Surface ultrastructure of SARS coronavirus revealed by atomic force microscopy

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1 lckwell Science, LtdOxford, UKCMICellulr Microiology lckwell Pulishing Ltd, Originl rticles. Lin et l.surfce ultrstructure of SRS coronvirus Cellulr Microiology (005) 7(1), doi: /j x Surfce ultrstructure of SRS coronvirus reveled y tomic force microscopy Shiming Lin, 1, * Chih-Kung Lee, Shih-Yun Lee, Chun-Ling Ko, Chii-Wnn Lin, 5 n-ng Wng, Su-Ming Hsu 6 nd Long-Sun Hung 1 Center for Optoelectronic iomedicine, Ntionl Tiwn University, Tipei, Tiwn. Institute of pplied Mechnics, Ntionl Tiwn University, Tipei, Tiwn. Deprtment of Chemistry, Tmkng University, Tipei, Tiwn. Deprtment of Medicl Technology, Ntionl Tiwn University, Tipei, Tiwn. 5 Institute of iomedicl Engineering, Ntionl Tiwn University, Tipei, Tiwn. 6 Deprtment of Pthology, Ntionl Tiwn University, Tipei, Tiwn. Summry tomic force microscopy hs een used to proe the surfce nnostructures of severe cute respirtory syndrome coronvirus (SRS-CoV). Single crown-like virion ws directly visulized nd quntittive mesurements of the dimensions for the structurl proteins were provided. coron of lrge, distinctive spikes in the envelope ws mesured fter tretment with hydroxyoctnoic cid. High-resolution imges reveled tht the surfce of ech single SRS-CoV ws surrounded with t lest 15 sphericl spikes hving dimeter of 7.9 ± 0.7 nm, which is in close greement with tht of S glycoproteins erlier predicted through the genomes of SRS-CoV. This study represents the first direct chrcteriztion of the surfce ultrstructures of SRS-CoV prticles t the nnometre scle nd offers new prospects for mpping virl surfce properties. Introduction novel coronvirus hs een identified s the prole cuse of the newly recognized severe cute respirtory syndrome (SRS) (Ksizek et l., 00; Drosten et l., 00; Peiris et l., 00). The sequences of the complete Received 1 pril, 005; revised 19 My, 005; ccepted 1 June, 005. *For correspondence. E-mil sml@ntumc.org; Tel. (+886) 156 ext. 858; Fx (+886) genomes of two isoltes of the SRS coronvirus (SRS-CoV) hve een reported (Rot et l., 00; Mrr et l., 00). Five mjor open reding frmes (ORFs) were determined through sequence similrity to known coronvirus proteins. This pproch identify two replicses 1 nd 1 tht undergo cotrnsltionl proteolytic processing nd four structurl proteins the spike (S), envelope (E), memrne (M) glycoproteins nd the nucleocpsid (N) protein (Holmes nd Enjunes, 00). Understnding pthogenesis of SRS requires knowledge of the structure nd physicochemicl properties of the SRS-CoV surfce t suprmoleculr scle. The ultrstructure of SRS-CoV virions hs een chrcterized y thin-section electron microscopy (Ksizek et l., 00); however, direct informtion on the ntive virl surfce ws inccessile. The tomic force microscopy (FM) (inning nd Qute, 1986), one of the scnning proe microscopy (inning et l., 198), hs proved useful to investigte the topogrphy of virl surfces (Kuznetsov et l., 00; 00; 005; Mlkin et l., 00; Nettikdn et l., 00, Hughes et l., 00; Negishi et l., 00), ecuse FM imges cn revel structurl detils with unprecedented resolution. While opticl microscopes re limited y diffrction (l/), FM cn chieve moleculr nd even tomic resolution for mny mterils (utt et l., 199; Miur nd Shukuy, 199; Liu nd Slmeron, 199; Xu et l., 1998). In ddition, three-dimensionl informtion cn e extrcted directly from FM topogrphic nd phse imges. These dvntges force us to pply the FM to the field of SRS-relted virology. Here, we used FM to determine the surfce ultr structure of ech single SRS-CoV virion nd oserve the surfce chrcteristics nd their topogrphy nd phse chnges fter tretments with hydroxyoctnoic cid or protese. This study provides new pproch to the mesurements of components of SRS-CoV virions for future ppliction in SRS proteomics. Results nd discussion Ntive SRS-CoV Figure 1 shows the FM height imges of the ntive SRS-CoV virions in the culture medium. In Fig. 1, the surfce ws uniformly covered with numer of sphericl 005 lckwell Pulishing Ltd

