key: cord-0033678-gfiqjgxg authors: Zhang, Yi; Wang, Wei; Gao, Jin-rong; Ye, Li; Fang, Xiao-nan; Zeng, Ying-chun; Wu, Zheng-hui; She, Ying-long; Ye, Lin-bai title: The functional motif of SARS-CoV S protein involved in the interaction with ACE2 date: 2009-09-16 journal: Virol Sin DOI: 10.1007/s12250-007-0054-8 sha: a08e6e3d8c3de886ae461ca0843a51d80b62e855 doc_id: 33678 cord_uid: gfiqjgxg SARS-CoV is a newly discovery pathogen causing severe acute respiratory problems. It has been established that the S protein in this pathogen plays an important rule in the adsorption and penetration of SARS-CoV into the host cell by interaction with the ACE2 receptor. To determinant which functional motif of the S protein was involved in the interaction with ACE2, seven truncated S proteins deleted from the N or C terminal were obtained by an E.coli expression system and purified by column chromatography to homogeneity. Each truncated S protein was fixed on to the well of an ELISA plate and an interaction was initiated with the ACE2 protein. The adsorption were quantified by ELISA, and the results indicated that amino acids from 388 to 496 of the S protein was responsible for the interaction with the ACE2 receptor, and the interaction could be completely disrupted by an antibody specific to these amino acids. Deletions adjacent to this domain did not appear to have a significant impact on the interaction with ACE2, suggesting that the S protein of SARS-CoV could be developed as a vaccine to prevent the spread of SARS-CoV. . The virus causes atypical pneumonia with diffuse alveolar damage with an overall mortality of 10% that ranges from 0% in children and 50% in persons over 65, seriously threatening public health worldwide. VIROLOGICA SINICA Vol.22 No.1 [ 2 The S protein of SARS-CoV is a large type I membrane glycoprotein projection from the viral envelope which is responsible for both binding to receptors on host cells and for membrane fusion. Angiotensin converting enzyme 2(ACE2) was found to be an efficient receptor for the S glycoprotein of SARS-CoV (9, 4, 12) . The S protein also contains important virus neutralizing epitopes that elicit neutralizing antibody in the host species (3, 2) . To determine the critical sequence of the S protein that interacts with the ACE2 receptor, the interactions between ACE2 and different truncated S proteins were investigated. Our study sheds some light on the interaction mechanism and provides useful insight into the development of a protein vaccine for SARS-CoV. Plasmids S1-1 encoding SARS-Cov S protein residues 262~606，S1-2 encoding 314~606，S1-3 encoding 388~606，S1-4 encoding 314~387，S1-5 encoding 262~496，S1-6 encoding 262~387，S1-7 encoding 388~496 (Fig. 1.) were constructed. The sequences of the primers (FP-forward primer, RPreberse primer) employed in the PCR reaction were： S1-1 FP：5′-gtcagggatccatgctcaagtatgatgaaaatggtac-3′ RP：5′-actcgagaattcctaaacatcagtgcagttaac-3′ S1-2 FP：5′-ctgcagggatccaccatggtgagattccctaatattac-3′ RP：5′actcgagaattcctaaacatcagtgcagttaac-3′ S1-3 FP：5′-ctgcagggatccaccatggtcaagggagatgatg-3′ RP：5′-actcgagaattcctaaacatcagtgcagttaac-3′ S1-4 FP：5′-ctgcagggatccaccatggtgagattccctaatattac-3′ RP：5′-actcgagaattcctatacaaaagaatctgc-3′ S1-5 FP：5′-gtcagggatccatgctcaagtatgatgaaaatggtac-3′ RP：5′-actcgagaattcctaaactctgtaaggttgg-3′ S1-6 FP：5′-gtcagggatccatgctcaagtatgatgaaaatggtac-3′ RP：5′-actcgagaattcctatacaaaagaatctgc-3′ S1-7 FP：5′-ctgcagggatccaccatggtcaagggagatgatg-3′ These forward primers carried a BamH Ⅰ 1.5 Preparation of rabbit anti-S1-1~ S1-7 sera 0.1 mg each of seven purified S proteins were mixed well with 1% Al(OH) 3 respectively, and used to immunized rabbit by injecting hypodermal at three point. Three weeks after first immunization. Booster shot were performed, 10 days later, the blood samples were collected and titers of antibodies were measured by ELISA. 