Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses.
Recent studies on coronavirus entry have been focused on those coronaviruses that use their S1-CTD as the receptor-binding domain. These studies have shown that S1-CTDs in those coronaviruses undergo a dynamic conformational change: lying down to evade immune surveillance and standing up for receptor binding. Receptor binding stabilizes S1-CTD in the standing up position, reducing the interface between S1 and S2. The weakened interactions between S1 and S2, plus two sequential protease cleavages (one at the S1/S2 boundary and the other at the S2’ site), allow S1 to dissociate from S2. Subsequently S2 undergoes the final conformational change and transitions to the post-fusion conformation. MHV differs from the above coronaviruses because it is the only coronavirus that uses its spike S1-NTD to bind a protein receptor. As a result of its unique receptor recognition pattern, the molecular mechanism for MHV entry is still elusive. In this study, we investigated the role of receptor binding by S1-NTD in the conformational changes of MHV spike, providing insight into the molecular mechanism for MHV entry.
We performed a combination of structural and biochemical studies on the receptor-associated activities of MHV spike. These studies included determination of cryo-EM structure of receptor-bound MHV spike ectodomain, receptor-dependent protease sensitivity analysis of virus-surface MHV spike, negative-stain EM analysis of the receptor-facilitated conformational changes of MHV spike, and receptor-facilitated MHV cell entry. Based on our results, we propose the following molecular mechanism for MHV entry. During MHV entry into host cells, MHV spike binds to CEACAM1a on host cell surface for viral attachment. One spike is capable of binding three CEACAM1a molecules. Receptor binding triggers conformational changes in MHV spike, weakening the S1/S2 interactions and positioning MHV spike for two sequential proteolyses (one at the S1/S2 boundary and the other at the S2’ site). CEACAM1a, which has flexible domain hinges, bends in order to approach S1-NTD on the side of the spike trimer. The receptor-induced conformational changes, receptor-facilitated proteolysis, and the potential bending of the receptor all contribute to the dissociation of S1 from S2. After S1 dissociates, S2 transitions to the post-fusion conformation through a hypothetical elongated intermediate state
The molecular mechanism for virus entry is one of the most fundamental questions in virology. Our study reveals the unique features of MHV entry, highlighting how receptor binding programs atomic level reorganization of MHV spike to promote membrane fusion. Hence MHV has adapted to its special need in receptor recognition and turns this need to its evolutionary advantage in cell entry. Our study demonstrates the diversity of cell entry by different coronaviruses and reveals new knowledge about this critical step in viral infection cycles
Materials and methods
Expression and purification of MHV spike ectodomain and mouse CEACAM1a
MHV spike gene (strain A59) was kindly provided by Dr. Zhaohui Qian from Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. MHV spike ectodomain (S-e) (residues 15–1227) was cloned into pFastBac vector (Life Technologies Inc.); the construct contained an N-terminal honeybee melittin signal peptide and C-terminal GCN4 and His6 tags. It was expressed in Sf9 insect cells using the Bac-to-Bac system (Life Technologies Inc.) and purified as previously described. Briefly, the protein was harvested from cell culture medium, and purified sequentially on Ni-NTA column and Superdex 200 size exclusion column (GE Healthcare). Mouse CEACAM1a ectodomain (residues 1–202) was expressed and purified as previously described; the construct contained a C-terminal His6 tag. Purified MHV S-e and CEACAM1a were mixed and incubate at 4°C for 2 hours. The MHV S-e/CEACAM1a complex was purified on Superdex 200 size exclusion column (GE Healthcare).
