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Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design., The spike protein of coronaviruses (S-protein) is an envelope-anchored trimeric type I transmembrane glycoprotein that mediates receptor binding and the fusion of the viral and host cell membranes. Here the authors report the conformational states of HCoV-229E S trimer and observe the conformational changes in S1 subunit from closed state to activated state for receptor binding.

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., Author summary Coronaviruses (CoVs) pose a severe threat to human and animal health. Identification of host genes essential for transmissible gastroenteritis virus (TGEV) infection may reveal novel therapeutic targets and strengthen our understanding of CoV disease pathogenesis. We performed genome-wide and sub-pooled CRISPR screens in porcine kidney cells with TGEV. We identified numerous candidate host factors for TGEV infection. Considering its extremely strong effect on TGEV infection, we focused our efforts on characterizing the roles that TMEM41B play in the virus replication cycle. Our results revealed that TMEM41B contributed to CoV internalization and the formation of replication organelles. TMEM41B is critical for the replication of TGEV and other RNA viruses. Our mouse infection model provides strong evidence that knocking out the TMEM41B gene can inhibit viral infection and delay the progression of a CoV disease. Our study identified potential therapeutic targets for common RNA viruses and reveals host factors that regulate susceptibility to highly pathogenic CoVs.

Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen of importance to the swine industry, causing high mortality in neonatal piglets. Efficient PEDV infection usually relies on the presence of trypsin, yet the mechanism of trypsin dependency is ambiguous. Here, we identified two PEDV strains, the trypsin-enhanced strain YN200 and the trypsin-independent strain DR13; the spike (S) protein of YN200 exhibits a stronger ability to induce syncytium formation and to be cleaved by trypsin than that of DR13. Using a full-length infectious YN200 cDNA clone, we confirmed that the S protein is a trypsin dependency determinant by comparison of rYN200 and rYN200-S(DR13). To explore the trypsin-associated sites of the YN200 S protein, we then constructed a series of mutations adjacent to the fusion peptide. The results show that the putative S2′ cleavage site (R892G) is not the determinant for virus trypsin dependency. Hence, we generated viruses carrying chimeric S proteins: the S1 subunit, the S2 subunit, and the S2(720∼892) domain (NS2′) were individually replaced by the corresponding DR13 sequences. Intriguingly, only the S2 substitution, not the S1 or NS2′ substitution, provides trypsin-independent growth of YN200. Additionally, the NS2′ recombinant virus significantly abrogated effective infection, indicating a vital role for NS2′ in viral entry. These findings suggest that the trypsin dependency of PEDV is controlled mainly by mutations in the S2 subunit rather than directly by a trypsin cleavage site. IMPORTANCE With the emergence of new variants, PEDV remains a major problem in the global swine industry. Efficient PEDV infection usually requires trypsin, but the mechanism of trypsin dependency is complex. Here, we used two PEDV strains, the trypsin-enhanced strain YN200 and the trypsin-independent strain DR13. By using a YN200 reverse genetic system, we showed that the S protein determined PEDV trypsin dependency. The S2 subunit was verified as the main portion of PEDV trypsin dependency, although the putative S2′ site mutation cannot provide trypsin-independent growth of YN200. Finally, these results provide new insight into PEDV trypsin dependency and might inspire vaccine development.

Outbreaks of coronaviruses, especially SARS-CoV-2, pose a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority., Coronaviruses that infect humans belong to the Alphacoronavirus (including HCoV-229E) and Betacoronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. The spike (S) homotrimers bind to their receptors via the receptor-binding domain (RBD), which is a major target to block viral entry. In this study, we selected Alphacoronavirus (HCoV-229E) and Betacoronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs exist two different conformational states (“lying” or “standing”) in the prefusion S-trimer structure. Then, the differences in the immune responses to RBDs from these coronaviruses were analyzed structurally and immunologically. Our results showed that more RBD-specific antibodies (antibody titers: 1.28 × 105 and 2.75 × 105, respectively) were induced by the S-trimer with the RBD in the standing state (SARS-CoV and SARS-CoV-2) than the S-trimer with the RBD in the lying state (HCoV-229E; antibody titers

Emerging coronaviruses (CoVs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. Here, A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (TGEV) infection in cell-based assays. Moreover, A9 exhibited potent antiviral activity against the replication of various CoVs, including murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV). We further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of A9 against TGEV infection in vitro. We specifically identified p38 and JNK1, which are the downstream molecules of receptor tyrosine kinases (RTKs) required for efficient TGEV replication, as A9 targets through plaque assays, qRT-PCR and Western blotting assays. p38 and JNK1 inhibitors and RNA interference further showed that the inhibitory activity of A9 against TGEV infection was mainly mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. All these findings indicated that the RTKI A9 directly inhibits TGEV replication and that its inhibitory activity against TGEV replication mainly occurs by targeting p38, which provides vital clues to the design of novel drugs against CoVs., Highlights • We screened inhibitors against coronavirus replication using TGEV as a surrogate model through a high-throughput assay. • A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, was identified as a robust inhibitor of TGEV. • A9 also exhibited potent antiviral activity against the replication of various coronaviruses. • The inhibitory activity of A9 against TGEV replication is mainly regulated by targeting p38.

Mycobacterium tuberculosis (Mtb) remains a leading cause of morbidity and mortality worldwide, as two billion people are latently infected with Mtb. To address Mtb drug resistance and the limitations of current vaccines, the characteristics of candidate Mtb vaccines need to be explored. Here, we report the three-dimensional structure of Rv0315 at 1.70 Å resolution, a novel immunostimulatory antigen of Mtb and demonstrate that Rv0315 is an inactive β-1,3-glucanase of the glycoside hydrolase 16 (GH16) family. Our study further elaborates the molecular basis for the lack of glucan recognition by Rv0315. Rv0315 has a large open groove and this particular topology cannot bind oligosaccharide chains in solution, thus explaining the lack of detectable hydrolytic activity towards its substrate. Additionally, we identified Glu-176, a conserved catalytic residue in GH16 endo-β-1,3-glucanases, as essential for Rv0315 to induce immunological responses. These results indicate that Rv0315 likely diverged from a broad-specificity ancestral GH16 glucanase and this inactive member of the GH16 family offers new insights into the GH16 glucanase. Together, our findings suggest that an inactive β-1,3-glucanase in Mtb drives T-helper 1 (Th1) immune responses, which may help develop more effective vaccines against Mtb infection.