6.1 Purified S1-1~S1-7 proteins ， dissolved respec-tively in coating buffer (0.016mol/L Na 2 CO 3 , In the interaction disruption assay, rabbit anti-S1-7 serum was added into the well before adding ACE2, the remainder of the steps were the same as above. The fragments of 1 035bp (S1-1), 879bp (S1-2), 657bp (S1-3), 222bp (S1-4), 705bp (S1-5), 128bp (S1-6) and 327bp (S1-7) were amplified by PCR using plasmid pGEM-S as template and were then cloned into prokaryotic expression vector pETHis. The corrected clones were confirmed by restriction analysis and sequencing. The results of agarose electrophoresis are shown in Fig.2 and Fig.3 . proteins S1-1~S1-7 S1-1~S1-7 proteins were successfully expressed in Fig.2 . Analysis of recombinant plasmid pETHis S1-1～S1-6 by BamHⅠ ﹠ EcoRⅠ digestion M, 1kb DNA ladder; 1,pETHis S1-1; 2, pETHis S1-2; 3, pETHis S1-3; 4, pETHis S1-4; 5, pETHis S1-5; 6, pETHis S1-6; 7, pETHis S1-5. Fig.3 . Analysis of recombinant plasmid pETHis S1-7. M, 1kb DNA ladder; 1, PCR product; 2, pETHis S1-7/BamHⅠ﹠ EcoRⅠ; 3,pET-his S1-2/ BamHⅠ. Fig.4 . Expression and purification of the recombinant protein S1-1～S1-7. M, protein marker; 1, S1-1; 2, S1-2; 3, S1-3; 4, S1-6; 5, S1-5; 6, S1-4; 7, S1-7; E.coli BL21(DE3) at a high level and formed insoluble inclusion bodies. As Fig.4 shows, the proteins were expressed and purified to a high purity. To investigate whether the induced proteins were the expected proteins, Western Blot analysis of purified proteins were processed. As Fig.5 showed, all purified ZHANG et al. The Functional Motif of SARS-CoV S Protein Involved in the Interaction with ACE2 5 S1-1~S1-7 proteins could react with the SARS pat ients sera specifically and the molecular weights were consistent with expectations, indicating the appropriate proteins were produced. The recombinant ACE2 protein was successfully Fig.5 . Western Blot of purified protein S1-1～S1-7. 1, S1-1; 2, S1-2; 3, S1-3; 4, S1-6; 5, S1-5; 6, S1-4; 7, S1-7. expressed in E.coli DH5. Fig.6 showed that ACE2 was induced and purified. The purified ACE2 protein could react with anti-ACE2 serum and the expected 92kDa band was observed in Western Blot (Fig.7) . Purified recombinant proteins S1-1~S1-7 were fixed in the 96-well plate by coating, and then purified ACE2 protein was added to allow their interaction. A well coated with purified ACE2 protein was used as positive control. Another coated with purified S1-1 protein without adding ACE2 protein was used as negative control. Then Anti-vero-ACE2 rabbit serum was added as first antibody and HRP labeled goat-anti-rabbit IgG was added as second antibody. ELISA showed that the results of S1-1, S1-2, S1-3, S1-5, S1-7 wells were positive, while the results of S1-4, S1-6 were negative (Table 1. ). Our data showed that the aa 388~396 of S1 protein was essential to the interaction between S protein and ACE2. To further confirm this result, an antibody induced by S1-7 which only contains aa 388~396 of S protein was used for disrupting the interaction between S and ACE2. The results show that all the interactions we re nearly completely blocked by this antibody (Table 2. ). Whereas the antibody induced by S1-4 which contained deletions aa 388~396 did not show ability to block this interaction. Table 1 . Interaction between truncated S protein and ACE2 S1-1 S1-2 S1-3 S1-4 S1-5 S1-6 S1-7 ACE2 Table 2 . Interaction blocking by antibody against S1-7 S1-1 S1-2 S1-3 S1-4 S1-5 S1-6 S1-7 ACE2 anti-S1-7 Engineering a protein