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JOURNAL OF VIROLOGY Nov 2007 p 12029 12039 Vol 81 No 21 0022 538X 07 08 00H110010 doi 10 1128 JVI 00315 07 Copyright 2007 American Society for Microbiology All Rights Reserved Specific Asparagine Linked Glycosylation Sites Are Critical for DC SIGN and L SIGN Mediated Severe Acute Respiratory Syndrome Coronavirus Entry H17188 Dong P Han 1 Motashim Lohani 1 and Michael W Cho 1 2 3 Departments of Medicine 1 Biochemistry 2 and Molecular Biology and Microbiology 3 Case Western Reserve University School of Medicine Cleveland Ohio 44106 Received 12 February 2007 Accepted 11 August 2007 Severe acute respiratory syndrome SARS is caused by a newly emerged coronavirus CoV designated SARS CoV The virus utilizes angiotensin converting enzyme 2 ACE2 as the primary receptor Although the idea is less clear and somewhat controversial SARS CoV is thought to use C type lectins DC SIGN and or L SIGN collectively referred to as DC L SIGN as alternative receptors or as enhancer factors that facilitate ACE2 mediated virus infection In this study the function of DC L SIGN in SARS CoV infection was examined in detail The results of our study clearly demonstrate that both proteins serve as receptors independently of ACE2 and that there is a minimal level of synergy between DC L SIGN and ACE2 As expected glycans on spike S glycoprotein are important for DC L SIGN mediated virus infection Site directed mutagenesis analyses have identified seven glycosylation sites on the S protein critical for DC L SIGN mediated virus entry They include asparagine residues at amino acid positions 109 118 119 158 227 589 and 699 which are distinct from residues of the ACE2 binding domain amino acids 318 to 510 Amino acid sequence analyses of S proteins encoded by viruses isolated from animals and humans suggest that glycosylation sites N227 and N699 have facilitated zoonotic transmission The etiological agent of severe acute respiratory syndrome SARS has been identified as a novel coronavirus CoV des ignated SARS CoV 9 20 32 35 SARS CoV represents one of several pathogens that have emerged in recent years The SARS epidemic during 2002 and 2003 had a major socioeco nomic impact globally Although SARS CoV infections have not been reported recently there is potential for the virus to reemerge in the future considering that humans often come into contact with animals that are susceptible to virus infection or that serve as reservoirs 11 21 29 Better understanding of the mechanism s of virus entry into host cells could facilitate development of a vaccine and antiviral agents The entry of CoVs into cells is mediated by spike S glyco protein S protein of SARS CoV is 1 255 amino acids aa long It has a 13 aa signal peptide a single ectodomain 1 182 aa and a transmembrane region followed by a short cytoplasmic tail 28 residues 28 37 Although S proteins of many CoVs are cleaved into and function as two separate subunits S1 and S2 1 17 31 S protein of SARS CoV is not 12 47 It is presumed nevertheless to have two functional domains and the border between them has been suggested to be around aa 680 27 41 The S1 domain is responsible for binding to cellular receptors and the S2 domain contains two heptad repeat regions HR1 and HR2 that form six helix bundles 5 16 43 50 51 and mediate fusion between viral and cellular membranes The receptor for SARS CoV has been identified as angio tensin converting enzyme related carboxypeptidase ACE2 24 The receptor binding domain RBD has been narrowed down to amino acid residues 318 to 510 3 46 47 A cocrystal structure of ACE2 bound to the RBD revealed that residues 424 to 494 form the receptor binding motif RBM that di rectly contacts ACE2 25 Not surprisingly site directed mu tagenesis studies have identified many residues within this re gion as critical to binding ACE2 6 46 Although it is clear that ACE2 serves as a receptor for SARS CoV other reports show that DC SIGN dendritic cell specific ICAM 3 grabbing nonintegrin and L SIGN for liver lymph node specific also called CD209L or DC SIGNR also are involved in virus entry 18 30 48 DC SIGN and L SIGN collectively referred to as DC L SIGN are members of a C type lectin family the interactions of which with ligands are carbohydrate dependent 2 14 26 they specifically recognize high mannose glycans 10 The exact role of these molecules in viral infection pathogenesis is unclear and somewhat con troversial While one study reported that L SIGN can serve as an alternative receptor 18 another study showed that DC L SIGN enhance only ACE2 mediated infections 30 Regard less the potential role of DC L SIGN in SARS CoV patho genesis is great since dendritic cells have been shown to transfer infectious viruses to susceptible target cells via DC SIGN 48 Moreover L SIGN is expressed in human lung tissue on type II alveolar cells which are important targets for SARS CoV infection 8 42 However results from genetic analyses seem to suggest that