Journal of the Formosan Medical Association (2014) 113, 143e147

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REVIEW ARTICLE

The receptor binding domain of MERS-CoV:The dawn of vaccine and treatmentdevelopment

Nan Zhou a, Yun Zhang a, Jin-Chun Zhang, Ling Feng, Jin-Ku Bao*

School of Life Sciences & Key Laboratory of Bio-resources, Ministry of Education, Sichuan University,Chengdu 610064, China

Received 15 September 2013; received in revised form 20 October 2013; accepted 13 November 2013

KEYWORDScoronavirus;drug design;Middle East;vaccines

Conflicts of interest: The authorsrelevant to this article.* Corresponding author. No. 29, Wa

Sciences & Key Laboratory of Bio-resoSichuan University, Chengdu 610064,

E-mail address: baojinku@scu.edua These authors contributed equally

0929-6646/$ - see front matter Copyrhttp://dx.doi.org/10.1016/j.jfma.201

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is becominganother “SARS-like” threat to the world. It has an extremely high death rate (w50%) as thereis no vaccine or efficient therapeutics. The identification of the structures of both the MERS-CoV receptor binding domain (RBD) and its complex with dipeptidyl peptidase 4 (DPP4), raisesthe hope of alleviating this currently severe situation. In this review, we examined the molec-ular basis of the RBD-receptor interaction to outline why/how could we use MERS-CoV RBD todevelop vaccines and antiviral drugs.Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

Introduction

On 20 September 2012, a novel coronavirus isolated from a60-year-old Saudi man with acute pneumonia and acuterenal failure was reported.1 In May 2013, the WHO adoptedthe virus name Middle East respiratory syndrome (MERS)

have no conflicts of interest

ngjiang Road, School of Lifeurces, Ministry of Education,China..cn (J.-K. Bao).to this work.

ight ª 2013, Elsevier Taiwan LLC3.11.006

coronavirus (MERS-CoV), which was defined by the Coro-navirus Study Group of the International Committee onTaxonomy of Viruses.2 As of 7 September 2013, the WHO hasbeen notified of 114 laboratory-confirmed cases in theMiddle East [Jordan, Saudi Arabia (KSA), the United ArabEmirates (UAE), and Qatar], Europe [France, Germany,United Kingdom (UK) and Italy], and North Africa (Tunisia),with 54 deaths.3

The world is facing a new challenge posed by a severeacute respiratory syndrome (SARS)-like infections inhumans caused by MERS-CoV. Its main clinical manifesta-tions in patients are pneumonia (or respiratory) failure andacute renal failure.4 The short-lived but alarming epidemicSARS-CoV killed nearly 10% of approximately 8000 cases inthe 2002e2003 outbreak.5 The data so far indicate that

& Formosan Medical Association. All rights reserved.

144 N. Zhou et al.

MERS-CoV possesses an unusually high crude mortality rateof approximately 50%, implying a big threat to people whoare infected. Clusters of cases including a UK family, andhospitals in KSA, France and UAE show epidemiologicalevidence of human-to-human transmission. Indeed, existingreports indicate person-to-person transmission occurs,raising significant concern on the possible emergence of aglobal epidemic in the near future.6e8 It is therefore ofutmost importance to pay worldwide attention to findantiviral drugs and effective vaccines to control its highdeath rate and spread.

Coronaviruses are a large family of enveloped, single-stranded RNA viruses that infect a number of different hostspecies, including humans. In people, coronaviruses cancause illnesses ranging in severity from the common cold toSARS. Coronaviruses can be categorized into four genera,Alphacoronavirus, Betacoronavirus, Gammacoronavirus,and Deltacoronavirus.9,10 The genus Betacoronavirus hasfour lineages: A, B, C, and D.11 MERS-CoV belongs to lineageC and is the first lineage C Betacoronavirus known to infecthumans.12

Coronaviruses infect animals and humans. The spike (S)entry protein binds to a cell surface receptor which pri-marily determines their tropism. Similar to other corona-viruses, MERS-CoV utilizes its large surface S protein tointeract with and enter the target cell.13,14 Raj et al13 haveidentified that dipeptidyl peptidase 4 (DPP4; also known asCD26) is its functional receptor. MERS-CoV binds to DPP4 viathe S protein and releases the RNA genome into the targetcell.2 The MERS-CoV S protein is a type-I membrane glyco-protein and contains S1 and S2 subunits.15 The S1 domaindetermines cellular tropism and interacts with the targetcell, while the S2 domain mediates membrane fusion.13,16

According to recent studies, the MERS-CoV receptor bind-ing domain (RBD) is mapped to the S1 region.17,18 MERS-CoVRBD binds to the receptor and induces significant neutral-izing antibody responses, indicating that it can be used as acandidate target for the development of vaccines andantiviral drugs.19 Herein, we review recent studies onMERS-CoV to make suggestions on antiviral vaccine and drugdevelopment.

