Michael J. Moore

The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318–510) bound ACE2 more efficiently than did the full S1 domain (residues 12–672). Smaller S protein fragments, expressing residues 327–510 or 318–490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC50 of less than 10 nm, whereas the IC50 of the S1 domain was ∼50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein. The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318–510) bound ACE2 more efficiently than did the full S1 domain (residues 12–672). Smaller S protein fragments, expressing residues 327–510 or 318–490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC50 of less than 10 nm, whereas the IC50 of the S1 domain was ∼50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein. A distinct coronavirus (SARS-CoV) 1The abbreviations used are: SARSsevere acute respiratory syndromeCoVcoronavirusSspikeACE2angiotensin-converting enzyme 2CEACAMcarcinoembryonic antigen-related cell adhesion moleculeMHVmouse hepatitis virusHCoVhuman coronavirusVSVvesicular stomatitis virusSIVsimian immunodeficiency virusGFPgreen fluorescent proteinHIV-1human immunodeficiency virus, type 1PBSphosphate-buffered salineBSAbovine serum albumin. has been identified as the etiological agent of SARS, an acute pulmonary syndrome characterized by an atypical pneumonia that results in progressive respiratory failure and death in close to 10% of infected individuals (1Ksiazek T.G. Erdman D. Goldsmith C.S. Zaki S.R. Peret T. Emery S. Tong S. Urbani C. Comer J.A. Lim W. Rollin P.E. Dowell S.F. Ling A.E. Humphrey C.D. Shieh W.J. Guarner J. Paddock C.D. Rota P. Fields B. DeRisi J. Yang J.Y. Cox N. Hughes J.M. LeDuc J.W. Bellini W.J. Anderson L.J. N. Engl. J. 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SARS-CoV does not belong to any of the three previously defined genetic and serological coronavirus groups; the SARS-CoV S protein, a surface glycoprotein that mediates coronavirus entry into receptor-bearing cells, is also distinct from those of other coronaviruses (5Marra M.A. Jones S.J. Astell C.R. Holt R.A. Brooks-Wilson A. Butterfield Y.S. Khattra J. Asano J.K. Barber S.A. Chan S.Y. Cloutier A. Coughlin S.M. Freeman D. Girn N. Griffith O.L. Leach S.R. Mayo M. McDonald H. Montgomery S.B. Pandoh P.K. Petrescu A.S. Robertson A.G. Schein J.E. Siddiqui A. Smailus D.E. Stott J.M. Yang G.S. Plummer F. Andonov A. Artsob H. Bastien N. Bernard K. Booth T.F. Bowness D. Czub M. Drebot M. Fernando L. Flick R. Garbutt M. Gray M. Grolla A. Jones S. Feldmann H. Meyers A. Kabani A. Li Y. Normand S. Stroher U. Tipples G.A. Tyler S. Vogrig R. Ward D. Watson B. Brunham R.C. Krajden M. Petric M. Skowronski D.M. Upton C. Roper R.L. Science. 2003; 300: 1399-1404Google Scholar, 6Rota P.A. Oberste M.S. Monroe S.S. Nix W.A. Campagnoli R. Icenogle J.P. Penaranda S. Bankamp B. Maher K. Chen M.H. Tong S. Tamin A. Lowe L. Frace M. DeRisi J.L. Chen Q. Wang D. Erdman D.D. Peret T.C. Burns C. Ksiazek T.G. Rollin P.E. Sanchez A. Liffick S. Holloway B. Limor J. McCaustland K. Olsen-Rasmussen M. Fouchier R. Gunther S. Osterhaus A.D. Drosten C. Pallansch M.A. Anderson L.J. Bellini W.J. Science. 2003; 300: 1394-1399Google Scholar). Reflecting this difference, SARS-CoV does not utilize any previously identified coronavirus receptors to infect cells. Rather, as we have recently demonstrated, angiotensin-converting enzyme 2 (ACE2) serves as a functional receptor for this coronavirus (7Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzeriaga C. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Google Scholar). severe acute respiratory syndrome coronavirus spike angiotensin-converting enzyme 2 carcinoembryonic antigen-related cell adhesion molecule mouse hepatitis virus human coronavirus vesicular stomatitis virus simian immunodeficiency virus green fluorescent protein human immunodeficiency virus, type 1 phosphate-buffered saline bovine serum albumin. The S proteins of some coronaviruses, for example, that of mouse hepatitis virus (MHV), can be cleaved into two subunits (S1 and S2) (8Sturman L.S. Holmes K.V. Adv. Exp. Med. Biol. 1984; 173: 25-35Google Scholar, 9Jackwood M.W. Hilt D.A. Callison S.A. Lee C.W. Plaza H. Wade E. Avian Dis. 2001; 45: 366-372Google Scholar). The S proteins of other coronaviruses, such as those of human coronavirus 229E (HCoV-229E) and SARS-CoV, are not cleaved by the virus-producing cell (10Gallagher T.M. Buchmeier M.J. Virology. 