Development of novel therapeutic strategies against the Middle East respiratory syndrome coronavirus

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus and has caused 2,090 laboratory-confirmed cases with 730 deaths (35% fatality) as of 13th October, 2017. Despite its high pathogenicity, no effective vaccines or antiviral regimens tested by randomized control clinical trials was currently unavailable. Therefore, in this study, we focused on the development of novel therapeutic strategies to combat against MERS-CoV infection. First, we performed phenotype-based screenings from a chemical library to seek for novel antiviral compounds that could directly inhibit virus replication. We identified 1-D10 as a promising lead compound that potently inhibited virus replication in a sub-micromolar range in vitro. We also assessed its in vivo antiviral efficacy and showed that the prophylactic treatment of 1-D10 significantly reduced the viral load in the lungs and brains of the hDPP4 transgenic mice. To identify the exact viral protein targeted by 1-D10, we raised escape mutants resistant to 1-D10 treatment and found anonymous mutations in the nsp3, nsp6 and orf4b genes. Taken together, we demonstrated 1-D10 as a potent antiviral compound that directly inhibited the replication of MERS-CoV. On the other hand, we also tried to improve the treatment outcome by alleviating pathological changes imposed by MERS-CoV infection. As previously reported, evidence of apoptosis was found in MERS patients and infected animals. Here, we further investigated how the apoptosis pathways were modulated by the virus. We demonstrated that MERS-CoV infection induced apoptosis through activating the extrinsic and the intrinsic apoptosis pathways. Interestingly, in an attempt to rescue cell death induced by virus infection, we found only the caspase-9, but not the caspase-8 inhibitor, significantly improved the viability of infected cells, implicating the initiative role of the intrinsic apoptosis pathway during virus infection. To reveal the underlying mechanisms of MERS-CoV-induced apoptosis, we screened MERS-CoV structural proteins on their capacity of inducing apoptosis and identified the membrane protein as a novel proapoptotic viral protein that significantly activated caspase-3. Moreover, the membrane protein colocalized with Grp78, which was the gatekeeper of the unfolded protein response (UPR). We further demonstrated that transient overexpression of the membrane protein led to the activation of both the IRE1α and the PERK ER stress pathways. Importantly, the finding that MERS-CoV-induced caspase activation could be inhibited with the PERK pathway inhibitor provided another solid evidence to support the involvement of the PERK pathway in MERS-CoV-induced apoptosis. Lastly, by using caspase inhibitors and stimulators, we elucidated that virus-induced apoptosis facilitated MERS-CoV propagation. Collectively, our data indicated that the apoptotic cell death induced by MERS-CoV infection not only contributed to its pathogenesis but also benefited virus propagation. Overall, we identified 1-D10 as a novel lead compound, which potently inhibited the MERS-CoV replication. In parallel, we described the therapeutic potential of inhibiting MERS-CoV-induced apoptosis to mitigate pathological changes as well as virus propagation. Taken together, our findings advance the knowledge of MERS-CoV pathogenesis and laid the foundations for future study on developing therapeutic strategies against MERS-CoV infection.