2 176 S. Lin et l. 0 Height (nm) Fig. 1. Topogrphic FM imges of the ntive SRS-CoV prticle on mic. The scnning res re mm nd mm for the low- () nd high-resolution () imges respectively. n imge cquired y zooming into the oxed res is displyed in. The high-resolution imge nd corresponding cursor profile () clerly revel the presence of SRS-CoV prticles. prticles in the medium environment. n imge cquired y zooming into the oxed re is displyed in Fig. 1. The higher-resolution imge shows tht mny individul sphericl protrusions exist on the surfce nd the corresponding cursor profile (Fig. 1) clerly revels the presence of SRS-CoV prticles nd ech single prticle is redily distinguishle. The corresponding cursor profile provides quntittive mesurements of the heights for the SRS-CoV prticles on the surfce. The profile displys tht the mesured heights of SRS-CoV prticles re 71.7, 90.9 nd 8.8 nm respectively. Over 0 single sphericl prticles were then exmined, the surfce trces re reproducile lthough some vritions exist in the height of surfce protrusions. The root men squre (RMS) vlue of heights of SRS-CoV prticles clculted ws pproximtely 81. ± 10.6 nm. fter verge height for ech virl prticle re clculted, it is found tht the RMS vlue of height (81. ± 10.6 nm) corresponds with tht ( nm in dimeter) otined from thin-section electron microscopy (Ksizek et l., 00). Crown-like SRS-CoV virions In previous study, typicl SRS-ssocited coronvirus hs een found y the use of negtive-stin electron microscopy which shows stin-penetrted coronvirus prticle with clu-shped surfce projections surrounding the periphery of the prticle (Ksizek et l., 00). To further investigte the surfce ultrstructure of single SRS-CoV virion y FM, we treted SRS-CoV smples with the surfce-modifying chemicl, hydroxyoctnoic cid (Fig. ). FM imges (scnning re mm ) of entire SRS-CoV fter tretment re displyed in Fig. (height imge) nd (phse imge) respectively. Higher-resolution height (Fig. nd C) nd phse (Fig. nd C ) imges (scnning re mm ) were otined y zooming into the oxed res displyed in Fig. nd respectively. These higher-resolution imges clerly revel the presence of crown-like SRS- CoV virions nd tht ech single virion with prtilly intct or completely disrupted ppernce is redily distinguishle. Ech surfce virion displyed in Fig. ws then profiled nd their dimensions mesured. It ws found tht, over 60 single prticles were exmined, the length, width nd height of treted virions vry in rod intervl of 6 7 nm, 77 5 nm nd nm respectively. Moreover, the phse imges otined were used to oserve the surfce chrcteristics. The phse imges re useful for compositionl mpping of surfces with different stiffness of the constituting sustnces (Mgonov et l., 1997). It llows us to identify immeditely hrd nd soft mteril, the right prts of the FM imge corresponding to hrd mteril (e.g. yellow is stiffer thn rown). s shown in Fig., the phse imge indicte the presence of some mteril (yellow, rrowheds in Fig. ) surround the virus tht ws of different composition thn either the virion itself (rown) or the mic sustrte (white). This my represent the chemicls (hydroxyoctnoic cid) intercted with the virions. In ddition, s shown in Fig. C, mny spheres (rown, rrowheds in Fig. C ) were lierted nd dispersed ner the virion itself. This might e ttriuted to the rekdown of virl envelope cused y hydroxyoctnoic cid. Hydroxyoctnoic cid is speculted to hve unusul effects on the surfce chemistry of virl envelope. Upon exposure to hydroxyoctnoic cid, the virl surfce of SRS-CoV ecme more heterogeneous (Fig. ) thn the ntive virus (Fig. 1). To investigte the surfce ultrstructure of single SRS-CoV virion, over 60 SRS-CoV virions displyed in 005 lckwell Pulishing Ltd, Cellulr Microiology, 7,

