Here, we present that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367C606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. of MERS-CoV infection. Introduction A novel human coronavirus, Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), was Rutaecarpine (Rutecarpine) identified as a pathogen causing a severe acute respiratory syndrome (SARS)-like disease in the Middle East and Europe in 2012 [1]. As of October 14, 2013, the World Health Organization (WHO) had been informed of 138 confirmed cases of MERS-CoV infection, including 60 deaths (a case fatality rate of 45%) (http://www.who.int/csr/don/2013_10_14/en/). Recent reports of family clusters and health care-associated transmission of Rutaecarpine (Rutecarpine) MERS-CoV through close contact have proven its capacity for human-to-human transmission [2]C[5]. Although its transmissibility is significantly lower than that of SARS coronavirus (SARS-CoV) [6]C[9], it may gain increased human-to-human transmissibility during its further evolution and potentially cause a pandemic in the future [10]. Accordingly, development of effective therapeutics and vaccines is critical for early intervention and prevention. Unlike SARS-CoV, which uses human angiotensin-converting enzyme 2 (ACE2) as its receptor for binding to ACE2-expressing cells [11], MERS-CoV utilizes a different receptor, dipeptidyl peptidase 4 (DPP4), for binding to DPP4-expressing cells [12]. Like the spike (S) protein of SARS-CoV, the S protein of MERS-CoV also plays important roles Rabbit Polyclonal to LIPB1 in virus entry and infection [13]. MERS-CoV S protein contains a S1 subunit that mediates virus binding to cells expressing DPP4 through its receptor-binding domain (RBD) region and an S2 subunit that mediates virus-cell Rutaecarpine (Rutecarpine) membrane fusion [12], [13]. Based on sequence alignment and homology modeling analysis and functional studies, we and Mou et al. have predicted that the RBD is located in residues 377-662 or 358-588 of the MERS-CoV S1 subunit [14]C[16] (Fig. 1A). Co-crystallographic analyses of the RBD/DPP4 complexes have confirmed that the RBD is attributed to residues 367-606 or 367-588 in MERS-CoV S1 [17]C[19] (Fig. 1A). Open in a separate window Figure 1 Construction and characterization of MERS-CoV S377-588-Fc.(A) Schematic structure of MERS-CoV S1 subunit and S377-588-Fc. RBM: the receptor-binding motif in the RBD. S377-588-Fc was constructed by fusing MERS-CoV residues 377-588 of S1 with Fc of human IgG. (B) SDS-PAGE and Western blot (WB) analysis of purified 377-588-Fc protein. Samples were either boiled for 10 min, or not boiled, followed by SDS-PAGE (left) and WB (right) analysis using a S1-specific polyclonal antibody. (C) Analysis of S377-588-Fc protein conformation by cross-linker. Samples were cross-linked with glutaraldehyde (with cross-linker at the final concentration of 4 M) or without cross-linker (w/o cross-linker), followed by SDS-PAGE (left) and WB (right) analysis as described above. The protein molecular weight marker (kDa) (Invitrogen) is indicated on the left. Previous studies have shown that the RBD of SARS-CoV S protein can significantly inhibit SARS-CoV infection [20] and is able to induce highly potent neutralizing antibodies protecting against SARS-CoV infection [20]. It is thus expected that the RBD of MERS-CoV, which belongs to the same betacoronavirus genus as SARS-CoV [21], [22], may also be effective in inhibiting MERS-CoV infection and inducing neutralizing antibody responses against infection of MERS-CoV in vaccinated animals. Indeed, our identified RBD (a 286-amino acid fragment spanning residues 377-662) could bind to DPP4 and induce neutralizing antibody response in immunized mice [15], while the RBD reported by Mou et al. (a 231-amino acid fragment spanning residues 358-588) could inhibit MERS-CoV infection at the 40 g/ml level and elicit effective neutralizing antibodies in vaccinated rabbits [16]. These results suggest that the overlapping region (residues 377-588) must contain the receptor-binding motif (RBM) and the major neutralizing epitope of the RBD. Crystallographic.