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. Coronaviruses recognize many receptors using their envelope-anchored spike protein. The role of receptor binding in coronavirus entry into host cells is a fundamental question in virology. Mouse hepatitis coronavirus (MHV) is unique among all coronaviruses in that it uses the N-terminal domain (NTD) of its spike protein to bind a protein receptor CEACAM1a. While extensive research has been performed on the cell entry mechanisms of coronaviruses that use a different domain of their spike protein for receptor binding, the cell entry mechanism for MHV is still elusive. Here we determined the cryo-EM structure of MHV spike protein complexed with CEACAM1a. The structure reveals unique features of receptor binding by MHV spike that facilitate the structural changes of MHV spike and promote cell entry of MHV. We further confirmed the structural results with biochemical and negative-stain EM analyses. These results suggest that receptor binding plays dual roles in MHV entry: it promotes both viral attachment to host cells and the fusion of host and viral membranes. Our study provides insight into the molecular mechanism of MHV entry, demonstrating how cell entry of MHV has been adapted to its unique way of receptor binding.

Porcine epidemic diarrhea virus (PEDV), a member of Alphacoronavirus, has caused huge economic losses for the global pork industry recently. The spike (S) protein mediates PEDV entry into host cells. Herein, we investigated the interactions between the S protein and its receptor porcine aminopeptidase N (pAPN) or co-receptor sugars. The C-terminal domain (CTD) of the S1 domain is bound to pAPN. The prototype strain demonstrated similar receptor-binding activity compared with the variant field isolate. Three loops at the tips of the β-barrel domains did not play crucial roles in the PEDV S-pAPN association, indicating that PEDV conforms to a different receptor recognition model compared with transmissible gastroenteritis virus (TGEV), porcine respiratory CoV (PRCV), and human coronavirus NL63 (HCoV-NL63). The N-terminal domain (NTD) of the PEDV S1 domain could bind sugar, a possible co-receptor for PEDV. The prototype strain exhibited weaker sugar-binding activity compared with the variant field isolate. Strategies targeting the receptor binding domain (RBD) may be helpful for developing vaccines or antiviral drugs for PEDV. Understanding the differences in receptor binding between the prototype and the variant strains may provide insight into PEDV pathogenesis.

Coronaviruses are enveloped, single-stranded RNA viruses that are distributed worldwide. They include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), many of which seriously endanger human health and well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host responses. The role of the C terminus of betacoronavirus nsp1 in virulence has been characterized, but the location of the alphacoronavirus nsp1 region that is important for virulence remains unclear. Here, using TGEV nsp1 as a model to explore the function of this protein in alphacoronaviruses, we demonstrate that alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal structure of full-length TGEV at 1.85-Å resolution and conducting several biochemical analyses, we observed that a specific motif (amino acids 91–95) of alphacoronavirus nsp1 is a conserved region that inhibits host protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9 technology to construct a recombinant TGEV in which this specific nsp1 motif was altered, we found that this mutation does not affect virus replication in cell culture but significantly reduces TGEV pathogenicity in pigs. Taken together, our findings suggest that alphacoronavirus nsp1 is an essential virulence determinant, providing a potential paradigm for the development of a new attenuated vaccine based on modified nsp1.

Coronaviruses cause widespread respiratory, gastrointestinal, and central nervous system diseases in humans and other animals, threatening human health and causing economic loss. Coronavirus nsp9, a member of the replication complex, is an important RNA binding subunit in the RNA-synthesizing machinery of all coronaviruses. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. In this study we determined the nsp9 crystal structures of PDCoV and PEDV. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. This study provides a structural and functional basis for understanding the mechanism of dimerization and shows that the diverse dimerization modes of coronavirus nsp9 proteins enhance their nucleic acid binding affinity. Importantly, these findings may provide a new insight for antiviral drug development.

Emerging coronaviruses (CoVs) pose a severe threat to human and animal health worldwide. To identify host factors required for CoV infection, we used α-CoV transmissible gastroenteritis virus (TGEV) as a model for genome-scale CRISPR knockout (KO) screening. Transmembrane protein 41B (TMEM41B) was found to be a bona fide host factor involved in infection by CoV and three additional virus families. We found that TMEM41B is critical for the internalization and early-stage replication of TGEV. Notably, our results also showed that cells lacking TMEM41B are unable to form the double-membrane vesicles necessary for TGEV replication, indicating that TMEM41B contributes to the formation of CoV replication organelles. Lastly, our data from a mouse infection model showed that the KO of this factor can strongly inhibit viral infection and delay the progression of a CoV disease. Our study revealed that targeting TMEM41B is a highly promising approach for the development of broad-spectrum anti-viral therapeutics.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV–host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R , an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔ I73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔ I73R can be a potent live-attenuated vaccine candidate.