3 Surfce ultrstructure of SRS coronvirus 1765 C C C C Fig.. FM imges of the SRS-CoV treted with hydroxyoctnoic cid. Height imge (top row) nd phse imge (ottom row). Imges of entire viruses re shown in left column (totl scnning re mm ). Imges cquired y zooming into the oxed re displyed in the second nd third columns (totl scnning re mm ) respectively. new tip ws used for the imges in the third columns. Fig. were exmined nd their higher-resolution imges (scnning re nm ) otined, nd some imges were shown in Fig. E for further oservtion nd mesurements. Ntive SRS-CoV virions in the environment of distilled wter were completely not dmged nd serve s control (Fig. ). When viruses were exposed to hydroxyoctnoic cid, the virl morphology ws ffected. s shown in Fig. E, SRS-CoV treted with hydroxyoctnoic cid displyed heterogeneous surfce fetures. The ntive virion ws roken down nd coron of lrge, distinctive protrusions surrounding the envelope re redily oserved in Fig.. The ppernce of pits s shown in Fig. C E indictes the relese of virl components, predomintely structurl proteins such s spike (S) protein, memrne (M) proteins, smll envelope (E) proteins or nucleocpsid (N) proteins, or non-structurl proteins (such s replicse 1 nd 1) (Ksizek et l., 00; Rot et l., 00; Mrr et l., 00; Holmes nd Enjunes, 00). The new structure fter tretment is not s tight s tht of the ntive virion, which leds to liertion of the virl content (Fig. E). fter tretment, intermoleculr interctions mong structurl proteins (S, M or E proteins) within the envelope re wekened. Desorption of structurl proteins occurs from the oundries of the memrne ptches, grdully leding to the relese of some structurl proteins. In ddition, lthough the whole virions in some imges (Fig. E) ppered disrupted to some extent, some prticles which initilly surround the periphery re still visile in FM imges, s indicted y the cursor profiles (Fig. e). Moreover, hydroxyoctnoic cid cused sustntil reduction in the surfce height nd irregulrity in the virl surfces. The prticles indicted y the cursor profiles (Fig. e) re proly lrger structurl proteins surrounding the virl periphery, such s spike proteins (vide infr). These regulr structures re unlikely to e rtifcts of either tip shpe or immoiliztion procedure ecuse they hve een oserved on t lest 00 SRS-CoV virions which hd een scnned in this work. The corresponding cursor profiles (Fig. e) provide quntittive mesurements of the dimensions for the surfce nnostructures. The profiles displyed in Fig. e shows tht the mximum height of prticles mesured re pproximtely 15.5, 9.78, 7.7, 7.19 nm respectively. s shown in Fig. nd C, ech sphericl protrusion on the cursor or c ws surrounded y other components in the envelope, while the sphericl protrusion on cursor d nd e in Fig. D nd E, respectively, my e nerly regrded s n individul prticle for further mesurement. Over 0 such prticles s displyed on the cursor d nd e were exmined, nd lthough some vritions exist in the exct height of such prticles, the surfce trces re reproducile. The clculted RMS vlue of heights of prticles is 7.9 ± 0.7 nm. 005 lckwell Pulishing Ltd, Cellulr Microiology, 7,

4 1766 S. Lin et l. C c D d E e C D E Height (nm) c d 7.19 e Fig.. Topogrphic FM imges of different single SRS-CoV virions efore (the first column) nd fter (the second to fifth column) tretment with hydroxyoctnoic cid. Imges cquired y zooming into the oxed res in Fig. re displyed in the E (two-dimensionl) nd E (three-dimensionl). Scle r = 100 nm for ech imge in the first row. The corresponding cursor profiles (the third row) provide quntittive mesurements of the dimensions for spike proteins. Chemicl composition of sphericl protrusions In order to get more informtion s to the chemicl composition of these sphericl protrusions in the envelope we scnned the re contining the SRS-CoV virions (in pired fshion) efore nd 15 min fter tretment with the proteolytic enzyme, protese. s illustrted in Fig. the imges ecme lurred nd the surfce crown-like structures