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Clathrin independent entry of SARS CoV 290 npg Cell Research Vol 18 No 2 February 2008 ORIGINAL ARTICLE SARS coronavirus entry into host cells through a novel clathrin and caveolae independent endocytic pathway Hongliang Wang 1 Peng Yang 1 Kangtai Liu 1 Feng Guo 1 Yanli Zhang 1 Gongyi Zhang 2 Chengyu Jiang 1 1 National Key Laboratory of Medical Molecular Biology Institute of Basic Medical Sciences Peking Union Medical College Tsin ghua University and Chinese Academy of Medical Sciences Beijing 100005 China 2 Department of Immunology National Jewish Medical and Research Center Denver CO 80206 USA Correspondence Chengyu Jiang Tel 86 10 65296908 Fax 86 10 65276551 E mail jiang Abbreviation angiotensin converting enzyme 2 ACE2 chlorpromazine CPZ cholera toxin subunit B CTB early endosome antigen 1 EEA1 human embryonic kidney 293E HEK293E methyl cyclodextrin M CD severe acute respiratory syndrome SARS severe acute respiratory syndrome coronavirus SARS CoV Received 27 November 2007 revised 29 November 2007 accepted 30 November 2007 published online 29 January 2008 While severe acute respiratory syndrome coronavirus SARS CoV was initially thought to enter cells through direct fusion with the plasma membrane more recent evidence suggests that virus entry may also involve endocytosis We have found that SARS CoV enters cells via pH and receptor dependent endocytosis Treatment of cells with either SARS CoV spike protein or spike bearing pseudoviruses resulted in the translocation of angiotensin converting enzyme 2 ACE2 the functional receptor of SARS CoV from the cell surface to endosomes In addition the spike bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes Further analyses using specific endocytic path way inhibitors and dominant negative Eps15 as well as caveolin 1 colocalization study suggested that virus entry was mediated by a clathrin and caveolae independent mechanism Moreover cholesterol and sphingolipid rich lipid raft microdomains in the plasma membrane which have been shown to act as platforms for many physiological signaling pathways were shown to be involved in virus entry Endocytic entry of SARS CoV may expand the cellular range of SARS CoV infection and our findings here contribute to the understanding of SARS CoV pathogenesis providing new information for anti viral drug research Keywords Severe Acute Respiratory Syndrome Coronavirus SARS CoV endocytosis angiotensin converting enzyme 2 ACE2 lipid rafts Cell Research 2008 18 290 301 doi 10 1038 cr 2008 15 published online 29 January 2008 npg Cell Research 2008 18 290 301 2008 IBCB SIBS CAS All rights reserved 1001 0602 08 30 00 Introduction Severe acute respiratory syndrome SARS emerged as an epidemic in Asia during the winter of 2002 2003 and quickly spread throughout the world The etiologic agent was soon identified as a new coronavirus SARS CoV 1 2 The plague ended in July 2003 with a death toll of more than 700 patients and 8 000 probable diagnoses http www who int csr sars country table2004 04 21 en index html This disease had a significant impact on international social and economic activities In addition to the likely reemergence of SARS there are serious concerns about its potential misuse as a biological weapon Four years after its identification however many pathological aspects of this virus have yet to be characterized The entry of enveloped viruses into cells is known to occur via two primary pathways some viruses deliver their genomes to the cytosol after their envelopes fuse with the plasma membrane at the cell surface whereas others take advantage of the cell s endocytic machinery In the latter mechanism the endocytosed virions are subjected to an activation step in the endosome which is typically mediated by the acidic endosomal pH resulting in fusion of the viral and endosomal membranes and release of the viral genome into the cytosol Therefore the endocytic mechanism is thought to be a pH sensitive process while direct membrane fusion is pH independent The endocytic pathways exploited by animal viruses to gain entry into host cells include macropinocytosis clathrin dependent endocytosis and caveolae dependent endocytosis as well as poorly characterized routes such as clathrin and caveo lae independent endocytosis 3 4 Although most viruses www cell Cell Research Hongliang Wang et al 291 npg use only one of these pathways to enter cells recent studies have shown that some viruses use multiple mechanisms to gain entry into host cells 5 8 The entry of SARS CoV into cells was first identified to occur by direct fusion at the plasma membrane 9 11 Some later studies have shown that entry of SARS CoV may be pH dependent 12 and that the endosomal protease cathepsin L 13 14 might be involved suggesting that this virus may employ endocytosis Because conflicting results have been obtained by various research groups using similar experimental methods such as fluorospectrometric monitoring of