Cryo-electron microscopy For sample preparation, aliquots of the MHV S-e/CEACAM1a complex (3 μl, 0.35 mg/ml, in buffer containing 2 mM Tris pH7.2 and 20 mM NaCl) were applied to glow-discharged CF-2/1-4C C-flat grids (Protochips). The grids were then plunge-frozen in liquid ethane using a Vitrobot system (FEI Company). For data collection, images were recorded using a Gatan K2 Summit direct electron detector in super resolution mode, attached to a FEI Titan-Krios TEM. The automated software SerialEM  was used to collect 2,250 total movies at 22,500x magnification and at a defocus range between 1 and 3 μm. Each movie had a total accumulated exposure of 77 e/Å2 fractionated in 50 frames of 10-second exposure. Data collection statistics are summarized
For data processing, whole frames in each movie were corrected for motion and dose compensation using MotionCor2. ~1,800 best images were manually selected. The final images were bin-averaged to reach a pixel size of 1.06 Å. The parameters of the microscope contrast transfer function were estimated for each micrograph using GCTF. Particles were automatically picked and extracted using RELION with a box size of 320 pixels. Initially, 842,337 particles were extracted and subjected to 2D alignment and clustering using RELION. The best classes were then selected for an additional 2D alignment. ~5,000 best particles were selected for creating the initial 3D model using RELION. 210,067 particles selected from 2D alignment were then subjected to 3D classification. The best class with 82,923 particles was subjected to 3D refinement to generate the final density map. The final density map was sharpened with modulation transfer function of K2 operated at 300keV using RELION. Reported resolutions were based on the gold standard Fourier shell correlation (FSC) = 0.143 criterion. Fourier shell correction curves were corrected for the effects of soft masking by high-resolution noise substitution. Data processing was concluded
Model building and refinement The initial model of the MHV S-e/CEACAM1a complex was obtained by fitting the cryo-EM structure of unliganded MHV S-e (PDB ID: 3JCL) and the crystal structure of MHV S1-NTD/CEACAM1a complex (PDB ID: 3R4D) into our cryo-EM density map using UCSF Chimera and Coot. Manual model rebuilding was performed using Coot based on the well-defined continuous density of the main chain. Side chain assignments were guided through the densities of N-linked glycans and bulky amino acid residues. The structural model of MHV S-e/CEACAM1a complex was refined using Phenix with geometry restrains and three-fold noncrystallographic symmetry constraints. Refinement and model rebuilding were carried out iteratively until no further improvements were achieved in geometry parameters and model-map correlation coefficient. The quality of the final model was analyzed with MolProbity and EMRinger. The validation statistics of the structural models are summarized
AlphaScreen protein-protein binding assay AlphaScreen protein-protein binding assay was carried out between recombinant MHV S1-NTD and recombinant CEACAM1a and between recombinant MHV S-e and recombinant CEACAM1a as described previously. Briefly, Fc-tagged CEACAM1a (at 6 nM final concentration) was incubated with either His6-tagged MHV S1-NTD or His6-tagged MHV S-e (at 100 nM final concentration) in ½ AreaPlate (PerkinElmer, Waltham, MA) at room temperature for 1 hour. AlphaScreen Nickel Chelate Donor Beads and AlphaScreen Protein A Acceptor Beads (PerkinElmer) were then added to one of the mixtures at final concentrations of 5 μg/mL each. The mixtures were then incubated at room temperature for 1 hour away from light. The AlphaScreen signals were measured using an EnSpire plate reader (PerkinElmer).
Packaging of MHV pseudoviruses MHV pseudoviruses were packaged as previously described. Briefly, full-length MHV spike gene (which contained a C-terminal C9 tag) was inserted into pcDNA3.1 (+) plasmid. Retroviruses pseudotyped with MHV spike and expressing a luciferase reporter gene were prepared through co-transfecting HEK293T cells with a plasmid carrying Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and the plasmid encoding MHV spike. The produced MHV pseudoviruses were harvested 72 hours post transfection.
MHV pseudovirus entry assay MHV pseudoviruses (strain A59) were generated as described above. The produced pseudoviruses with indicated treatment were then used to enter HEK293T cells expressing CEACAM1a. After incubation at 37°C for 5 hours, medium was changed and cells were incubated for an additional 60 hours. Cells were then washed with PBS and lysed. Aliquots of cell lysates were transferred to Optiplate-96 (PerkinElmer), followed by addition of luciferase substrate. Relative light unites (RLUs) were measured using EnSpire plate reader (PerkinElmer). All the measurements were carried out in triplicates.
Proteolysis assay MHV pseudoviruses were purified using a 10–30% sucrose gradient ultracentrifugation at 250,000×g at 4°C for 2 hours. Purified MHV pseudoviruses were incubated alone or with recombinant CEACAM1a (which is in excess) at 37°C for 30 minutes. Then MHV pseudoviruses were incubated with different concentrations of trypsin at 4°C for 30 minutes. Subsequently soybean trypsin inhibitor (which is in excess) was added to stop the reaction. Samples were then applied for Western blot analysis using an antibody targeting the C-terminal C9 tag of MHV spike.