vaccine for SARS, therefore, is a relatively safer option and has great potential as a future direction in SARS vaccine development. The spike protein of SARS-CoV is composed of two parts: S1 and S2. S1 is involved in viral infection, pathogenesis, host ranges and binding to the receptors (5, 6) . So this study on the interaction between S1 protein and the receptor ACE2 may shed light on the development of engineering protein vaccines of SARS. In this study, seven truncated S1 sequences (S1~S7) were cloned in the prokaryotic expression vectors and induced to express at a high level. Our results showed that S1-1, S1-2, S1-3, S1-5 and S1-7 can interact with purified ACE2 protein specifically, but S1-4 and S1-6 cannot interact with this receptor. S1-1, S1-2 and S1-3 are sequential truncated proteins of the N-terminal S protein. They can both bind to ACE2, suggesting aa 262~387 is not essential for S1-ACE2 interaction. S1-5 can bind to ACE2, while S1-6 can't and S1-7 can bind to ACE2 while S1 4 can t, both of which suggested that aa 388~496 is essential for the S1 ACE2 interaction. In this study, two methods were used for investiga-ting the binding of various truncated S1 proteins to the ACE2. The first one was the Far-Western Blot. Various truncated S1 proteins were resolved on a 15% SDS-PAGE gel and then transferred onto N.C membrane. The blots were overlayed with ACE2 and the binding of ACE2 protein was detected by X-ray with anti-ACE2 rabbit serum as the first antibody and goat-anti-rabbit IgG as the second antibody. However, no ACE2 protein binding to the membrane was detected. The second method was performed as follows. Truncated S1 proteins were fixed onto the plates well by coating without denaturing, and then purified ACE2 protein solution was added to allow the interaction to proceed.Anti-ACE2 rabbit serum was used as the first antibody and goat-anti-rabbit IgG was used as the second antibody. HRP activities were assayed and our results showed that S1-1, S1-2, S1-3, S1-5, S1-7 can all bind to purified ACE2 protein specifically, while S1-4 and S1-6 can not interact with ACE2. The results in these two experiments are different suggesting that denaturing of various truncated S1 proteins in SDS-PAGE in the first experiment disrupt the conformation of S1 proteins. But in the second experiments, all these proteins maintained their activated conformation. Our data indicated that the ZHANG et al. The Functional Motif of SARS-CoV S Protein Involved in the Interaction with ACE2 7 interaction of S1 protein and receptor ACE2 perhaps depend on the conformation of S1 protein. Our data showed that anti-S1-7 protein rabbit antiserum could block the interactions between ACE2 and truncated S1 proteins (S1-1, S1-2, S1-3, S1-5, S1-7). So the recombinant S1-7 protein could induce protective neutralizing antibody against SARS-CoV. Our study sheds light on the development of engineering protein vaccines for SARS. Inactivated SARS-CoV vaccine elicits high titers of spike protein specific antibodies that block receptor binding and virus entry Antigenicity and receptor-binding ability of recombinant SARS coronavirus spike protein Identification of two antigenic epitopes on SARS-CoV spike protein Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2 Receptor and viral determinants of SARS coronavirus adaptation to human ACE2 The Genome sequence of the SARS-associated coronavirus The severe acute respiratory syndrome A model Pf the ACE2 structure and function as a SARS-CoV-receptor Characteri-zation of a novel coronavirus associated with severe acute respiratory syndrome Expression cloning of functional receptor used by SARS coronavirus