incubated with HR1 a lanes 8 and 9 The absence of complex formation between HR2 and HR1 a is consistent with the absence of inter chain interactions between HR2 and the N terminal region of HR 1 reported previously Liu et al 2004 Supekar et al 2004 Tripet et al 2004 Xu et al 2004a b Hence the C terminal sequ ences of HR1 Ile916 Ser950 contains sufficient determinant for interaction with HR2 Circular dichroism CD analysis showed that HR1 b is a random coil while HR2 peptide displayed a typical CD spectrum for a helical peptide with double minima at 208 and 222 nm Equal molar mixture of HR1 b HR2 complex showed enhancement of the total helical content Fig 3A suggesting that the interactions between HR1 b and HR2 promote the helical content of HR 1b This is consistent with the results reported by Tripet et al 2004 where enhanced helical content was observed when 50 TFE or equal molar quantity of a peptide equivalent to HR2 was added to a peptide with the same sequence of HR1 b No significant changes of ellipticity was observed when equal molar HR1 a and HR2 were mixed result not shown Thermal denaturation experiments revealed that specific inter chain interactions were present between HR1 b and HR2 The melting temperature T m of HR2 was 36 38C Fig 3B When equal molar of HR1 b was added to HR2 the T m increased by 23 2 59 58C Fig 3C Similar to the HR1 b HR2 interaction His 6 HR1 and GST removed HR2 interacted and acquired enhanced thermostability His 6 HR1 which spans the combined length of HR1 a and HR1 b displayed a CD spectrum typical for Fig 1 Amino acid sequences of recombinant proteins and synthetic peptides used in this study His 6 HR1 residues 899 iV 958 and GST removed HR2 1 145 iV 1 192 are recombinant proteins whereas HR1 a residues 899 iV 926 HR1 b residues 916 iV 950 and HR2 residues 1 151 iV 1 185 are synthetic peptides The conserved hydrophobic amino residues a and d positions of the predicted heptad repeat patterns are shown above the amino acid sequences Fig 2 HR1 b interacts with HR2 in vitro HR1 a or HR1 b was incubated with HR2 at the indicated concentration before subjected to Native PAGE analysis HR1 a and HR1 b are negatively charged in the running pH and do not migrate into the gel on their own lanes 2 5 A high mobility species was found when HR2 was incubated with HR1 b lanes 6 and 7 but not with HR1 a lanes 8 and 9 Inhibitors Targeting SARS CoV S Protein Fusion Core 2337 helices with double minima at 208 and 222 nm GST removed HR2 however appeared as a random coil Fig 3D An equal molar mix ture of His 6 HR1 GST removed eHR2 showed enhancement of the total helical content Fig 3D which reflected interactions between His 6 HR1 and GST removed HR2 The inter action also resulted in enhanced thermal sta bility with an increase of Tm by 23 38C from 62 98C of His 6 HR1 to 89 28C of the equal molar mixture of His 6 HR1 and GST removed HR2 Fig 3E F Laser light scattering LS analysis was used to study the stoichiometry of HR1 HR2 and their complex In Figure 4 elution of the proteins was traced by the refractive index of the solution and molar mass distribution plot of relevant peaks was shown on top of the peaks concerned In each of the traces the soluble aggregate of the protein loaded was eluted at C2410 ml On its own His 6 HR1 molecular weight of monomer 9 9 kDa appeared tetrameric with a mass of 39 kDa Fig 4A whereas GST removed HR2 has a Fig 3 Folding and thermostability of the peptides and recombinant proteins A The CD spectra of HR1 b blue HR2 magenta and HR1 b HR2 complex yellow B Thermal denaturation curve of HR2 The Tm was 36 38C C Thermal denaturation curve of HR1 b HR2 complex The Tm was 59 58C D The CD spectra of His 6 HR1 blue GST removed HR2 magenta and the His 6 HR1 GST removed HR2 complex yellow E Thermal denaturation curve of His 6 HR1 The Tm was 62 98C F Thermal denaturation curve of His 6 HR1 GST removed HR2 complex The Tm was 86 28C 2338 Chu et al mass of 6 1 kDa suggesting a monomeric form Fig 4B When mixed in equal molar ratio His 6 HR1 GST removed HR2 appeared as a monodispersed species with a molecular weight of 49 kDa Fig 4C This is consistent with a six helix bundle fusion core conformation with 3 sets of each polypeptides 3C29 910 His 6 HR1 3C26 430 GST removed HR2 49 020 Da of S proteins Liu et al 2004 Supekar et al 2004 Tripet et al 2004 Xu et al 2004a b The mini coiled coil HR 2 Helix was de signed by linking sequences covered by GST removed HR2 and HR1 by a 10 amino acid linker GGSGEFGGSG Fig 1 It was charac terized using CD spectroscopy and laser LS The CD profile of HR 2 Helix showed a spectrum typical to helices with double minima at 208 and 222 nm Fig 5A The T m of HR 2 Helix was 101 008C Fig 5B indicating strong interactions between the linked HR1 and HR2 HR 2 Helix appeared as a monodispersed peak in LS analysis with an average MW of 40 kDa Fig 5C It is consistent with the expected molecular weight for a trimer of HR 2 Helix 3C213 170 77 39 512 31 Da indicat ing that the HR 2 Helix self associated to form a six helix bundle as reported by Xu et al 2004a These properties suggested that the HR 2 Helix trimeric coiled coil bundle represents the SARS CoV S protein fusion core structure of the most stable post fusion hairpin state HIV luc SARS Pseudotyped Virus Entry Inhibition Studies HIV luc SARS pseudotyped virus that ex pressed luciferase and SARS CoV S protein was constructed and