JOURNAL OF VIROLOGY Feb 2008 p 1899 1907 Vol 82 No 4 0022 538X 08 08 00H110010 doi 10 1128 JVI 01085 07 Copyright 2008 American Society for Microbiology All Rights Reserved Difference in Receptor Usage between Severe Acute Respiratory Syndrome SARS Coronavirus and SARS Like Coronavirus of Bat Origin H17188 Wuze Ren 1 Xiuxia Qu 2 Wendong Li 1 Zhenggang Han 1 Meng Yu 3 Peng Zhou 1 Shu Yi Zhang 4 Lin Fa Wang 3 Hongkui Deng 2 and Zhengli Shi 1 State Key Laboratory of Virology Wuhan Institute of Virology Chinese Academy of Sciences Wuhan China 1 Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education College of Life Sciences Peking University Beijing China 2 CSIRO Livestock Industries Australian Animal Health Laboratory and Australian Biosecurity Cooperative Research Center for Emerging Infectious Diseases Geelong Australia 3 and School of Life Science East China Normal University Shanghai China 4 Received 20 May 2007 Accepted 15 November 2007 Severe acute respiratory syndrome SARS is caused by the SARS associated coronavirus SARS CoV which uses angiotensin converting enzyme 2 ACE2 as its receptor for cell entry A group of SARS like CoVs SL CoVs has been identified in horseshoe bats SL CoVs and SARS CoVs share identical genome organiza tions and high sequence identities with the main exception of the N terminus of the spike protein S known to be responsible for receptor binding in CoVs In this study we investigated the receptor usage of the SL CoV S by combining a human immunodeficiency virus based pseudovirus system with cell lines expressing the ACE2 molecules of human civet or horseshoe bat In addition to full length S of SL CoV and SARS CoV a series of S chimeras was constructed by inserting different sequences of the SARS CoV S into the SL CoV S backbone Several important observations were made from this study First the SL CoV S was unable to use any of the three ACE2 molecules as its receptor Second the SARS CoV S failed to enter cells expressing the bat ACE2 Third the chimeric S covering the previously defined receptor binding domain gained its ability to enter cells via human ACE2 albeit with different efficiencies for different constructs Fourth a minimal insert region amino acids 310 to 518 was found to be sufficient to convert the SL CoV S from non ACE2 binding to human ACE2 binding indicating that the SL CoV S is largely compatible with SARS CoV S protein both in structure and in function The significance of these findings in relation to virus origin virus recombination and host switching is discussed The outbreaks of severe acute respiratory syndrome SARS in 2002 2003 which resulted in over 8 000 infections and close to 800 deaths was caused by a novel coronavirus CoV now known as the SARS associated CoV SARS CoV 12 25 33 36 The association of SARS CoV with animals was first re vealed by the isolation and identification of very closely related viruses in several Himalayan palm civets Paguma larvata and a raccoon dog Nyctereutes procyonoides at a live animal mar ket in Guangdong China A very high genome sequence iden tity more than 99 exists between the SARS CoV like virus from civets and SARS CoV from humans supporting the no tion that SARS CoV is of animal origin 18 However subse quent studies showed that palm civets on farms and in the field were largely free from SARS CoV infection 23 40 These results suggested that palm civets played a role as an interme diate host rather than as a natural reservoir Subsequent sur veillance studies among different bat populations revealed the presence in several horseshoe bat species genus Rhinolophus of a diverse group of CoVs which are very similar to SARS CoV in genome organization and sequence These viruses are designated SARS like CoVs SL CoVs or SARS CoV like vi ruses 26 29 Such discoveries raised the possibility that bats are the natural reservoirs of SARS CoV 26 29 38 and trig gered a surge in the search for CoVs in different bat species in different geographic locations 39 43 44a Phylogenetic analysis based on different protein sequences suggested that SL CoVs found in bats and SARS CoVs from humans and civets should be placed in a separate subgroup group b in CoV group 2 G2b to differentiate them from other group 2 CoVs in the genus Coronavirus 17 26 29 43 G2b CoVs display major sequence differences in the N termi nal regions of their S proteins The S proteins of CoVs play a key role in virus entry into host cells including binding to host cell receptors and membrane fusion 4 10 24 Angiotensin converting enzyme 2 ACE2 has been identified as the func tional receptor of SARS CoV and the molecular interaction between ACE2 and the SARS CoV S protein has been well characterized 27 28 31 42 A 193 residue fragment amino acids aa 318 to 510 in the SARS CoV S protein was dem onstrated to be the minimal receptor binding domain RBD which alone was able to efficiently bind to ACE2 1 42a 45 Furthermore it was shown that minor changes in amino acid