A novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), has caused outbreaks of a SARS-like illness with high case fatality rate. The reports of its personto-person transmission through close contacts have raised a global concern about its pandemic potential. Here we characterize the six-helix bundle fusion core structure of MERS-CoV spike protein S2 subunit by X-ray crystallography and biophysical analysis. We find that two peptides, HR1P and HR2P, spanning residues 998-1039 in HR1 and 1251-1286 in HR2 domains, respectively, can form a stable six-helix bundle fusion core structure, suggesting that MERS-CoV enters into the host cell mainly through membrane fusion mechanism. HR2P can effectively inhibit MERS-CoV replication and its spike protein-mediated cell-cell fusion. Introduction of hydrophilic residues into HR2P results in significant improvement of its stability, solubility and antiviral activity. Therefore, the HR2P analogues have good potential to be further developed into effective viral fusion inhibitors for treating MERS-CoV infection.
Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.A s of June 16, 2014, the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) had infected 701 people, with a fatality rate of ∼36% (www.who.int/csr/don/ 2014_06_16_mers/en/), and had demonstrated the capability for human-to-human transmission (1, 2). Alarmingly, coronavirus surveillance studies have suggested that MERS-CoV originated from animals, with bats as the likely natural reservoir and camels as the likely intermediate hosts (3-6). Hence, cross-species transmission of MERS-CoV from bats to humans, either directly or through camels, poses a constant and long-term threat to human health. Phylogenetic analysis has revealed that MERS-CoV is genetically related to two bat coronaviruses, HKU4 and HKU5 (7-9). Understanding the pathogenesis and potential cross-species transmissibility of these bat coronaviruses is critical for evaluating long-term emerging disease potentials and for preventing and controlling the spread of bat-originated coronaviruses in humans. This study investigates the receptor usage and cell entry mechanisms of HKU4 and HKU5, providing insight into how MERSCoV and MERS-related bat coronaviruses can cross species barriers, adapt to human cells, and gain infectivity in humans.Receptor recognition has been established as an important determinant of the host range and tropism of coronaviruses (10, 11). An envelope-anchored spike protein mediates coronavirus entry into host cells by first binding to a host receptor through its S1 subunit and then fusing the host and viral membranes via its S2 subunit. Coronaviruses recognize a wide range of receptors, including proteins and si...
An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367–606) 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. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients’ lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.
The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is currently spreading among humans, making development of effective MERS vaccines a high priority. A defined receptor-binding domain (RBD) in MERS-CoV spike protein can potentially serve as a subunit vaccine candidate against MERS-CoV infections. To identify an ideal vaccine candidate, we have constructed five different versions of RBD fragments, S350-588-Fc, S358-588-Fc, S367-588-Fc, S367-606-Fc, and S377-588-Fc (their names indicate their residue range in the spike protein and their C-terminal Fc tag), and further investigated their receptor binding affinity, antigenicity, immunogenicity, and neutralizing potential. The results showed that S377-588-Fc is among the RBD fragments that demonstrated the highest DPP4-binding affinity and induced the highest-titer IgG antibodies in mice. In addition, S377-588-Fc elicited higher-titer neutralizing antibodies than all the other RBD fragments in mice, and also induced high-titer neutralizing antibodies in immunized rabbits. Structural analysis suggests that S377-588-Fc contains the stably folded RBD structure, the full receptor-binding site, and major neutralizing epitopes, such that additional structures to this fragment introduce non-neutralizing epitopes and may also alter the tertiary structure of the RBD. Taken together, our data suggest that the RBD fragment encompassing spike residues 377-588 is a critical neutralizing receptor-binding fragment and an ideal candidate for development of effective MERS vaccines, and that adding non-neutralizing structures to this RBD fragment diminishes its neutralizing potential. Therefore, in viral vaccine design, it is important to identify the most stable and neutralizing viral RBD fragment, while eliminating unnecessary and non-neutralizing structures, as a means of “immunofocusing”.
A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine. In 2003, Farzan and colleagues successfully identified the receptor of SARS-CoV, angiotensin-converting enzyme 2 (ACE2) (7), and a 193-amino-acid fragment in the spike (S) protein (residues 318 to 510) as the receptor-binding domain (RBD) (8). We found that SARS-CoV S-RBD contains a critical neutralizing site (9) which induces potent neutralizing antibodies and protection against SARS-CoV infection in an animal model (10).Since MERS-CoV is genetically related to SARS-CoV (1), we compared their S protein sequences and predicted that the RBD of MERS-CoV might be located in the region spanning residues 377 to 662 of the S1 subunit (Fig. 1). Using the Swiss-Model Workplace homology modeling server (11) and basing our work on the X-ray crystal structure of the SARS-CoV S-RBD (Protein Data Bank [PDB] identification no. 2DD8) (12), we predicted the conformational structure of the region consisting of residues 377 to 662 in the S1 subunit of the MERS-CoV S protein (13). We noticed that the SARS-CoV S-RBD and the predicted MERS-CoV S-RBD possessed similar core structures but had an extended secondary structure consisting predominantly of the receptor-binding motifs (RBM) (12,14). The extended region in MERS-CoV S-RBD is much longer than that in SARS-CoV S-RBD, suggesting that MERS-CoV and SARS-CoV use different receptors. Indeed, it has been proven that dipeptidyl peptidase-4 (DPP4; also known as CD26) is the functional receptor of MERS-CoV (15).We then constructed MERS-CoV S-RBD based on the synthesized codon-optimized MERS-CoV S sequences (GenBank accession no. AFS88936.1) and fused it to Fc of human IgG using pFUSE-hIgG1-Fc2 expression vector (here named Fc) (InvivoGen, San Diego, CA). The SARS-CoV S-RBD-Fc was constructed by fusing RBD of codon-optimized SARS-CoV S sequence into the Fc vector referred to above as a control (Fig. 1) (16). The S-RBD-Fc proteins were expressed in 293T cell culture supernatant and purified by protein A affinity chromatography (GE Healthcare, Piscataway, NJ) (17). We found that both MERS-CoV and SARS-CoV S-RBD-Fc proteins were highly purified from transfected culture supernatants ( Fig. 2A, panel a). MERS-CoV S-RBD-Fc could be recognized by an MERS-CoV S-specific polyclonal antibody (1:1,000), while SARS-CoV S-RBD-Fc could not react with this antibody, as detected by Western blotting (Fig. 2A, panel b).Using analysis performed by Western blotting, we found that DPP4 was highly express...
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