Structure of MERS-CoV RBD

Lu et al15 and other research teams have identified thecrystal structure of MERS-CoV RBD after the discovery of itsreceptor.17,20 Structural topology considers a sequence ofsecondary structure elements making protein structureeasy to interpret by laying out the 3D structural informationin two dimensions in a manner that makes the structureclear. Therefore we used Pro-origami to automaticallygenerate the schematic representation of the MERS-CoVRBD topology.21 The MERS-CoV RBD has a core and anexternal subdomain (Fig. 1A and B). The core subdomain isa five-stranded antiparallel b sheet (b1, b3, b4, b5, andb10) decorated by the connecting helices (a1e4 and h1, 2)and two small b-strands (b2 and b11) (Fig. 1B). Three di-sulfide bonds stabilize the fold by connecting C383 to C407,C425 to C478, and C437 to C585 (Fig. 1B). The externalreceptor binding subdomain reveals a four-stranded anti-parallel b sheet with three large strands (b6, b8, and b9)

and one small strand (b7) between strands b5 and b10 ofthe core domain (Fig. 1B). The b5/6, b7/8 and b9/10intervening loops touch the core subdomain and anchor theexternal to the core (Fig. 1B). There is a long loop con-taining h3 crosses perpendicular to the b sheet connectingb7 and b8 strands, and a disulfide bond between C503 andC526 links the h3 helix to strand b6 (Fig. 1B).

Mechanism of MERS-CoV binding to DPP4

Multifunctional DPP4 plays a major role in glucose meta-bolism, T-cell activation, chemotaxis modulation, celladhesion, and apoptosis.22,23 In humans, it is primarilyexpressed on the epithelial cells in the lungs, liver, smallintestine, kidney, and prostate, and on activated leuko-cytes, while it also occurs in a soluble form in the circula-tion.23,24 The structure of DPP4, as shown in previousstudies, is composed of an N-terminal eight-bladed b-pro-peller domain (S39 to D496) and a C-terminal a/b-hydrolasedomain (N497 to P766).25,26 The b-propeller domain con-tains eight blades, each made up of four antiparallel b-strands. The receptor-binding subdomain of MERS-CoV RBDbinds to the DPP4 b-propeller, contacting blades four andfive and a small bulged helix in the blade-linker.15,20

Structural analysis and mutational analysis by Lu et al15

and Wang et al20 have identified Y499, L506, W533, andE513 in the RBD to be critical for receptor binding and viralentry, and mutations of these significantly abrogate itsinteraction with DPP4.

MERS-CoV RBD-based vaccine design

One reason for the exceptionally high crude mortality rateof nearly 50% in MERS-CoV infection is the lack of vaccines.Therefore, it is necessary to develop efficient and safevaccines to control MERS quickly. MERS-CoV infects a widevariety of host species whereas coronaviruses generallytend to have a narrow host tropism.1 DPP4 sequencealignment demonstrates that its orthologs are highlyconserved for MERS-CoV acquiring cross-species trans-missibility by binding to an evolutionarily conserved re-ceptor.27 Minor mutations within the RBD domain candisturb the lock-and-key interaction of the RBD-receptorbinding interface and then places a barrier for cross-species transmission.

The roles of MERS-CoV RBD in receptor binding indicatethat vaccines based on it could induce antibodies to blockvirus binding or infection. Among structural proteins ofMERS-CoV, S protein is known to be the main antigeniccomponent to induce significant neutralizing antibody re-sponses up to now.19 Du et al19 found that MERS-CoV RBDbinds to the receptor and induces significant neutralizingantibody responses. Mou et al18 revealed that MERS-CoVRBD can efficiently elicit neutralizing antibodies. Besides,previous studies have shown that the related RBD of SARS-CoV strongly reacts with antisera from patients with SARSand the depletion of the RBD-specific antibodies results insignificant elimination of the neutralizing activity.28 Luet al2 also proposed that SARS vaccines based on SARS-CoVRBD are more effective and safer than vaccine candidatesbased on the inactivated virus, DNA or viral vectors. In

Figure 1 Structure of MERS-CoV RBD (PDB: 4KQZ). (A) A cartoon illustration of the structure of MERS-CoV RBD. The core sub-domain is shown in yellow and the external subdomain is shown in green. (B) A schematic representation of MERS-CoV RBD topology.The N and C termini are labeled. Arrows denote b strands and cylinders denote a helices. Purple sticks represent the disulfidebonds. Connected residues are also labeled.