in the envelope considerly decresed pproximtely 70% in height over 15 min of protese tretment. This indictes tht imged sphericl prticles on the surfce hve een hydrolysed y protese ction. This oservtion lso shows tht these sphericl prticles on viril Fig.. FM imges of SRS-CoV prticle fter tretment with protese. Height imge nd phse imge of n individul virion is shown in nd respectively. Scle r = 50 nm for nd. surfce re minly composed of mteril which contin protein molecules. Spike proteins In previous studies the SRS-CoV genome ws reported to contin six mjor ORFs through sequence similrity tht encode two replicse polyproteins; nd four structurl proteins, the spike (S) protein, the smll envelope (E) protein, the memrne (M) glycoprotein, nd the nucleocpsid (N) protein (Rot et l., 00; Mrr et l., 00). The numers of mino cids of six coronvirus proteins identified re 8, 68, 155, 76, 1 nd, respectively (Mrr et l., 00), nd thus the moleculr weights of proteins predicted re pproximtely 56, 15, 151, 9, 7, nd 51 kd respectively. To investigte whether the sphericl prticles oserved ove re spike (S) proteins or other proteins in the virl envelope, immunogloulin G (IgG) molecules (mol. wt. 150 kd) which hve the sme moleculr weight with the spike protein (pproximtely 151 kd) were prepred nd scnned for FM imging under the sme conditions with SRS-CoV smple. Multiple cross-sections of the individul IgG imges y FM were mde nd men dimeter nd height of ech single molecule were mesured. fter verge height for ech IgG molecule is clculted, it ws found tht the RMS vlue of heights for IgG molecules is pprox- 005 lckwell Pulishing Ltd, Cellulr Microiology, 7,

5 Surfce ultrstructure of SRS coronvirus 1767 f C c d e c e Height (nm) d f Fig. 5. () Two- nd () three-dimensionl FM imges nd contour mp (C) of single SRS-CoV virion. Scle r = 100 nm in nd C. The corresponding cursor profiles (middle nd ottom row) provide quntittive mesurements of the dimensions for the spike proteins (1 15) displyed in C. imtely 7.1 ± 0.1 nm (dt not shown). This vlue is out consistent with the vlue (7.9 ± 0.7 nm) otined for sphericl prticles (Fig. ) surrounding on the surfce of SRS-CoV. Thus, the sphericl prticles visile on the virl surfce re speculted to e possily spike (S) proteins ecuse of their size, 7.9 ± 0.7 nm. For those SRS-CoV virions tht mintin their crown-shped morphology, the high-resolution imges ( mm) shown in Fig. 5 revel tht coron of lrge, distinctive spikes surrounding the envelope of SRS-CoV. These spikes in the envelope mkes possile the identifiction of coronviruses y FM. Figure 5 shows the two- (Fig. 5) nd three-dimensionl (Fig. 5) FM imges nd their contour mp (Fig. 5C) of single SRS-CoV virion. The corresponding cursor profiles (Fig. 5 f) revel couples of umps nd dents formed y the spikes proteins on the surfce nd lso provide quntittive mesurements of the dimensions for these spike proteins. It ws found tht from the contour mp (Fig. 5C) 15 spike proteins (rrowheds) surround the envelope of single SRS-CoV virion. It is thus speculted tht t lest 15 spike proteins form the whole coron of single SRS-CoV virion. In ddition, s the virion displyed in Fig. 5 does not represents n intct one, the thickness of its coron surrounding the periphery rnges pproximtely from 1 to 17 nm (Fig. 5 f), which siclly corresponds to the dimension (0- to 0-nm) of the coron of SRS-CoV otined previously from electron microscopic study (Ksizek et l., 00). Smll sphericl prticles relesed This work hs lso creted wy to rek down new infectious gent, SRS-CoV, into smll components. s shown in Fig. 6, fter the intct virion ws rek down y chemicl tretment, numer of smll sphericl prticles (lso see Fig. C, rrowheds) were relesed nd redily oserved from the height (Fig. 6) nd phse (Fig. 6) imge. The phse imge provides mesure of smple heterogeneity nd lso indictes tht the smll sphericl prticles (rrowheds) come from the SRS-CoV virion itself (Fig. 6). The cursor profiles displyed in Fig 6 nd cn provide quntittive mesurement of the dimension for ech smll sphericl prticle oserved in Fig lckwell Pulishing Ltd, Cellulr Microiology, 7,

