Structural And Functional Insights Into MERS-CoV And SARS-CoV Spike Glycoproteins And Their Interaction With Receptors

The Middle East respiratory syndrome coronavirus(MERS-CoV)was first isolated from the sputum of a patient in Saudi Arabia in 2012,and quickly spread among the world.It has caused a total of 1917 laboratory confirmed infections and 684 related deaths in 27 countries until March 2017.It is the second highly pathogenic coronavirus after SARS-CoV emerging from 2002.Coronaviruses are enveloped,positive-sense,single-strand RNA viruses.Based on genotypic and serological characterisitics,coronaviruses can be divided into four genera:?,?,? and 8.Thus far,six coronaviruse spicies are reported capable of infecting humans:hCoV-NL63 and hCoV-229E,which belong to ? genus,while hCoV-OC43,hCoV-HKU1,SARS-CoV,MERS-CoV belong to ? genus.Both MERS-CoV and SARS-CoV can cause severe pulmonary diseases in humans,posing great threat to public health and economy.The spike glycoprotein(S)is an important structural protein of all coronaviruses.It is a homotrimer embedded in the virus envelope,which is involved in the proccess of receptor binding and membrane fusion.Therefore,it is a key determinant of host specificity and the major target of neutralizing antibodies.The S protein can be cleaved by host proteases into an N-terminal S1 subunit and C-terminal S2 subunit.The S1 subunit contains the receptor binding domain(RBD),while the S2 subunit is responsible for fusion of viral envelope and host membrane.The S1 subunit has two separate domains:N-terminal domain(NTD)and C-terminal domain(CTD).The RBD of MERS-CoV is located in the CTD and recognizes DPP4(dipeptidyl peptidase 4,or the so-called CD26)as the receptor.In order to uncover the mechanisms of coronavirus entry and host tropism,we should fully understand the structure of S protein and its interactions with receptors.Recently,the spike protein of mouse hepatitis coronavirus(MHV)was solved by cryo-EM single particle reconstruction,which is the first S protein structure within the family of Coronaviridae.However,little is known about the S protein structure of MERS-CoV and SARS-CoV,two highly pathogenic coronaviruses that can cause severe human infections.Thus,we set up the structural studies on the S proteins of these two viruses.Besides,the MERS-CoV is believed to be a zoonotic pathogen originated from animals,but its epidemiology and transmitting mechanism from natural reservoir to human beings are poorly understood.By comparing the CD26 molecules from different animal species,we can find the potential intermediate hosts and explain the molecular basis of cross-species transmission of MERS-CoV.So we conducted the following experiments.First of all,we present the near atom resolution structures of the trimeric MERS-CoV and SARS-CoV spike proteins in their pre-fusion conformation determined by single particle cryo-EM reconstruction.The overall structures of MERS-CoV and SARS-CoV S trimers are similar to other CoV S proteins reported during this study,but their receptor binding domains adopted different conformations.We captured two states of RBD:one is buried(lying state)and the other is exposed(standing state).All of the reported S protein RBDs share a same lying state,but the MERS-CoV and SARS-CoV RBDs showed both the lying and standing states.The standing RBDs can readily be recognized by the receptor while the lying ones are inaccessible with buried receptor binding sites.In addition,some coronaviruses,such as bovine coronavirus(BCoV)and mouse hepatitis virus(MHV),utilize NTDs to bind glycans or other receptors,thus facilitating the attachment and invasion into host cells.So far,little is known about the structural and functional information of MERS-NTD and SARS-NTD.Therefore,we also solved the crystal structures of MERS-NTD and SARS-NTD.Our results showed that the glycan-binding site on the top of NTD is occupied by a short helix and an N-linked glycan,which might prevent the attachment of viruses to recognize the cell surface sugar molecules.Further analysis on six human-infecting coronaviruses revealed that the fusion peptide,the hepatad repeat 1(HR1)region and the central helix are the most conserved in the entire S protein structure,indicating the potential to be targeted for broadly-neutralizing antibodies and inhibitors.Many viruses,including MERS-CoV and SARS-CoV,are believed to transmit from the natural host,probably bats,to other species,like human beings.In order to study the potential infection of MERS-CoV in bats,we expressed CD26s of seven bat species and tested their binding affinities to MERS-RBD by SPR(Surface Plasmon Resonance)assay.Among the seven CD26s,the CD26 of Myotis davidii showed the highest binding affinity with MERS-RBD.We further performed FACS(Fluorescence-activated cell sorting)and pseudovirus infection assays and confirmed that MERS-CoV can enter cells using CD26s from certain bat species as receptors.We also crystallized MERS-RBD in complex with the CD26 of Myotis davidii and determined the structure.Based on the complex structure,the CD26 of Myotis davidii binds to MERS-RBD in a similar mode as that of hCD26.The T295 and K336 on batCD26 contribute pivotal interactions with MERS-RBD.Our findings indicate that MERS-CoV can potentially infect many kinds of bats,therefore special care should be taken to different bat species in order to prevent the transmission of MERS-CoV.Besides.the host spectrum of MERS-CoV is not clear yet,so CD26s of different species were studied to explore the potential hosts of MERS-CoV.By SPR and pseudo virus infection assay,we found that MERS-RBD can bind to CD26s of humans,monkeys,dromedaries and pigs,but not cats,dogs,mice or rats.Although the binding affinity of pigCD26 to MERS-RBD is lower than that of hCD26,the MERS-CoV pseudovirus can infect cells expressing pigCD26.We further determined the complex structure of pigCD26 bound to MERS-RBD to investigate the molecular basis of their interactions.According to the complex structure,the binding interface between pigCD26 and MERS-RBD is much smaller compared with that of hCD26,which might explain their weak binding affinity.It is interesting that S339 in pigCD26 does not directly interact with MERS-RBD but can significantly influence their interactions.A S339C single mutation can induce the formation of a disulfide bond with C328,which favors the interaction with MERS-RBD and enhances the binding affinity by roughly 1000 folds as determined by SPR-These results imply that pig might be susceptible to MERS-CoV infectioa To validate the deduction,we challenged Danish Landrace piglets with MERS-CoV and monitored their physical signs of infection.Despite no obvious clinical signs of disease,the replication process of viral RNA and antibodies against MERS-CoV could be detected in experimental animals.In summary,we solved the structures of MERS-CoV and SARS-CoV S proteins by cryo-EM reconstruction.These structures demonstrate their RBDs have to undertake a conformational change before exposing the receptor binding sites to interact with receptors,which intensified our understanding of the mechanism of corona virus invading host cells.Besides,we as also determined the structures of MERS-RBD bound to batCD26 or pigCD26 by X-ray crystallography.Combined with biochemical analysis and cell based assays,we showed that MERS-RBD can bind CD26s from different species with distinct affinities,implying the potential of MERS-CoV to transmit among certain host species.Further animal experiment confirmed MERS-CoV can infect pigs,however the infection did not cause obvious clinical symptoms,indicating pigs may play an important role in the transmission of MERS-CoV.These findings also provide important insights into the cross-species transmission of MERS-CoV,which would be of great value in guiding the prevention and treatment of coronaviruse infections.