The receptor binding domain is a target 145

addition, Zhu et al29 even proposed to design an RBD-basedvaccine against MERS-CoV following an experimental paththe same as that of the RBD-based SARS vaccine. Therefore,we strongly believe that MERS-CoV RBD is an importantcandidate target for developing MERS vaccines.

MERS-CoV RBD-based drug design

Up to now, no effective antiviral drugs against MERS-CoVhave been discovered, which is another factor contrib-uting to the high death rate. Fortunately, the structure ofMERS-CoV RBD and the mechanism of MERS-CoV binding toits cell surface receptor have been revealed. They arevery important information for computer-aided drugdesign, which is a promising approach to finding noveldrugs for MERS. With that, virtual screening (VS) and

structure-based drug design (SBDD) can be implementedto find candidate drugs from molecular databases such asZINC, PubChem, DrugBank, and so on.30e32 The structuralcomparison between MERS-CoV and SARS-CoV RBDs showsthat although their core subdomains are homologous andsimilar in structure, their receptor binding subdomainsare clearly different. Thus, the core subdomain is aneffective candidate target not only for anti-MERS-CoVtherapeutics, but also for anti-Betacoronavirus treat-ments.15

Generally, the identification of pockets and cavities ofthe protein structure is essential for VS and SBDD, whilethe binding pocket of MERS-CoV RBD remains unknown. Inpractice, the current situation is not so bad. Lu et al15

found that although the engagement of the receptordoes not induce significant conformational changes inreceptor binding motifs, the h2-a4 loop in the RBD core

Figure 2 Ligands binding pocket of MERS-CoV RBD (PDB: 4KQZ). (A) A structural alignment between the free and the receptor-bounded MERS-CoV RBD. Significant structural variance is observed for the h2-a4 loop in the core subdomain, which is marked witha black arrow. Yellow: free RBD, the core subdomain; green: free RBD, the external subdomain; blue: the bounded RBD. (B) Surfacerepresentation of MERS-CoV RBD. The orange spheres represent the h2-a4 loop. The candidate binding pocket predicted byPocketPicker is represented by darker spheres.

146 N. Zhou et al.

exhibits a surprisingly large conformational differencebetween the free and the bounded conditions (Fig. 2A).That is to say the RBDeDPP4 interaction is largelydependent on the conformational variant of the loop.Therefore, utilizing ligands binding to that region, we caninterfere with the essential conformational change,consequently preventing MERS-CoV infection. With thehelp of PocketPicker, a ligand pocket detection tool, wefound computationally that the correct binding pocket ofMERS-CoV RBD with a buried and a solvent exposed part isclose to the loop (Fig. 2B).33 Therefore, the h2-a4 loopand its peripheral region are important for VS and SBDD tofind novel efficient antiviral drugs.

Summary

Generally speaking, MERS-CoV, the first lineage C Betacor-onavirus known to infect human beings, has attractedworldwide attention as it causes SARS-like infections inhumans. To date, neither vaccines nor virus-specific drugsare available, making MERS-CoV a threat to global publichealth. Although the limited data cannot confirm human-to-human transmission, two new cases that have familycontacts with confirmed patients show an increasing prob-ability.3,34 MERS-CoV utilizes DPP4 as its cell receptor toenter the target cell. The RBD is located in the S1 domain ofMERS-CoV S protein and its crystal structure has already

The receptor binding domain is a target 147

been determined. The structure exhibits the mechanism ofMERS-CoV binding to DPP4. The identified MERS-CoV RBDmay also facilitate the development of vaccines and effi-cient treatment and ultimately lower the high crude mor-tality rate and prevent global spread.

Acknowledgments

We are grateful to our colleagues for their critical reviews andexcellent suggestions regarding thismanuscript. Thisworkwassupported in part by grants from the National Natural ScienceFoundation of China (No. 81373311, No. 31300674, No.81173093, No. 30970643, and No. J1103518) and Special Pro-gram for Youth Science and Technology Innovative ResearchGroup of Sichuan Province, China (No. 2011JTD0026).

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