GFP labeled pseudovirus entry into host cells we decided to use a different approach We labeled the SARS CoV functional receptor angiotensin converting enzyme 2 ACE2 with GFP by stable transfection of hu man embryonic kidney 293E HEK293E cells Receptor recycling was then tracked after the cells were treated with pseudoviruses or the spike protein a membrane component of SARS CoV that mediates membrane fusion and is re quired for viral entry 11 12 This new approach greatly improves experimental stability and reliability The results showed that SARS CoV entered cells via receptor depen dent pH sensitive endocytosis We also showed that the specific endocytic pathway used by SARS CoV to enter cells is clathrin and caveolae independent Moreover lipid rafts were found to play an important role in this process Our results provide further insight into the pathological characteristics of this newly emerged virus Results Spike protein induced translocation of the SARS CoV re ceptor ACE2 from the plasma membrane to cytoplasmic compartments In order to examine the entry of SARS CoV into cells cell lines were first generated which stably expressed ACE2 fused to either a Myc tag or GFP ACE2 expression in the 293E ACE2 Myc cell lines was confirmed by western blot ting Supplementary information Figure S1 while ACE2 receptor expression in the 293E ACE2 GFP cell lines was directly visualized by fluorescence Figure 1A shows that ACE2 GFP is localized primarily at the cell surface sug gesting that expression and localization of ACE2 in this cell line is similar to that in Vero E6 cells The 293E ACE2 GFP cell line allowed for direct tracking of the movement of the receptor under a fluorescence microscope In agreement with a previous report which showed that ACE2 expres sion in some nonpermissive cells renders them permissive to infection 15 both of these stable cell lines can be infected by spike bearing pseudoviruses Supplementary information Figure S1 We first tested whether spike protein alone could enter Figure 1 SARS CoV receptor ACE2 translocates from the plasma membrane to cytoplasmic compartments following treat ment with spike protein A In HEK293E ACE2 GFP cells ACE2 is primarily located on the cell surface B When HEK293E ACE2 GFP cells were treated with the S1190 Fc protein for 3 h at 37 C ACE2 GFP was internalized from the cell surface C When HEK293E ACE2 GFP cells were treated with Fc protein alone for 3 h at 37 C no translocation of the SARS CoV receptor ACE2 was observed D Spike protein colocalizes with ACE2 GFP in cytoplasmic vesicles after 3 h incubation at 37 C Spike Fc protein was probed with Alexa 568 goat anti human IgG E ACE2 receptor recycling was observed after a 14 h incubation with spike Fc at 37 C few green vesicles were visible in the cells after 14 h F HEK293E ACE2 GFP cells were treated with NH 4 Cl before they were incubated with S1190 Fc protein ACE2 was trapped in cytoplasmic vesicles even after 14 h Scale bar 20 m A B C D E F 20 m 20 m 20 m 20 m 20 m 20 m Clathrin independent entry of SARS CoV 292 npg Cell Research Vol 18 No 2 February 2008 cells using the 293E ACE2 GFP cell line since spike protein was believed to mediate virus entry Purified recom binant spike protein fused to the human IgG Fc fragment S1190 Fc was added to 293E ACE2 GFP cells After incubation at 37 C for 3 h a number of GFP containing vesicles were observed in the perinuclear area Figure 1B whereas vesicles were not observed in the control cells treated with Fc protein alone Figure 1C suggesting that spike protein specifically induced translocation of ACE2 GFP from the cell surface to the interior of the cells Sta tistical analysis of the percentage of cells in which vesicles accumulated showed that there was a significant difference between the effects of these treatments on virus entry Sup plementary information Figure S2 A dual staining assay for spike protein and ACE2 GFP showed colocalization of these two proteins Figure 1D which suggests that spike protein was bound to ACE2 in the vesicles and that spike protein was taken up by the cell most likely by endocytosis The fact that spike protein induced translocation of ACE2 from the cell surface to intracellular compartments is in agreement with our previous finding that surface expres sion of ACE2 in Vero E6 cells decreased after incubation with spike protein at 37 C for 3 h 16 After an additional incubation at 37 C receptor con taining vesicles were no longer visible within the cells and green signals were instead seen clustered near the cell surface suggesting that the receptors were recycled Fig ure 1E This process can be blocked by lysosomotropic agents which are reported to function by elevating the pH of acidic compartments thereby inhibiting dissociation of the ligand from the receptor and trapping the receptor in the endosome 17 We found that after treatment with ammonium chloride bafilomycin A1 or chloroquine the viral receptor was trapped within perinuclear vacuoles