homozygosity for L SIGN plays a protective role in SARS CoV infection by promoting higher levels of proteasome mediated virus degradation 7 In light of these conflicting observations the role of DC L SIGN in SARS CoV infections needs to be further examined In this study SARS pseudoviruses were utilized to charac Corresponding author Mailing address Case Western Reserve University School of Medicine Department of Medicine Division of Infectious Diseases 10900 Euclid Ave Cleveland OH 44106 4984 Phone 216 368 1221 Fax 216 368 0069 E mail mcho case edu H17188 Published ahead of print on 22 August 2007 12029 on March 8 2015 by VETERINARY MED LIB E http jvi asm org Downloaded from terize and to compare virus infections mediated by DC L SIGN and by ACE2 Here we demonstrate unambiguously that DC L SIGN indeed serve as receptors for SARS CoV and that they function independently of ACE2 As expected gly cans on S protein play an important role in DC L SIGN me diated infections Moreover site directed mutagenesis analy ses revealed that carbohydrate moieties on specific asparagine N linked glycosylation sites are critical The results of our study provide a better understanding of SARS CoV entry and identify another potential target for development of antiviral agents against the virus MATERIALS AND METHODS Plasmids and site directed mutagenesis A plasmid encoding full length wild type human ACE2 was generously provided by Michael Farzan 24 Plasmids encoding DC SIGN or L SIGN 33 34 were obtained from the NIH AIDS Research and Reference Reagent Program catalog nos 5444 and 6746 respec tively Site directed mutagenesis of SARS CoV S protein pHCMV S 12 was performed using a QuikChange XL site directed mutagenesis system Strat agene with PfuTurbo DNA polymerase Seventeen pairs of primers were used to generate mutants Sense strand primer sequences are shown in Table 1 Some primers included silent mutations for introducing restriction sites All of the mutations were verified by sequencing Pseudovirus production and infections Cell lines TELCeB6 38 HeLa and Vero E6 were maintained in Dulbecco s modified Eagle s medium DMEM supplemented with 5 to 7 fetal bovine serum 2 mM L glutamine and penicil lin streptomycin antibiotics Cells were cultured in 5 CO 2 incubators at 37 C Pseudoviruses that encode H9252 galactosidase were produced as we have previously described 12 Briefly TELCeB6 cells which continuously release murine leu kemia virus particles were cotransfected with plasmids encoding S glycoprotein pHCMV S and pcDNA3 1 Invitrogen using Lipofectin Invitrogen per the manufacturer s protocol To generate TELCeB6 cells that stably express S gly coprotein cotransfected cells were selected in the growth medium containing Geneticin 0 4 H9262g ml Invitrogen Geneticin resistant clones were isolated and expanded and those able to produce high titers of SARS pseudoviruses were selected Although cells subsequently were maintained in the absence of Gene ticin they continuously produced pseudoviruses with titers of 5 H11003 10 3 to 6 H11003 10 3 per ml The virus titer was determined in Vero E6 cells Mutant pseudoviruses were produced by transient transfections with plasmids encoding mutant S pro teins Pseudovirus infections were done using Vero E6 cells 96 well plates or HeLa cells 24 well plates transfected with plasmid encoding ACE2 DC SIGN or L SIGN Cells were transfected with 1 H9262g or indicated amounts of plasmid DNA per well using Lipofectin After overnight incubation culture medium was replaced Approximately 24 h posttransfection cells were infected with 100 to 150 infectious units of pseudoviruses Pseudoviruses were allowed to adsorb onto cells for about 60 min Cells subsequently were washed with serum free DMEM to remove unadsorbed viruses and fresh medium was added Infections were allowed to proceed for an additional 1 5 days at which time infected cells were stained with 5 bromo 4 chloro 3 indolyl H9252 D galactopyranoside and quantified as previously described 12 Unless specified results of virus infections are shown as absolute titers i e number of infectious foci or normalized as a percentage of the wild type control or no inhibitor or neutralizing antibodies for the given receptor Pseudovirus inhibition and neutralization assays ACE2 derived inhibitory peptide P6 was previously described 13 The peptide or mannan Sigma Aldrich dissolved in phosphate buffered saline at the indicated concentrations was preincubated with 100 infectious units of SARS CoV pseudoviruses for 20 min at 37 C Subsequently the virus inhibitor mixture was added to HeLa cells transfected with either ACE2 or L SIGN Cells were incubated at 37 C for an additional 1 5 days and were stained for H9252 galactosidase activity as described above Neutralizing mouse monoclonal antibodies MAbs against SARS CoV S pro tein 44 were generously provided by Lia M Haynes at the Centers for Disease Control and Prevention Polyclonal mouse anti SARS CoV S protein antiserum 22 was