isolated from 293 T cells Pseudotyped virus entry inhibition assays were done by incubating isolated pseudotyped virus with peptide or small molecule inhibitors before infecting Vero E6 cells in triplicate see Experimental Methods Section Luciferase activity assays were performed on the Vero E3 cells after overnight incubation with the treated pseudotyped virus Peptides HR1 a HR1 b and HR2 and re combinant protein His 6 HR1 and GST removed HR2 were tested for their entry inhibi tion activity We found that for the peptide candidates derived from the HR1 region only HR1 a showed antiviral activity with an EC 50 of 1 61mM Table I For the peptides derived from the HR2 region both GST removed HR2 and HR2 peptide demonstrated antiviral activities EC 50 of GST removed HR2 was 2 15 mM while that of HR2 was 0 34 mM Table I We have also tested the pseudotyped virus entry inhibition activities of 39 small molecule drugs Amongst these 39 small molecule drugs only 5 of them Table I were found to have relatively potent inhibitory activities when compared to quceretin EC 50 64 8 mM HKC 5 EC 50 38 38 mM HKC 21 EC 50 35 9 mM HKC 24 EC 50 32 60 mM HKC 25 EC 50 18 74 mM and ADS J1 EC 50 3 89 mM Because ADS J1 showed the highest potency it was selected for further studies Fig 4 Laser light scattering analysis of the stoichiometry of the HR1 HR2 complex Molar mass distribution plots of His 6 HR1 A GST removed HR2 B His 6 HR1 GST removed HR2 com plex C The solid horizontal bar indicates the average molecular weights for each peak Inhibitors Targeting SARS CoV S Protein Fusion Core 2339 Fig 5 Characterization of HR 2 Helix CD spectrum A and thermal denaturation curve B are shown The Tm was 101 008C Laser light scattering analysis showed that HR 2 Helix forms a monodispersed species of 40 110 Da The solid horizontal bar indicates the average molecular weight for the HR 2 Helix peak C ADS J1 Inhibits HR1 HR2 Interactions In Vitro GST HR2 and His 6 HR1 were mixed in the presence of ADS J1 XXT or PBS followed by immobilization onto glutathione resin The resin was then washed with PBS and eluted with 10 mM reduced glutathione The fractions were separated by SDS PAGE and analyzed by Western blot using anti His tag antibodies When incubated with PBS only majority of His 6 HR1 was detected in the elution fractions Fig 6A showing that His 6 HR1 is bound to GST HR2 In the presence of ADS J1 His 6 HR1 was only found in the flow through but not in the elution fractions Fig 6A whereas XTT had no effect on the interactions and His 6 HR1 was found only in the elution fractions Fig 6B It indicated that ADS J1 interfered with the interactions between His 6 HR1 and GST HR2 In Silico 3D Molecular Modeling Drug target The hydrophobic deep pocket in the SARS CoV S protein fusion core According to our pseudotyped virus entry inhibition assays ADS J1 exhibited the highest entry inhibition activity In vitro bind ing interference assays showed that ADS J1 interfered with the interactions between HR1 and HR2 From the X ray crystal structures of the SARS CoV S protein fusion core there is a deep pocket located within the relatively deep grooves on the surface of central HR1 coiled coil Phe909 to Leu927 Liu et al 2004 Supekar et al 2004 Tripet et al 2004 Xu et al 2004a Duquerroy et al 2005 This structural pocket is conserved among heptad repeat region of class I membrane fusion proteins It has been suggested that the presence of the pocket is important for adapting alternative conforma tions during fusogenesis Duquerroy et al 2005 Anti HIV drugs such as T20 have been developed to target this deep groove Kilby et al 1998 The X ray crystal structures of SARS CoV S core peptides showed that four hydrophobic residues from HR2 Leu1168 Val1171 Leu1175 and Leu1179 penetrate into the deep pocket consisting of Phe909 to Leu920 of HR1 and make extensive hydrophobic con tacts with each other Fig 7 This region was chosen to be the drug target site for our in silico molecular modeling studies Docking of ADS J1 to the HR1 hydro phobic deep pocket ADS J1 was indi vidually positioned in such a way that its hydrophobic groups phenyl and naphthalene groups can penetrate into the deep pocket and interact with the hydrophobic residues Phe909 Ile913 Ile916 and Leu920 within the deep pocket and with Ala906 outside the pocket The docking simulations were shown in Figure 7B Hydrophobic groups of ADS J1 interact with the hydrophobic residues in the deep pocket and its SO 3 H was in close proximity to Lys911 Fig 8 The total docking energy for the simulated complex was monitored accordingly as an indicator of the final ligand placement The in silico generated van der Waals energy showed a substantial favorable energy for the complex C037 05 kcal mol C01 suggesting that TABLE I Viral Entry Inhibition Activity of Heptad Repeat Peptides and Small Molecules Peptide EC 50 mM a His 6 HR1 ND GST removed HR2 2 15 HR1 a 1 16 HR1 b ND HR2 0 34 Small molecule EC 50 mM HKC 5 38 38 HKC 21 35 94 HKC 24 32 60 HKC 25 18 74 ADS J1 3 89 Quercetin 64 80 XXT ND ND not detectable a All measurements were average values of triplicates Fig 6 ADS J interferes with the interactions between HR1 and HR2 in vitro His 6 HR1 was preincubated in PBS control lanes 1 4 panel A and lanes 1 3 panel B 1 500 mM ADS J1 lanes 5 8 panel A or 1 500 mM XXT lanes 4 6 panel B before loading into the glutathione resin captured GST HR2 The elution fractions w