2001; 279: 371-374Google Scholar). Nonetheless, S1 and S2 domains of these latter S proteins can be identified through their homology with the S1 and S2 subunits of cleaved coronavirus S proteins. The S1 domain of all characterized coronaviruses, including that of SARS-CoV, mediates an initial high affinity interaction with a cellular receptor (11Bonavia A. Zelus B.D. Wentworth D.E. Talbot P.J. Holmes K.V. J. Virol. 2003; 77: 2530-2538Google Scholar, 12Breslin J.J. Mork I. Smith M.K. Vogel L.K. Hemmila E.M. Bonavia A. Talbot P.J. Sjostrom H. Noren O. Holmes K.V. J. Virol. 2003; 77: 4435-4438Google Scholar, 13Kubo H. Yamada Y.K. Taguchi F. J. Virol. 1994; 68: 5403-5410Scopus (0) Google Scholar). Independently folded receptor-binding domains of two coronaviruses have been described. The first 330 amino acids of the 769-residue S1 subunit of the MHV S protein is sufficient to bind carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), the cellular receptor for MHV (13Kubo H. Yamada Y.K. Taguchi F. J. Virol. 1994; 68: 5403-5410Scopus (0) Google Scholar, 14Dveksler G.S. Pensiero M.N. Cardellichio C.B. Williams R.K. Jiang G.S. Holmes K.V. Dieffenbach C.W. J. Virol. 1991; 65: 6881-6891Google Scholar, 15Dveksler G.S. Dieffenbach C.W. Cardellichio C.B. McCuaig K. Pensiero M.N. Jiang G.S. Beauchemin N. Holmes K.V. J. Virol. 1993; 67: 1-8Google Scholar). A very different region of the S1 domain of HCoV-229E, between residues 407 and 547, is sufficient to associate with the cellular receptor for this coronavirus, aminopeptidase N (APN, CD13) (11Bonavia A. Zelus B.D. Wentworth D.E. Talbot P.J. Holmes K.V. J. Virol. 2003; 77: 2530-2538Google Scholar, 12Breslin J.J. Mork I. Smith M.K. Vogel L.K. Hemmila E.M. Bonavia A. Talbot P.J. Sjostrom H. Noren O. Holmes K.V. J. Virol. 2003; 77: 4435-4438Google Scholar, 16Yeager C.L. Ashmun R.A. Williams R.K. Cardellichio C.B. Shapiro L.H. Look A.T. Holmes K.V. Nature. 1992; 357: 420-422Google Scholar). Here we show that a 193-amino acid fragment of the SARS-CoV S protein, residues 318–510, binds the SARS-CoV receptor ACE2 and blocks S protein-mediated infection more efficiently than does the full-length S1 domain. This region includes seven cysteines, five of which are essential for expression or ACE2 association. Point mutations within this domain, at glutamic acid 452 or aspartic acid 454, interfere with or abolish association with ACE2. These data identify a domain of the SARS-CoV S protein that may be a critical target for neutralizing antibodies against the virus. Construction of S1-Ig, Truncation Variants, and Mutants—A plasmid encoding S1-Ig was generated by amplifying a region encoding residues 12–672 from an expression vector containing a codon-optimized form of the full-length S protein gene (7Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzeriaga C. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Google Scholar) and ligating this region into a previously described vector encoding the signal sequence of CD5 and the Fc domain of human IgG1 (17Farzan M. Mirzabekov T. Kolchinsky P. Wyatt R. Cayabyab M. Gerard N.P. Gerard C. Sodroski J. Choe H. Cell. 1999; 96: 667-676Google Scholar). Truncation variants were generated by inverse PCR amplification, using the S1-Ig plasmid as a template. Mutations within S1-Ig, or within a truncation mutant thereof expressing residues 318–510, were generated by site-directed mutagenesis using the QuikChange method (Stratagene). Two independent plasmids were generated for each variant, sequenced within their coding regions and assayed. Purification of S1 Protein Variants—293T cells were transfected with plasmids encoding S1-Ig or S1-Ig variants. One day post-transfection, cells were washed in PBS and subsequently incubated in 293 SFM II medium (Invitrogen). Medium was harvested after 48 h and proteins precipitated with protein A-Sepharose beads at 4 °C for 16 h. Beads were washed in PBS, 0.5 M NaCl, eluted with 50 mm sodium citrate/50 mm glycine (pH 2), and neutralized with NaOH. Purified proteins were concentrated with Centricon filters (Amicon) and dialyzed in PBS. Binding and Flow Cytometry—293T cells were transfected with a previously described plasmid encoding ACE2 (7Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzeriaga C. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Google Scholar) or with vector (pcDNA3.1, Invitrogen) alone. Three days post-transfection, cells were detached in PBS, 5 mm EDTA and washed with PBS, 0.5% BSA. S1-Ig or variants thereof were added to 106 cells to a final concentration of 250 nm, and the mixture was incubated on ice for 1 h. Cells were washed three times with PBS, 0.5% BSA, then incubated for 30 min on ice with anti-human IgG FITC conjugate (Sigma; 1:50 dilution). Cells were again washed with PBS, 0.5% BSA. Binding of IgG-tagged viral proteins to 293T cells transfected with ACE2-expressing plasmid was detected by flow cytometry. The mean value of the binding of S1-Ig or variants with the ACE2-transfected cells was subtracted from that of the mock-transfected cells and normalized to that of S1-Ig. Immunoprecipitation of Soluble ACE2—293T cells transfected with a previously described plasmid expressing soluble ACE2 (7Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzeriaga C. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Google Scholar) were metabolically labeled with [35S]cysteine and -methionine. Labeled medium was harvested 3 days post-transfection. 0.5 ml of soluble ACE2-containing medium was incubated for 15 min on ice with 25 pmol of purified S1-Ig or variants, to a final concentration of 50 nm. 20 μl of protein A-Sepharose was added to the mixture, which was then incubated for 1 h at room temperature. Protein A-Sepharose beads were washed three times with PBS, 0.1% Nonidet P-40 and once with PBS. Protein was analyzed by SDS-PAGE and quantified by phosphorimaging using ImageQuant software. Infection Assay with S Protein-Pseudotyped Virus—–293T cells were transfected with a plasmid encoding SARS-CoV S protein or VSV-G, together with a previously described plasmid encoding the genome of simian immunodeficiency virus (SIV), modified by deletion of the env gene and by replacement of the nef gene with that for green fluorescent protein (GFP) (18Bannert N. Schenten D. Craig S. Sodroski J. J. Virol. 2000; 74: 10984-10993Google Scholar). Supernatants of transfected cells were harvested, and viral reverse transcriptase activity was measured. Supernatants containing S protein- or VSV-G-pseudotyped SIV were added to ACE2- or mock-transfected 293T cells in the presence or absence of the indicated concentrations of S1-Ig or of the 12–327 or 318–510 variants thereof. Media were changed the following day and GFP expression in infected cells was measured 2 days later by flow cytometry. A protein in which the S1 domain of the SARS-CoV S protein was fused to the Fc region of human IgG1 has been shown to associate with ACE2-expressing cells and to precipitate ACE2 (7Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzeriaga C. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Google Scholar). To identify the receptor-binding domain of the S protein, this fusion protein, S1-Ig, was sequentially deleted at the N and C termini of the S1 domain to make a total of 12 additional variants. Each variant expressed efficiently and could be readily purified using protein A-Sepharose beads (Fig. 1A, top). S1-Ig and truncation variants thereof were used to precipitate a metabolically labeled and soluble form of ACE2. In contrast to an analogous MHV S protein truncation variant, which efficiently binds the MHV receptor CEACAM1 (13Kubo H. Yamada Y.K. Taguchi F. J. Virol. 1994; 68: 5403-5410Scopus (0) Google Scholar), the S1-Ig variant containing S1 domain residues 12–327 did not associate with ACE2. Neither did another expressing residues 12–481, whereas variants expressing residues 12–510 and 12–572 efficiently bound soluble ACE2 (Fig. 1A). These data indicate that residues 511–672 at the C terminus of the S1 domain do not contribute significantly to ACE2 association. Removal of residues 12–260 from the S1-Ig N terminus had no effect on ACE2 association (Fig. 1A). Variants expressing residues 298–510 and 318–510 efficiently bound S protein. The 318–510 variant precipitated ACE2 more efficiently than did the full S1 domain. However, two variants expressing slightly smaller fragments of the S1 domain (residues 318–490 and 327–510) did not detectably precipitate ACE2. These data imply that some residues from 318 to 326 and from 491 to 509 contribute either directly to the association of the S1 domain with ACE2 or to the correct folding of the receptor-binding domain. Fig. 1B compares the ability of each S1-Ig truncation variant to precipitate soluble ACE2 over several experiments (gray bars) with its ability to bind ACE2-expressing 293T cells, as measured by flow cytometry (black bars). A good correlation is observed between these two binding assays. We note that, under conditions used here, flow cytometry more sensitively detects low affinity associations