residues of the receptor binding motif RBM of SARS CoV S Corresponding author Mailing address for Z Shi State key Lab oratory of Virology Wuhan Institute of Virology Chinese Academy of Sciences Wuhan Hubei 430071 China Phone 86 27 87197240 Fax 86 27 87197240 E mail zlshi Mailing address for L F Wang CSIRO Livestock Industries Australian Animal Health Laboratory P O Bag 24 Geelong Victoria 3220 Australia Phone 61 3 52275121 Fax 61 3 52275555 E mail Linfa wang csiro au W R and X Q contributed equally to this work Present address School of Life Science Heilongjiang University Harbin 150080 China H17188 Published ahead of print on 12 December 2007 1899 on June 4 2015 by SOUTHERN ILLINOIS UNIV http jvi asm org Downloaded from protein could abolish the entry of SARS CoV into cells ex pressing human ACE2 huACE2 7 31 In the corresponding RBD region of the SL CoV S proteins there is significant sequence divergence from those of the SARS CoV S proteins including two deletions of 5 and 12 or 13 aa From crystal structural analysis of the S ACE2 complex it was predicted that the S protein of SL CoV is unlikely to use huACE2 as an entry receptor 30 although this has never been experimen tally proven due to the lack of live SL CoV isolates Whether it is possible to construct an ACE2 binding SL CoV S protein by replacing the RBD with that from SARS CoV S proteins is also unknown In this study a human immunodeficiency virus HIV based pseudovirus system was employed to address these issues Our results indicated that the SL CoV S protein is unable to use ACE2 proteins of different species for cell entry and that SARS CoV S protein also failed to bind the ACE2 molecule of the horseshoe bat Rhinolophus pearsonii However when the RBD of SL CoV S was replaced with that from the SARS CoV S the hybrid S protein was able to use the huACE2 for cell entry implying that the SL CoV S proteins are structurally and functionally very similar to the SARS CoV S These results suggest that although the SL CoVs discovered in bats so far are unlikely to infect humans using ACE2 as a receptor it remains to be seen whether they are able to use other surface molecules of certain human cell types to gain entry It is also conceivable that these viruses may become infectious to humans if they undergo N terminal sequence variation for example through recombination with other CoVs which in turn might lead to a productive interaction with ACE2 or other surface proteins on human cells MATERIALS AND METHODS Cell lines and antibodies The human cell lines 293T and HeLa were grown in Dulbecco s modified Eagle s medium supplemented with 10 fetal calf serum Gibco Goat polyclonal antibody against the huACE2 ectodomain was pur chased from R palm civet AY881174 RpACE2 EF569964 mouse NM 027286 and rat NM 001012006 Black shading 100 identity gray shading 75 identity VOL 82 2008 RECEPTOR USAGE BY DIFFERENT SARS CORONAVIRUSES 1901 on June 4 2015 by SOUTHERN ILLINOIS UNIV http jvi asm org Downloaded from contact with the SARS CoV S protein 27 there are seven changes in RpACE2 compared to the cognate human protein Q24R Q in huACE2 and R in RpACE2 at aa 24 Y41H Q42E M82D Q325E E329N and G354D Fig 1 A stable HeLa cell line that continuously expressed the bat RpACE2 protein was established Western blot analysis showed that RpACE2 was expressed efficiently in HeLa cells and was recognized by anti RpACE2 antibodies Fig 2 Moreover huACE2 and pcACE2 could also be recognized by anti RpACE2 antibodies The levels of expression for the three different ACE2 molecules were very similar On SDS PAGE RpACE2 has mobility very similar to those of the other two ACE2 proteins with an apparent mass of approximately 90 to 100 kDa which is within the size range of the predicted mass of 93 kDa The localization of expressed ACE2 on the cell surface was demonstrated by immunofluorescence confocal microscopy us ing a commercial antibody raised against the ectodomain of huACE2 Fig 3 The surface location as well as ACE2 func tionality was further assessed by an enzyme activity assay using an ACE2 specific peptide substrate QFS As shown in Fig 4 membrane fractions prepared from three ACE2 expressing HeLa cell lines displayed substantially higher protease activities toward QFS than the control HeLa cells Furthermore the protease activity was largely abolished in the presence of 0 1 mM EDTA a known inhibitor of ACE2 proteases 11 These data indicated that all three ACE2 molecules were expressed as a functional protease associ ated with cellular membranes Pseudovirus packaging The expression of functional S pro teins and their correct incorporation into pseudoviruses were monitored by three approaches i e Western blotting of cell lysates with an S specific MAb Western blotting of pelleted virions using both S specific and p24 specific MAbs and direct examination of virions by EM The results are summarized in Fig 5 Both the BJ01 S and Rp3 S genes were efficiently ex pressed in transfected 293T cells and