even after a 14 h incubation a period that would allow the receptor to be recycled to the cell surface under normal conditions Figure 1F and unpublished data These results reflect the normal traffic of membrane flow that is cargos are first internalized by a besieged membrane which fuses with the early endosome and then the late endosome where cargos are dissociated from the receptors Receptors are usually recycled back to the cell membrane while cargos are targeted to lysosomes SARS CoV spike bearing pseudoviruses enter cells via endocytosis Due to the highly contagious nature of SARS CoV we used spike protein bearing pseudoviruses to study the virus entry route Pseudoviruses are often used to mimic the entry of real viruses such as hepatitis C virus 18 19 Ebola virus and Marburg virus 20 into host cells This strategy is a powerful tool for studying early events in the life cycle of a virus Therefore we used retroviral pseudoviruses bearing the SARS CoV spike protein to infect ACE2 GFP expressing HEK293E cells After a 3 h incubation at 37 C GFP containing vesicles were detected within the cells Figure 2A while there were few intracellular vesicles when the same cell lines were treated with a control pseudovirus bearing VSV G protein on the surface Figure 2B Dual labeling of spike protein and ACE2 also showed colocalization Figure 2C which indicates that the pseudovirus may be contained in the vesicles After an additional 10 h incubation few vesicles were observed Figure 2D for both treatments This result was similar to that observed following treatment with spike protein alone Moreover after treatment with ammonium chloride bafilomycin A1 or chloroquine GFP containing vesicles were detected even after a 12 h incubation Figure 2E and unpublished data suggesting that these reagents inhibited receptor recycling These results were nearly identical to those obtained following spike protein treat ment which indicated that SARS CoV may enter cells via endocytosis Although the pseudovirus can express GFP the green vesicles observed in these cells at this time point resulted from cellular ACE2 GFP rather than from virally expressed GFP as expression from the viral GFP gene was not observed at 12 h after infection This notion was verified with ACE2 Myc expressing HEK293E cells in which no GFP expression was observed at 12 h postinfection with the pseudovirus Supplementary information Figure S3 The endocytic pathway is usually thought to be pH dependent Therefore if SARS CoV can enter cells via endocytosis lysosomotropic agents should inhibit virus infection Vero E6 cells have been reported to be naturally permissive for SARS CoV We infected Vero E6 cells with spike bearing pseudovirus in the presence or absence of lysosomotropic agents Since the pseudovirus expresses GFP infected cells can be distinguished from uninfected cells by viral GFP expression Figure 2F shows that pseudovirus infection of Vero E6 cells leads to viral GFP expression while the lysosomotropic agents inhibited GFP expression This result suggests that successful virus entry is pH dependent To provide further confirmation that SARS CoV enters host cells via endocytic pathways dual immunofluores cence labeling with antibodies specific for the SARS CoV spike protein and the early endosome marker protein early endosome antigen 1 EEA1 was performed followed by confocal microscopy analysis After 1 h of infection a punctate pattern characterized by strong colocalization of the spike protein and EEA1 was observed confirming that SARS CoV was targeted to early endosomes Figure 2J L Alexa labeled transferrin was used as a positive control Figure 2G I since the endocytosis of transferrin has been www cell Cell Research Hongliang Wang et al 293 npg 10 m 10 m 10 m 10 m10 m 10 m 10 m10 m 10 m Figure 2 SARS CoV spike bearing pseudoviruses can enter cells via endocytosis A When HEK293E ACE2 GFP cells were treated with spike bearing pseudovirus for 3 h translocation of the ACE2 receptor was observed B When HEK293E ACE2 GFP cells were treated with spike minus pseudovirus VSV G pseudovirus for 3 h no translocation of the ACE2 receptor was observed C Colocalization of SARS CoV spike protein and ACE2 GFP in cytoplasmic vesicles 3 h after treatment with spike bearing pseudovirus Spike protein was probed with primary antibody and then detected with Alexa 568 goat anti mouse secondary antibody D Twelve hours after treatment with spike bearing pseudovirus HEK293E ACE2 GFP cells showed few cytoplasmic vesicles E After chloroquine treatment cytoplasmic vesicles were detected even at 12 h after spike bearing pseudovirus infection F NH 4 Cl chloroquine and bafilomycin A1 treatments inhibit viral GFP expression Vero E6 cells were mock treated or pretreated with 50 mM NH 4 Cl 100 M chloroquine or 80 nM bafilomycin A1 for 1 h before pseudovirus infection After 48 h the cells were lysed and GFP was measured as described in the Materials and Methods section Double asterisks indicate a