kindly provided by Chul Joong Kim at Chung Nam National University South Korea Pseudoviruses were incubated with antibodies for1hat37 C Subsequently the antibody virus mixture was added to HeLa cells transfected with either ACE2 or L SIGN Cells were incubated at 37 C for 1 5 days and stained for H9252 galactosidase activity Assays were done in duplicate To evaluate effects of endoglycosidase H Endo H New England Biolabs on SARS CoV pseudovirus infectivity about 100 infectious units were treated with 500 U of enzyme for various times 0 to 4 h in a nondenaturing condition For controls pseudoviruses were incubated with either normal culture medium or buffer only 50 mM sodium citrate pH 5 5 Western blotting Freshly seeded TELCeB6 cells less than 1 day old were transfected as previously described 12 13 with plasmids encoding either the wild type pHCMV S or mutant pHCMV S H9262 S gene using Lipofectin In vitrogen Three days posttransfection culture medium was used as a source of SARS pseudovirus and cells were lysed with a hypotonic buffer containing nonionic detergent 10 mM Tris pH 7 5 10 mM NaCl 1 5 mM MgCl 2 and 1 NP 40 for protein analysis Nuclei were removed by brief centrifugation Post nuclear cell extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electrotransfer to nitrocellulose membranes for Western blot analyses SARS S proteins were detected with rabbit anti S poly clonal antibodies 1 200 dilution of serum generously provided by Shan Lu 45 followed by goat anti rabbit immunoglobulin G conjugated with horseradish peroxidase Pierce Protein bands were visualized with SuperSignal chemilumi nescent substrates Pierce according to the manufacturer s protocol RESULTS DC SIGN and L SIGN both serve as alternative receptors for SARS CoV entry independently of ACE2 To determine whether DC SIGN or L SIGN could serve as alternative re ceptors for SARS CoV rather than simply as enhancer factors infectivity of SARS pseudoviruses murine leukemia virus pseudotyped with S glycoprotein was examined by using HeLa cells transfected with plasmids encoding these proteins As shown in Fig 1A HeLa cells transfected with pcDNA empty vector were completely refractory to SARS pseudovirus infec tion In contrast cells transfected with ACE2 expressing plas mid efficiently supported pseudovirus infection HeLa cells expressing either DC SIGN or L SIGN also were susceptible to infection albeit considerably less so than those expressing ACE2 Although the difference was minimal and not statis tically significant cells expressing L SIGN were consistently more susceptible to infection than those expressing DC SIGN TABLE 1 Primers used for generating mutant S glycoproteins a Amino acid Sense strand sequence 5H11032 to 3H11032 position of Asn 29 gat gtt caa gct cct CaG tac act caa cat ac 65 ctt cca ttt tat tct CaG gtt aca ggg ttt c 73 ggg ttt cat act att CaG cat acg ttt ggc aac 109 gg gtt ttt ggA tcC acc atg aac CaG aag tca cag tcg gtg 118 cg gtg att att att CaG aat tct act aat g 119 gtg att att att aac CaG tct act aat gtt g 158 c gat aat gca ttt CaG tgc act ttc gag tac 227 g cct ctt ggt att CaG att aca aat ttt ag 269 ctc aag tat gat gaa CaG ggt aca atc aca g 318 gtt gtg aga ttc cct CaG att aca aac ttg tg 330 gga gag gtt ttt CaG gct act aaa ttc cc 357 c tct gtg ctc tac CaG tcG aca ttt ttt tca acc 589 agt gta att aca ccC ggG aca CaG gct tca tct gaa gtt gct gtt c 602 cta tat caa gat gtt CaG tgc act gat gtt tct 691 tca att gct tac tct CaG aac acc att gct ata 699 att gct ata cct act CaG ttt tca att agc att 783 acc cca act ttT aaa tat ttt ggt ggt ttt CaG ttt tca caa ata tta a Capital letters indicate mutated nucleotides Antisense primers are comple mentary to the sense primers Bold type indicates where changes were made In case of an amino acid change the entire codon is boldfaced 12030 HAN ET AL J VIROL on March 8 2015 by VETERINARY MED LIB E http jvi asm org Downloaded from We have previously described the development of an ACE2 derived peptide P6 that potently inhibits SARS pseudovirus infections of Vero E6 or HeLa cells expressing ACE2 with a 50 inhibitory concentration of approximately 100 nM 13 This peptide consists of two discontinuous segments of ACE2 amino acid residues 22 to 44 and 351 to 357 artificially linked by a single glycine residue These determinants have been shown to interact biochemically and structurally with the RBM of S protein and are critical for mediating SARS CoV infection 23 25 To unequivocally demonstrate that pseudovirus infections of HeLa cells expressing L SIGN are not mediated through ACE2 infectivity was examined in the presence of the P6 peptide As shown in Fig 1B infection mediated by ACE2 was potently inhibited by P6 peptide in a dose dependent manner as previously described 13 In con trast no inhibition was observed for L SIGN mediated infec tion even in the presence of 100 H9262M Similar results were observed for DC SIGN mediated infections data not shown These results not only indicate that DC L SIGN serve