with ACE2, whereas precipitation better reveals differences among efficiently binding variants. The truncation variants assayed in Fig. 1, A and B, are represented in Fig. 1C. We further examined the ability of the S1-Ig variant containing residues 318–510 to bind ACE2 with higher affinity than does full-length S1-Ig. A 50 nm concentration of S1-Ig was compared with varying concentrations of the 318–510 variant. As shown in Fig. 2A, the same concentration (50 nm) of 318–510 precipitated more than twice as much ACE2 as did S1-Ig. A 25 nm concentration of 318–510 precipitated the same amount of soluble ACE2 as did 50 nm S1-Ig. The results of two such experiments are summarized in Fig. 2B. These data imply that the 318–510 variant binds ACE2 at least twice as efficiently as does S1-Ig. We also investigated the ability of S1-Ig and the 318–510 variant to block S protein-mediated infection. To do so, we utilized a system we recently developed in which a lentivirus expressing green fluorescent protein and pseudotyped with the SARS-CoV S protein infects 293T cells stably expressing ACE2. 2M. J. Moore, T. Dorfman, W. Li, S. K. Wong, T. C. Greenough, M. Farzan, and H. Choe, manuscript in preparation. Incubation of 293T cells with the 12–327 variant had no effect on infection, consistent with the inability of this variant to bind ACE2 (Fig. 2C). In this assay, S1-Ig inhibited infection by S protein-pseudotyped lentivirus with an IC50 of ∼50 nm, whereas the 318–510 variant blocked infection by the same virus with an IC50 of less than 10 nm (Fig. 2C). The 318–510 variant did not substantially interfere with infection of lentivirus pseudotyped with the VSV-G protein, which mediates entry independently of ACE2 (Fig. 2C). Fig. 2D displays fluorescent microscopic fields of view in the presence of 250 nm of the 12–327 or 318–510 variants. Many fields lacked observable green cells in the presence of the 318–510 variant. We asked whether the difference in the abilities of the 318–510 and 327–510 variants to bind ACE2 was a consequence of the loss of cysteine 323 in the latter variant. Fig. 3A demonstrates that this is not the case. A series of point mutations was made in which each of the seven cysteines within 318–510 was altered to alanine. The variant in which cysteine 323 was altered bound ACE2 as efficiently as 318–510 itself. Alteration of cysteine 378 also had little effect on binding; however, a combination of mutations at residues 323 and 378 resulted in a construct with decreased ability to bind ACE2 (Fig. 3A, right panel). Alteration of cysteine 366 or substantially expression of the 318–510 variant. of cysteines and precipitation of ACE2 a effect on These data indicate that between 318 and 326 other than cysteine 323 contribute directly or to ACE2 association. we the ability of some residues between 318 and to contribute to ACE2 on a region among coronavirus S proteins. acid 452 and aspartic acids 454, and were altered to in the 318–510 variant and These 318–510 variants were assayed for their ability to bind ACE2 (Fig. panel). effect was observed with the The and 318–510 variants precipitated and of the ACE2 precipitated by the 318–510 variant. The full S1 domain, at precipitated ACE2 with an to that of the 318–510 variant the same mutation (Fig. right panel). The abolished ACE2 association in the 318–510 variant (Fig. and in the full-length S1 domain (Fig. right expression of either protein. These data that ACE2 with the SARS-CoV S domain in the of aspartic acid Fig. the 318–510 region within the SARS-CoV S protein, with the S proteins of and As is from this each of the receptor-binding domains of these S proteins is in a different region of the S1 domain, consistent with the that each of these coronaviruses to a distinct serological and genetic The described the S protein receptor-binding domain. A series of truncation variants of the S1 domain, fused to the Fc region of human were assayed for their ability to associate with ACE2 on the surface of transfected cells and to soluble ACE2. The fragment that ACE2 association was of residues 318–510 and bound ACE2 more efficiently than did the full-length S1 domain, whereas slightly smaller fragments did The higher affinity of the 193-residue fragment the that other regions of the S protein this receptor-binding domain. the receptor-binding domain described may be more soluble or better folded than the S1 protein, which includes regions that may the S2 domain or other S proteins in the The 193-amino acid