significant difference from controls P 0 01 t test Error bars represent the SD of three independent experiments G I Alexa 594 transferrin G and EEA1 H colocalized in Vero E6 cells after a 1 h incubation at 37 C EEA1 was detected with its specific antibody followed by Alexa 488 secondary antibody J L Vero E6 cells were infected with spike bearing pseu dovirus for 1 h before they were fixed and immunolabeled with primary antibodies specific for SARS CoV spike protein J and the early endosome marker EEA1 K These two proteins were found to colocalize Spike protein was detected by Alexa 568 secondary antibody while EEA1 was detected with Alexa 488 secondary antibody A B C D E 10 m G H I F 1 2 1 0 8 0 6 0 4 0 2 0 Control NH 4 Cl Chloroquine Bafilomycin A1 Relative infectivity J K L 10 m Clathrin independent entry of SARS CoV 294 npg Cell Research Vol 18 No 2 February 2008 well characterized After binding of transferrin to its recep tor the complex was shown to be internalized in coated vesicles followed by fusion with endosomes 21 23 Thus the spike bearing pseudovirus like spike protein can cause ACE2 receptor translocation in a pH dependent manner and the pseudovirus infects Vero E6 cells in a pH dependent manner In addition the pseudovirus is targeted to the early endosome after it enters the cell Taken together these results indicate that the spike bearing pseudovirus can enter cells via endocytosis SARS CoV can enter cells in the absence of clathrin medi ated endocytosis After determining that SARS CoV can enter cells via endocytosis we attempted to identify the specific en docytic pathway exploited by this virus The endocytic pathways exploited by animal viruses to enter host cells include macropinocytosis the clathrin dependent pathway and the caveolae dependent pathway as well as routes that are not as well characterized such as clathrin and caveolae independent pathways The clathrin dependent pathway is the most common of these pathways Because drugs can produce pleiotropic effects we employed sev eral complementary approaches to determine the role of clathrin mediated endocytosis in the entry of SARS CoV into cells 4 Chlorpromazine CPZ a drug commonly used to inhibit clathrin mediated endocytosis causes clathrin lattices to assemble on endosomal membranes and prevents the as sembly of coated pits at the cell surface 24 The effect of CPZ on clathrin mediated endocytosis was first tested with transferrin labeled with Alexa 594 Alexa 594 Tf using Vero E6 cells In mock treated cells transferrin appeared to cluster in the perinuclear region while in the CPZ treated cells transferrin uptake was blocked leaving transferrin at the cell surface Figure 3A and 3B This result confirms the effectiveness of CPZ We then tested the effect of CPZ on spike pseudovirus entry using two different methods confocal microscopy and spectrofluorometer measurement After Vero E6 cells were treated with CPZ and incubated with spike bearing pseudoviruses for 1 h the ability of spike bearing pseudovirus to enter Vero E6 cells was examined using confocal microscopy Figure 3C and 3D showed that the spike bearing pseudovirus can enter Vero E6 cells despite CPZ treatment In order to quantify the ef fect of CPZ on viral entry Vero E6 cells were treated with the indicated amounts of CPZ and were then infected with spike bearing pseudoviruses The relative infectivity of the viruses was determined by measuring the level of GFP expression at 60 h postinfection using a spectrofluorometer Our results demonstrated that CPZ did not significantly inhibit virus entry Figure 3E We then used the more specific siRNA approach to further explore the role of clathrin mediated endocytosis in SARS CoV entry HEK293E ACE2 Myc cells were used because of their high transfection efficiency Cells were Figure 3 The effect of CPZ treatment and siRNA knockdown of clathrin on SARS CoV entry Vero E6 cells were mock treated A and C or pretreated with CPZ 10 M B and D and then incubated with Alexa 594 transferrin A and B or spike bearing pseudovirus C and D for 1 h at 37 C Spike protein was de tected with anti spike antibody followed by Alexa 488 secondary antibody E Vero E6 cells were treated with the indicated amount of chlorpromazine CPZ before they were incubated with GFP spike bearing pseudovirus Virus infectivity was measured using a spectrofluorometer at 60 h post infection see details in Materials and methods Virus entry was not inhibited by this treatment F HEK293 cells engineered to express ACE2 Myc were treated with siRNA specific for clathrin HC Western blotting revealed that the siRNA markedly reduced the level of clathrin HC Cells were then treated with GFP spike bearing pseudoviruses and the infectivity was measured using a spectrofluorometer at 48 h post infection The siRNA treatment did not inhibit virus entry Error bars represent the SD of three independent experiments 5 M 10 M 15 M 1 4 1 2 1 0 8 0 6 0 4 0 2 0 A B C D 20 m 20 m 20 m20 m Control Chlorpromazi