as al ternative receptors but also indicate that the binding site s of these proteins on S protein is distinct from the site that ACE2 binds DC L SIGN are members of a C type lectin family the in teractions of which with ligands are carbohydrate dependent 2 14 26 they specifically recognize high mannose glycans 10 Mannan a carbohydrate composed of high mannose inhibits binding of ligands to DC L SIGN To demonstrate that infections of HeLa cells expressing DC L SIGN by our SARS pseudoviruses are indeed mediated by DC L SIGN infection of HeLa cells expressing either ACE2 or L SIGN were carried out in the presence of various amounts of mannan Fig 1C As expected L SIGN mediated infections were inhibited by mannan in a dose dependent manner In contrast ACE2 me diated infections were not affected by mannan To further characterize virus entry mediated by ACE2 and DC L SIGN sensitivity of pseudoviruses to neutralizing MAbs was evaluated Four MAbs CDC 336 CDC 341 CDC 523 and CDC 540 obtained from Lia Haynes at the Centers for Disease Control and Prevention were evaluated These anti bodies were generated from mice immunized with whole inac tivated SARS CoV particles 44 The epitope recognized by CDC 341 is amino acid residues 490 to 510 which is at the C terminal end of the RBD The epitopes of the other MAbs have not yet been determined Regardless all four MAbs in hibited ACE2 mediated virus entry Fig 1D This is not sur prising since they were screened for their ability to block virus infection of Vero E6 cells In contrast none of the MAbs inhibited L SIGN mediated virus entry This is not because L SIGN mediated infections are intrinsically difficult to in hibit since polyclonal antiserum from mice immunized with Lactobacillus casei expressing S protein fragments 22 was able to inhibit infections mediated by both receptors To gether these results demonstrate that DC L SIGN can medi ate SARS CoV infections independently of ACE2 DC L SIGN minimally enhance ACE2 mediated infections Although the results of our study indicated that DC L SIGN function as alternative receptors for SARS CoV there re mained a possibility that they also enhance ACE2 mediated infections To evaluate whether there is a synergistic relation ship between ACE2 and DC SIGN or L SIGN pseudovirus infectivity in HeLa cells expressing various amounts of the latter proteins in the presence or the absence of ACE2 was evaluated As expected transfection of greater amounts of plasmid expressing DC SIGN Fig 2A or L SIGN Fig 2B increased pseudovirus infectivity However infectivity reached a plateau at about 0 5 H9262g of plasmid DNA similar to what was observed with ACE2 13 Consistent with results shown in Fig 1A L SIGN was more efficient than DC SIGN in supporting SARS pseudovirus entry When ACE2 expressing plasmid was cotransfected 0 25 H9262g a low level of synergy was observed The maximal synergistic effect 1 6 fold over the additive level was observed when 0 25 H9262g of plasmids expressing DC SIGN or L SIGN was used The synergy was lost when 1 H9262gof plasmid was used This is likely due to the fact that as DC L SIGN concentrations increase they are removing a pool of viruses that can bind ACE2 rendering the virus infection less efficient These results indicated that infections mediated by ACE2 and DC L SIGN are minimally synergistic and that the receptors likely function independently Glycans play an important role in SARS CoV infections mediated by DC L SIGN Since mannan inhibited SARS pseudovirus infections mediated by L SIGN glycans on S gly FIG 1 DC SIGN and L SIGN serve as alternative receptors A HeLa cells transfected with plasmids expressing ACE2 DC SIGN or L SIGN were infected with SARS pseudoviruses An empty vector pcDNA was used as a negative control B ACE2 and L SIGN mediated infections were examined in the presence of various concentrations of an inhibitory peptide P6 derived from ACE2 13 C L SIGN mediated but not ACE2 mediated infections are inhibited by mannan in a dose dependent manner D Specific inhibition of ACE2 mediated infections by MAbs A polyclonal anti S protein antiserum poly Ab from mice is able to inhibit infections mediated by both ACE2 and L SIGN VOL 81 2007 ROLE OF GLYCANS IN SARS CoV ENTRY 12031 on March 8 2015 by VETERINARY MED LIB E http jvi asm org Downloaded from coprotein most likely play an important role To demonstrate this more directly effects of removing carbohydrate moieties on pseudovirus infectivity were examined We have previously shown that carbohydrate moieties on S protein are high man nose and or hybrid oligosaccharides and that they can be re moved using Endo H under a mild condition 12 To remove glycans on S protein pseudoviruses were treated with Endo H for different durations from 0 to 4 h Their infectivity was examined in HeLa cells expressing DC SIGN L SIGN or ACE2 Fig 3 As expected infectivity of SARS pseudoviruses treated with Endo H decreased drastically in cells expressing DC SIGN or L SIGN in a time d