the expressed S proteins were incorporated into the respective pseudoviruses as ex pected Fig 5A When examined by EM the pseudoviruses containing the Rp3 S or BJ01 S protein displayed a morphol ogy characteristic of CoVs Fig 5B On the other hand con trasting results were obtained for two CS proteins For CS 14 608 the expressed S protein was incorporated into the pseudovirus as observed for the other two nonchimeric constructs How ever for CS 424 494 although the expression of the S protein was normal the pseudovirus seemed unable to assemble the CS protein at a level detectable by Western blot analysis Fig 5A and EM Fig 5B whereas p24 expression and assembly appeared to be normal Fig 5A FIG 2 Western blot analysis of bat ACE2 RpACE2 expressed in HeLa cells using rabbit anti RpACE2 antibodies Lane 1 HeLa cell lysate used as a negative control lanes 2 to 4 lysates from HeLa huACE2 HeLa pcACE2 and HeLa RpACE2 respectively The mo lecular masses in kDa of prestained protein markers Fermentas Canada are given on the left FIG 3 Detection of ACE2 expression by immunofluorescence con focal microscopy Cells were incubated with goat anti huACE2 antibody followed by probing with FITC conjugated donkey anti goat IgG The top three rows from the top down show HeLa cells expressing huACE2 pcACE2 and RpACE2 The bottom row shows HeLa cells as a negative control The columns from left to right show staining of expressed ACE2 green fluorescence of FITC staining of cell nuclei blue fluorescence of Hoechst 33258 and the merged double stained image FIG 4 Determination of ACE2 activity The protease activities from different ACE2 expressing cell membrane fractions were determined us ing the ACE2 specific substrate QFS see Materials and Methods The liberated fluorescence in relative fluorescence units RFU determined at 320 to 420 nm in the absence A and presence B of EDTA is shown HeLa cells with no exogenous ACE2 gene and PBS buffer were used as negative controls The error bars indicate standard deviations 1902 REN ET AL J VIROL on June 4 2015 by SOUTHERN ILLINOIS UNIV http jvi asm org Downloaded from Use of huACE2 pcACE2 and bat RpACE2 for cell entry by different pseudoviruses When HIV Rp3 S pseudovirus was used to infect HeLa cells expressing huACE2 pcACE2 or RpACE2 only a low level of luciferase activity approximately 100 relative light units the same as the background level gen erated by the vector control was obtained Fig 6 In contrast when the BJ01 S typed pseudovirus was used high levels of luciferase activity more than 10 5 relative light units were detected in the cell lysates of HeLa huACE2 and HeLa pcACE2 Fig 6A and B but not in HeLa RpACE2 Fig 6C Interestingly the pseudovirus packaged with a CS protein HIV CS 14 608 displayed a level of luciferase activity similar to that of HIV BJ01 S Fig 6A As expected from the above mentioned Western blot and EM analyses HIV CS 424 494 in which the RBM of BJ01 S was transferred into the Rp3 S backbone failed to produce luciferase activity above the back ground level in any of the three ACE2 expressing HeLa cell lines Mapping of the minimal BJ01 S region required to convert the non ACE2 binding Rp3 S to a chimeric ACE2 binding S protein Based on the results mentioned above it is evident that the Rp3 S protein is capable of mediating cell entry as long as the ACE2 binding component is incorporated into the N terminal region of its molecule To define the minimal re gion required for this conversion from non huACE2 binding to huACE2 binding a series of CS proteins was constructed and the exact amino acid locations are summarized in Fig 7A S protein expression in 293T cells and its incorporation into pseudovirus for each construct were analyzed by Western blot ting using S specific and p24 specific antibodies as described above Fig 7B Although the levels of S protein expression were similar for all constructs significant differences in incor poration were observed The S protein was undetectable in pseudoviruses derived from CS 371 608 and was only weakly de tected for the chimeras CS 310 608 CS 45 608 and CS 310 518 The infectivities of all the CS constructs in HeLa huACE2 were examined and several observations were made Fig 7C With the exception of the chimeras CS 417 608 and CS 371 608 which produced a background level of luciferase activity all of the chimeras exhibited significant levels of luciferase activity sug gesting that pseudoviruses containing these CS proteins were able to use huACE2 for cell entry From these results it was deduced that the region from aa 310 to 518 of BJ01 S was necessary and sufficient to convert Rp3 S into a huACE2 FIG 5 Analysis of S protein expression and incorporation into pseudovirus A Western blot analysis S proteins expressed in trans fected 293T cells were probed with MAb F26G8 or S MAb top the middle and bottom panels are Western blots of pelleted pseudoviruses using MAbs F26G8 and p24 respectively B EM examination of pseudovirus morphology