6 1768 S. Lin et l Height (nm) Fig. 6. Imges cquired y zooming into the oxed res in Fig. C re displyed in (height imge) nd (phse imge). Scle r = 00 nm. In the height imge () section line is plced through two smll sphericl prticles. The resulting profiles ( nd ) re shown in the right pnel. The corresponding cursor profiles ( nd ) provide quntittive mesurements of the dimensions for the prticles relesed from virion itself. Over 0 vrious smll sphericl prticles lierted were then exmined, the heights of smll sphericl prticles rnges from.8 to.57 nm. The structurl proteins of SRS-CoV hve een reported to include spike (S) protein (mol. wt. 151 kd), memrne (M) protein (mol. wt. 7 kd), smll envelope (E) protein (mol. wt. 9 kd) nd nucleocpsid (N) protein (mol. wt. 51 kd) (Mrr et l., 00). lthough SRS-CoV lso encode numer of non-structurl proteins tht re of unknown function nd locted etween S, nd E, etween M nd N, or downstrem of N, moleculr weights of these non-structurl proteins re predicted to e less thn kd. (Rot et l., 00; Mrr et l., 00). Thus, vrious sizes of smll sphericl prticles (.8.57 nm) relesed when SRS-CoV roke down represents the different types of proteins of SRS-CoV reported previously. Therefore, the study reported here provides n pproch to mesurement of some components of single SRS-CoV virion for future ppliction in SRS proteomics-relted studies. Conclusions We hve shown tht FM is useful technique in providing structurl informtion for individul SRS-CoV virions. In ddition, three-dimensionl informtion on components in the envelope cn e extrcted from the topogrphic imges. However, it should e noted tht it is not possile to e solutely definite in ttriuting topogrphicl nd phse fetures in FM imges to virions or virl deris. Whilst removing/voiding impurities in smple preprtion reduces the possiility of imging third-prty deris other methods re necessry to confirm the identifiction. Further work will concentrte on the use of ntiodynnogold conjugtes for the identifiction of SRS-CoV virion prticles. In this work, we lso reveled for the first time the surfce imge of individul SRS-CoV virions efore nd fter tretment. Quntittive mesurements of the dimensions for the spike (S) proteins in the envelope hve een provided. The physicl mesurements of the S proteins in the SRS-ssocited coronvirus my suggest how S proteins ffect the pthogenesis of SRS. We hve lso creted wy to rek down the SRS-CoV into smll components. Then, people cn quickly isolte nd test the components to find out which one stimultes the est immune response for the future development of vccines or new drugs for SRS. Experimentl procedures Severe cute respirtory syndrome coronvirus preprtion Severe cute respirtory syndrome coronvirus (TW-1) (Gen- nk ccession no. Y915) used in this study ws originlly isolted from the throt sw specimens of one ptient in Tiwn. Throt sw specimens were inoculted into Vero E6 cells, cultured, nd monitored (Hsueh et l., 00; Yeh et l., 00). Once the virus-induced cytopthic effects ppered, the culture cell superntnt contining viruses ws hrvested nd impurities in smple preprtion were removed s descried (Hsueh et l., 00; Yeh et l., 00). ll division into liquots, pipetting, culture, tretments, nd ttchment ttempts were performed in lminr-flow sfety cinets in iosfety level lortory; fter these steps, the smple preprtions were removed to iosfety level lortory for completion of the FM imging. ttchment of SRS-CoV to mic For FM studies, SRS-CoV smple ws ttched through electrosttic interctions y plcing in contct with freshly cleved mic tht hd een coted with poly L-lysine hydroromide, positively chrged compound. fter clening the mic with methnol nd Milli-Q wter, drop of 1% (wt/vol) poly L-lysine hydroromide solution ws pplied nd incuted for 0 min. The mic surfce ws then wshed with Milli-Q wter efore introduc- 005 lckwell Pulishing Ltd, Cellulr Microiology, 7,