receptor-binding region also more efficiently blocked S protein-mediated infection of ACE2-expressing cells than did the full S1 domain, to its affinity for ACE2. of this fragment may into of that block SARS-CoV infection. We also investigated the of cysteines and some residues within the 193-residue We that of the seven cysteines to expression or to ACE2 association and were to identify or cysteines within this variant. in this We however, identify two glutamic acid 452 and aspartic acid 454, that to make an to S1 protein interaction with ACE2. to of these residues be the that variants containing these mutations expressed as efficiently as those and that these mutations had on the 318–510 variant and the full-length S1 domain, that or of these residues contribute directly to ACE2 association. this have of SARS-CoV, whether as a to human several that the of a against this virus be less for example, the of an SARS-CoV is more than an can this that, in contrast to the the S protein may do little to its receptor-binding domain. with this of the antibodies that for S protein association with ACE2 have been again in contrast to and either to the of the or to the of little has been observed in S protein from these that a subunit that includes the S protein receptor-binding domain described may be in the of virus

The ability of engineered biological nanomachines to communicate with biological systems at the molecular level is anticipated to enable future applications such as monitoring the condition of a human body, regenerating biological tissues and organs, and interfacing artificial devices with neural systems. From the viewpoint of communication theory and engineering, molecular communication is proposed as a new paradigm for engineered biological nanomachines to communicate with the natural biological nanomachines which form a biological system. Distinct from the current telecommunication paradigm, molecular communication uses molecules as the carriers of information; sender biological nanomachines encode information on molecules and release the molecules in the environment, the molecules then propagate in the environment to receiver biological nanomachines, and the receiver biological nanomachines biochemically react with the molecules to decode information. Current molecular communication research is limited to small-scale networks of several biological nanomachines. Key challenges to bridge the gap between current research and practical applications include developing robust and scalable techniques to create a functional network from a large number of biological nanomachines. Developing networking mechanisms and communication protocols is anticipated to introduce new avenues into integrating engineered and natural biological nanomachines into a single networked system. In this paper, we present the state-of-the-art in the area of molecular communication by discussing its architecture, features, applications, design, engineering, and physical modeling. We then discuss challenges and opportunities in developing networking mechanisms and communication protocols to create a network from a large number of bio-nanomachines for future applications.

Effective prophylaxis and antiviral therapies are urgently needed in the event of reemergence of the highly contagious and often fatal severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) infection. We have identified eight recombinant human single-chain variable region fragments (scFvs) against the S1 domain of spike (S) protein of the SARS-CoV from two nonimmune human antibody libraries. One scFv 80R efficiently neutralized SARS-CoV and inhibited syncytia formation between cells expressing the S protein and those expressing the SARS-CoV receptor angiotensin-converting enzyme 2 (ACE2). Mapping of the 80R epitope showed it is located within the N-terminal 261-672 amino acids of S protein and is not glycosylation-dependent. 80R scFv competed with soluble ACE2 for association with the S1 domain and bound S1 with high affinity (equilibrium dissociation constant, Kd=32.3 nM). A human IgG1 form of 80R bound S1 with a 20-fold higher affinity of 1.59 nM comparable to that of ACE2 (Kd=1.70 nM), and neutralized virus 20-fold more efficiently than the 80R scFv. These data suggest that the 80R human monoclonal antibody may be a useful viral entry inhibitor for the emergency prophylaxis and treatment of SARS, and that the ACE2-binding site of S1 could be an attractive target for subunit vaccine and drug development.