The name of the S protein construct in each pseudovirus is shown at the top left corner of the electron micrograph FIG 6 Measurement of pseudovirus infectivity by determining the reporter luciferase activity Cell lysates were prepared 48 h p i from HeLa huACE2 A HeLa pcACE2 B and HeLa RpACE2 C and luciferase activity was determined as described in Materials and Meth ods The error bars indicate standard deviations VOL 82 2008 RECEPTOR USAGE BY DIFFERENT SARS CORONAVIRUSES 1903 on June 4 2015 by SOUTHERN ILLINOIS UNIV http jvi asm org Downloaded from binding molecule For CS 310 608 CS 45 608 and CS 310 518 the levels of luciferase activity detected were much higher than that predicted from the level of S protein incorporated into the virion suggesting that the infection assay is more sensitive than Western blotting It is also important to note that although a low level of S protein was present in the virion of CS 417 608 it did not lead to a productive infection DISCUSSION The CoV spike glycoproteins are responsible for cellular receptor recognition cell tropism and host specificity 7 9 19 25a Our previous results showed that the SL CoVs identified in bats are similar to SARS CoV in genome sequence and organization The key difference between these two groups of closely related viruses lies in their S protein sequences specif ically the RBM in which there are two deletions in the bat SL CoV S sequences 29 From previous published studies indicating the importance of the structural fit between the receptor ACE2 and the S protein of SARS CoV variants and the sensitivity of S proteins to point mutations in the RBM region it was speculated that SL CoV S is unlikely to use ACE2 as a receptor for cell entry unless the bat ACE2 ho molog is significantly different from those of other mammals To address these unanswered questions we cloned and ex pressed the bat R pearsonii ACE2 gene and examined the abilities of ACE2 proteins from human palm civet and R FIG 7 Construction and functional analysis of pseudoviruses derived from different CS protein constructs A Schematic presentation of constructs of human SARS CoV S protein BJ01 S bat SL CoV S Rp3 S and different CS proteins The numbers in the subscripts indicate the amino acid locations of the BJ01 S sequences used to replace the corresponding region of Rp3 S The open box indicates the location of the RBM TPA signal peptide from tissue plasminogen activator TM transmembrane domain derived from the fusion protein of Sendai virus B Western blot analysis S proteins in transfected 293T cells top or purified pseudoviruses middle were probed with MAb F26G8 S MAb at the bottom is a blot of different pseudoviruses probed with p24 MAb as a control to determine the relative quantity of HIV pseudovirus for each construct C Measurement of pseudovirus infectivity by determining luciferase activity Cell lysates were prepared 48 h p i from HeLa huACE2 infected with pseudoviruses containing different S proteins as indicated below the bar graph The error bars indicate standard deviations 1904 REN ET AL J VIROL on June 4 2015 by SOUTHERN ILLINOIS UNIV http jvi asm org Downloaded from pearsonii to support infection by HIV based pseudoviruses containing different S protein constructs Our results indicated that the bat SL CoV Rp3 S protein is unable to use ACE2 for cell entry regardless of the origin of the ACE2 molecule We also demonstrated that the human SARS CoV S cannot use bat RpACE2 as a functional receptor On the other hand we demonstrated that after replacement of a small segment aa 310 to 518 of Rp3 S by the cognate sequence of BJ01 S the CS protein mimics the function of BJ01 S in regard to receptor usage in the HIV pseudovirus assay system Although we cur rently have no way of confirming that the Rp3 S protein is functional in binding its cognate receptor due to the lack of a horseshoe bat cell line our chimeric construct analysis sug gests that the Rp3 S gene was intact and would be functional if an appropriate receptor was identified It is also worth noting that although we have no experimental data to demonstrate direct binding of different S proteins to huACE2 on the HeLa cell surface we did confirm that either anti huACE2 or anti SARS CoV polyclonal antibodies were able to neutralize in fection by different pseudoviruses data not shown The significance of these findings is as follows First the failure of SARS CoV S protein to use bat RpACE2 as a re ceptor suggests that despite the presence of a diverse group of SL CoVs in horseshoe bats they are unlikely to be the natural reservoir of the immediate progenitor virus for SARS CoV It is therefore important to continue the search for the reservoir of SARS CoV in other bat and wildlife species It is also important to conduct such searches in different geographical locations because live animal markets in southern China source their animals from all over China and from foreign countries 26 29 39 and it is possible that the na