7 Surfce ultrstructure of SRS coronvirus 1769 tion of the SRS-CoV specimens. The specimen ws pplied onto the poly L-lysine-treted mic for 5 min t room temperture, followed y wshing with distilled wter (50 ml, three times) nd drying in ir prior to the FM experiments. Chemicl tretment with hydroxyoctnoic cid The mic sustrte dsored with SRS-CoV virions ws wshed with Milli-Q wter efore introduction of the chemicl, hydroxyoctnoic cid. 10 ml portion of the ove chemicl (0.1 mg ml -1 ) in 0 mm PS uffer, ph 7., ws dded to the mic sustrte, followed y incution for 5 10 min, removing of the residul chemicl solution, nd drying in ir prior to the FM experiments. ccurte in detiling the height of fetures on the surfce. Phse imges hve proved to yield etter resolution in detiling the lterl dimensionl dt of surfce fetures (Kopp-Mrsudon et l., 000) ut s the different fctors tht cuse phse shift cnnot e seprted, interprettion of phse imges cn e much more difficult. Phse imging is useful s complement to topogrphic imging for providing informtion on SRS-CoV smple heterogeneity. cknowledgements This work ws supported in prt y ME 9-EC S1-0017, NSC Grnt nd NSC Grnt Protese tretment Protese from Streptomyces griseus (P517, Sigm, US) ws dissolved in 10 mm sodium cette uffer with 5 mm clcium cette, ph 7.5 t concentrtion of 50 mg ml ml portion of the ove protese solution (0 units ml -1 ) ws dded to the mic sustrte immoilized with SRS-CoV virions, followed y wshing with Milli-Q wter three times nd ir-drying prior to the FM experiments. tomic force microscopy experiments tomic force microscopy experiments in tpping mode of opertion were crried out using multimode scnning proe microscope (SPI00 HV, Seiko Instruments, Chi, Jpn) in comintion with light microscope (Mitotoyo, Jpn). FM Tips were 00 mm long nd hd typicl resonnt frequency of 10 khz (Nnosensor, Wetzlr-lnkenfeld, Germny). Clirtion of the FM cntilever tip were crried out using stndrd diffrction grting (Seiko Instruments, Chi, Jpn). Light tpping ws used, which involved mintining high mplitude reference reltive to the free mplitude of the cntilever. Typiclly, we egn y scnning 0 mm 0 mm re tht would contin hundreds of SRS-CoV virions. Grdully, the imge size ws reduced to isolte individul virion (500 nm 500 nm). SRS- CoV specimen were scnned in oth directions severl times efore cpturing n imge, to help ensure tht tip rtifcts, such s hysteresis, were not ltering the imges. Over 00 high-qulity imges were cptured for ech SRS-CoV virion, nd numer of imges were selected from these. Tips were replced when there ws ny indiction of rtifcts present in the imges. fter imges were collected, n offline section nlysis ws performed on ech imge in order to gin informtion on virl topogrphy. When line ws drwn cross the imge, the topogrphy of the smple s function of distnce ws displyed. These height trces were performed to provide more informtion on how ech tretment ffected the surfce topogrphy. Roughness nlyses were lso performed on some smples, conducted ccording to the mnufcturer s softwre progrm (SPI 800N). The RMS verge of the surfce roughness vlue ws clculted s the stndrd devition of ll the height vlues within the given re. Phse imges were cptured simultneously with height (topogrphic) imges. In phse imging, the phse shift of the oscillting cntilever is mesured s function of tip position on the surfce. Height imges revel surfce topogrphy nd re more References inning, G., nd Qute, C.F. (1986) tomic force microscope. Phys Rev Lett 56: inning, G., Rohrer, G.H., Gerer, C.H., nd Weiel, E. (198) Surfce studies y scnning tunneling microscopy. Phys Rev Lett 9: utt, H.J., Seifert, K., nd merg, E. (199) Imging moleculr defects in lknethiol monolyers with n tomic force microscope. 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