Double proteolysis assay Recombinant MHV S-e molecules (3 μg) were first treated with low concentration of trypsin at room temperature for 10 min. The reactions were stopped using soybean trypsin inhibitor. The products from this first proteolysis assay were analyzed using silver staining. They were then divided into two halves: one half was incubated with CEACAM1a at 37°C for 2 hours, and the other half was not incubated with CEACAM1a. Subsequently both halves were treated with proteinase K (final concentration for the assay: 1 μM) on ice for 20 min. The products from the second proteolysis assay were analyzed using silver staining. Purified MHV pseudoviruses were also subjected to the same double proteolysis assay, except that Western blot (using an antibody targeting the C-terminal C9 tag of MHV spike) replaced silver staining in analyzing the proteolysis products from both proteolysis assays.
Cleavage of MHV spike using lysosomal extracts Lysosomal extracts from HEK293T cells were prepared according to the lysosome isolation kit procedure (Sigma-Aldrich) as previously described. Briefly, HEK293T cells were harvested and washed with PBS buffer and then resuspended in 2.7 packed cell volumes (PCV) of extraction buffer. The cells were then broken in a 7-ml Dounce homogenizer using a loose pestle (i.e., pestle B) until 80% to 85% of the cells were broken (protease inhibitors from the kit were omitted in our procedure). The samples were centrifuged at 1,000 × g for 10 min, and the supernatants were transferred to a new tube and centrifuged at 20,000 × g for another 20 min. The supernatants were removed, and the pellets were resuspended in extraction buffer as the crude lysosomal fraction (CLF). The CLF was diluted in buffer containing 19% Optiprep density gradient medium solution and further purified using density gradient centrifugation at 150,000 × g for 4 hours to produce lysosomal extracts. For cleavage of MHV spike using lysosomal extracts, purified MHV pseudoviruses were incubated with membrane-bound CEACAM1a (i.e., HEK293T cells expressing CEACAM1a on the surface) for 1 hour and then were treated with lysosomal extracts at 37°C for 20 min. Subsequently, samples were denatured and analyzes using SDS-PAGE gel. Cleaved MHV spike molecules were detected by Western Blot using an anti-C9 tag antibody.
Live MHV infection assay MHV live virus particles (strain A59) were generated from an infectious clone, which is comprised of seven fragments maintained in pSMART (Lucigen) or pCR-XL-TopoA (Invitrogen) vectors and was amplified according to previously published protocols. Viral stock was propagated in delayed brain tumor (DBT) cells and viral titers were determined using plaque titration. For live MHV infection, viruses with indicated treatment were used to infect DBT cells with a multiplicity of infection (MOI) of 0.05 PFU/cell for a one-hour adsorption period, followed by three washes with phosphate-buffered saline (PBS). Fresh medium was then added to each culture, and the infection was maintained at 37°C. Each condition was performed in triplicate. Microscope images were obtained 7 hours post infection.
Negative-stain electron microscopy The MHV S-e/CEACAM1a complex treated under different conditions was diluted to a final concentration of 0.02 mg/mL in 2 mM Tris-HCl pH7.2 and then loaded onto glow-discharged 400-mesh carbon grids (Electron Microscopy Sciences). Subsequently the grids were stained with 0.75% uranyl formate. All micrographs were collected at the University of Minnesota using a Tecnai G2 Spirit BioTWIN at 120 keV (FEI Company) and an Eagle 3.1 mega pixel CCD camera at 6,000 × nominal magnification. For 2D image averaging, particles were picked and extracted using RELION.
Calculation of interface area The total surface area and buried surface area of pre-fusion MHV S-e and MHV S-e/CEACAM1a complex were calculated using the PISA server at the European Bioinformatics Institute. For each trimeric S-e (unliganded or receptor-bound), a PDB file containing both S1 subunits and S2 subunits was submitted to the PISA server, and the total surface area and buried surface area on S1 and S2 were individually calculated.
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