DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

Diversity for endoribonuclease nsp15-mediated regulation of alpha-coronavirus propagation and virulence.

IMPORTANCE: Understanding the role of the endoribonuclease non-structural protein 15 (nsp15) (EndoU) in coronavirus (CoV) infection and pathogenesis is essential for vaccine target discovery. Whether the EndoU activity of CoV nsp15, as a virulence-related protein, has a diverse effect on viral virulence needs to be further explored. Here, we found that the transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV) nsp15 proteins antagonize SeV-induced interferon-ß (IFN-ß) production in human embryonic kidney 293 cells. Interestingly, compared with wild-type infection, infection with EnUmt-TGEV or EnUmt-FIPV did not change the IFN-ß response or reduce viral propagation in immunocompetent cells. The results of animal experiments showed that EnUmt viruses did not reduce the clinical presentation and mortality caused by TGEV and FIPV. Our findings enrich the understanding of nsp15-mediated regulation of alpha-CoV propagation and virulence and reveal that the conserved functions of nonstructural proteins have diverse effects on the pathogenicity of CoVs.

Genome-scale CRISPR screen identifies TRIM2 and SLC35A1 associated with porcine epidemic diarrhoea virus infection.

Porcine epidemic diarrhoea (PED) caused by the porcine epidemic diarrhoea virus (PEDV) is the most devastating disease in the global pig industry due to its high mortality rate in piglets. The host factors critical for PEDV replication are poorly understood. Here, we designed a pooled African green monkey genome-scale CRISPR/Cas9 knockout (VeroCKO) library containing 75,608 single guide RNAs targeting 18,993 protein-coding genes. Subsequently, we use the VeroCKO library to identify key host factors facilitating PEDV infection in Vero E6 cells. Several previously unreported genes associated with PEDV infection are highly enriched post-PEDV selection. We discovered that knocking out the tripartite motif 2 (TRIM2) and the solute carrier family 35 member A1 (SLC35A1) inhibited PEDV replication. Virtual screening and molecular docking approaches showed that chem-80,048,685 (M2) s ignificantly inhibited PEDV attachment and late replication by impeding SLC35A1. Furthermore, we found that knocking out SLC35A1 in Vero E6 cells upregulated a disintegrin and metalloprotease protein-17 (ADAM17) by splicing porcine aminopeptidase N (pAPN) and angiotensin-converting enzyme 2 (ACE2) ectodomains to reduce PEDV-infection in a CMP-Sialic Acid (CMP-SA) cell entry-independent manner. These findings provide a new perspective for a better understanding of host-pathogen interactions and new therapeutic targets for PEDV infection.

mLST8 is essential for coronavirus replication and regulates its replication through the mTORC1 pathway.

Coronaviruses (CoVs), which pose a serious threat to human and animal health worldwide, need to hijack host factors to complete their replicative cycles. However, the current study of host factors involved in CoV replication remains unknown. Here, we identified a novel host factor, mammalian lethal with sec-13 protein 8 (mLST8), which is a common subunit of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), and is critical for CoV replication. Inhibitor and knockout (KO) experiments revealed that mTORC1, but not mTORC2, is essential for transmissible gastroenteritis virus replication. Furthermore, mLST8 KO reduced the phosphorylation of unc-51-like kinase 1 (ULK1), a factor downstream of the mTORC1 signaling pathway, and mechanistic studies revealed that decreased phosphorylation of the mTORC1 downstream factor ULK1 promoted the activation of autophagy, which is responsible for antiviral replication in mLST8 KO cells. Then, transmission electron microscopy indicated that both mLST8 KO and autophagy activator inhibited the formation of double-membrane vesicles in early viral replication. Finally, mLST8 KO and autophagy activator treatment could also inhibit the replication of other CoVs, indicating a conserved relationship between autophagy activation and CoV replication. In summary, our work reveals that mLST8 is a novel host regulator of CoV replication, which provides new insights into the mechanism of CoV replication and can facilitate the development of broad-spectrum antiviral drugs. IMPORTANCE CoVs are highly variable, and existing CoV vaccines are still limited in their ability to address mutations in CoVs. Therefore, the need to improve our understanding of the interaction of CoVs with the host during viral replication and to find targets for drugs against CoVs is urgent. Here, we found that a novel host factor, mLST8, is critical for CoV infection. Further studies showed that mLST8 KO inhibited the mTORC1 signaling pathway, and we found that autophagy activation downstream of mTORC1 was the main cause of antiviral replication in mLST8 KO cells. Autophagy activation impaired the formation of DMVs and inhibited early viral replication. These findings deepen our understanding of the CoV replication process and provide insights into potential therapeutic applications.

Genome-scale CRISPR screen identifies TMEM41B as a multi-function host factor required for coronavirus replication.

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.

The trypsin-enhanced infection of porcine epidemic diarrhea virus is determined by the S2 subunit of the spike glycoprotein.

Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen in 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, trypsin-enhanced YN200 and trypsin-independent DR13, in which the spike (S) protein of YN200 exhibits a stronger ability to induce syncytium formation and 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-SDR13 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, S2 subunit, and S2720∼892 aa domain (NS2') were individually replaced by the corresponding DR13 sequences. Intriguingly, only the S2 substitution, not the S1 or NS2' substitutions, 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 mainly controlled by mutations in the S2 subunit rather than directly trypsin cleavage site.ImportanceWith the emergence of new variants, PEDV remains a major problem in the global swine industry. Efficient PEDV infection usually requires trypsin, while the mechanism of trypsin dependency is complex. Here, we used two PEDV strains, trypsin-enhanced YN200 and trypsin-independent DR13, and results showed that the S protein determined PEDV trypsin dependency by using a reverse genetic system of YN200. The S2 subunit was verified as the main portion of PEDV trypsin dependency, though the putative S2' site mutation cannot render trypsin-independent growth of YN200. Finally, these results provide some different insight to the PEDV trypsin dependency and might inspire vaccine development.

Cryo-EM analysis of the HCoV-229E spike glycoprotein reveals dynamic prefusion conformational changes.

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.

Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins.

Coronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (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 Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (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; 2.75×105) 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: <500), and more S-trimer-specific antibodies were induced by the RBD in the SARS-CoV and SARS-CoV-2 (antibody titers: 6.72×105; 5×105) than HCoV-229E (antibody titers:1.125×103). Besides, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was lower than S-trimer, and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus Our findings will provide important implications for future development of coronavirus vaccines.Importance Outbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in "lying" and "standing" states in the prefusion S-trimer structure. Here, we evaluated the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that the S-trimer and RBD are both candidates for subunit vaccines in Beta-coronavirus (SARS-CoV and SARS-CoV-2) with a RBD "standing" state. However, for Alpha-coronavirus (HCoV-229E) with a RBD "lying" state, the S-trimer may be more suitable for subunit vaccines than the RBD. Our results will provide novel ideas for the development of vaccines targeting S protein in the future.

Receptor tyrosine kinase inhibitors block proliferation of TGEV mainly through p38 mitogen-activated protein kinase pathways.

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.

Crystal structural basis for Rv0315, an immunostimulatory antigen and inactive beta-1,3-glucanase of Mycobacterium tuberculosis.

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 ß-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-ß-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 ß-1,3-glucanase in Mtb drives T-helper 1 (Th1) immune responses, which may help develop more effective vaccines against Mtb infection.