Ependymal polarity defects coupled with disorganized ciliary beating drive abnormal cerebrospinal fluid flow and spine curvature in zebrafish.

Idiopathic scoliosis (IS) is the most common spinal deformity diagnosed in childhood or early adolescence, while the underlying pathogenesis of this serious condition remains largely unknown. Here, we report zebrafish ccdc57 mutants exhibiting scoliosis during late development, similar to that observed in human adolescent idiopathic scoliosis (AIS). Zebrafish ccdc57 mutants developed hydrocephalus due to cerebrospinal fluid (CSF) flow defects caused by uncoordinated cilia beating in ependymal cells. Mechanistically, Ccdc57 localizes to ciliary basal bodies and controls the planar polarity of ependymal cells through regulating the organization of microtubule networks and proper positioning of basal bodies. Interestingly, ependymal cell polarity defects were first observed in ccdc57 mutants at approximately 17 days postfertilization, the same time when scoliosis became apparent and prior to multiciliated ependymal cell maturation. We further showed that mutant spinal cord exhibited altered expression pattern of the Urotensin neuropeptides, in consistent with the curvature of the spine. Strikingly, human IS patients also displayed abnormal Urotensin signaling in paraspinal muscles. Altogether, our data suggest that ependymal polarity defects are one of the earliest sign of scoliosis in zebrafish and disclose the essential and conserved roles of Urotensin signaling during scoliosis progression.

Immune evasion strategy involving propionylation by the KSHV interferon regulatory factor 1 (vIRF1).

Post-translational modifications (PTMs) are essential for host antiviral immune response and viral immune evasion. Among a set of novel acylations, lysine propionylation (Kpr) has been detected in both histone and non-histone proteins. However, whether protein propionylation occurs in any viral proteins and whether such modifications regulate viral immune evasion remain elusive. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral interferon regulatory factor 1 (vIRF1) can be propionylated in lysine residues, which is required for effective inhibition of IFN-ß production and antiviral signaling. Mechanistically, vIRF1 promotes its own propionylation by blocking SIRT6's interaction with ubiquitin-specific peptidase 10 (USP10) leading to its degradation via a ubiquitin-proteasome pathway. Furthermore, vIRF1 propionylation is required for its function to block IRF3-CBP/p300 recruitment and repress the STING DNA sensing pathway. A SIRT6-specific activator, UBCS039, rescues propionylated vIRF1-mediated repression of IFN-ß signaling. These results reveal a novel mechanism of viral evasion of innate immunity through propionylation of a viral protein. The findings suggest that enzymes involved in viral propionylation could be potential targets for preventing viral infections.

Drug screening identified that handelin inhibits feline calicivirus infection by inhibiting HSP70 expression in vitro.

Feline calicivirus (FCV) is considered one of the major pathogens of cats worldwide and causes upper respiratory tract disease in all cats. In some cats, infection is by a highly virulent strain of FCV (vs.-FCV), which can cause severe and fatal systemic disease symptoms. At present, few antiviral drugs are approved for clinical treatment against FCV. Therefore, there is an imminent need for effective FCV antiviral agents. Here, we used observed a cytopathic effect (CPE) assay to screen 1746 traditional Chinese medicine monomer compounds and found one that can effectively inhibit FCV replication, namely, handelin, with an effective concentration (EC50) value of approximately 2.5 µM. Further study showed that handelin inhibits FCV replication via interference with heat shock protein 70 (HSP70), which is a crucial host factor and plays a positive role in regulating viral replication. Moreover, handelin and HSP70 inhibitors have broad-spectrum antiviral activity. These findings indicate that handelin is a potential candidate for the treatment of FCV infection and that HSP70 may be an important drug target.

Correction: Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway.

[This corrects the article DOI: 10.1371/journal.ppat.1008730.].

The CDE region of feline Calicivirus VP1 protein is a potential candidate subunit vaccine.

BACKGROUND: Feline calicivirus (FCV) infection causes severe upper respiratory disease in cats, but there are no effective vaccines available for preventing FCV infection. Subunit vaccines have the advantages of safety, low cost and excellent immunogenicity, but no FCV subunit vaccine is currently available. The CDE protein is the dominant neutralizing epitope region of the main antigenic structural protein of FCV, VP1. Therefore, this study evaluated the effectiveness of the CDE region as a truncated FCV VP1 protein in preventing FCV infection to provide a strategy for developing potential FCV subunit vaccines. RESULTS: Through the prediction of FCV VP1 epitopes, we found that the E region is the dominant neutralizing epitope region. By analysing the spatial structure of VP1 protein, 13 amino acid sites in the CD and E regions were found to form hydrogen bonding interactions. The results show the presence of these interaction forces supports the E region, helping improve the stability and expression level of the soluble E protein. Therefore, we selected the CDE protein as the immunogen for the immunization of felines. After immunization with the CDE protein, we found significant stimulation of IgG, IgA and neutralizing antibody production in serum and swab samples, and the cytokine TNF-α levels and the numbers of CD4+ T lymphocytes were increased. Moreover, a viral challenge trial indicated that the protection generated by the CDE subunit vaccine significantly reduced the incidence of disease in animals. CONCLUSIONS: For the first time, we studied the efficacy of the CDE protein, which is the dominant neutralizing epitope region of the FCV VP1 protein, in preventing FCV infection. We revealed that the CDE protein can significantly activate humoral, mucosal and cellular immunity, and the resulting protective effect can significantly reduce the incidence of animal disease. The CDE region of the FCV capsid is easy to produce and has high stability and excellent immunogenicity, which makes it a candidate for low-cost vaccines.

Identification of a Novel Neutralizing Epitope on the N-Terminal Domain of the Human Coronavirus 229E Spike Protein.

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. IMPORTANCE Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

Paper-based sensor based on lead-free manganese halide for the determination of water content in organic solvents.

A highly stable and luminescent lead-free manganese(II) halide hybrid MnBr4(TMN)2 (C34H42Br4MnN4) was designed and synthesized by introducing a large cationic organic spacer. The MnBr4(TMN)2 displays high luminescence with quantum yields up to 77% and possesses turn-off fluorescence behavior (Ex/Em=365/546 nm) for water. These properties make the MnBr4(TMN)2 a promising candidate as an alternative indicator for the detection of water with potential applications for the fabrication of LEDs. Herein, a paper-based sensor based on MnBr4(TMN)2 is described for the determination of water content in organic solvents. The mechanism of water sensing can be tentatively explained by fluorescence quenching originating from the destruction of water due to the Mn-Br bonds of MnBr4(TMN)2. The MnBr4(TMN)2-based paper sensor exhibits an excellent discrimination ability of water content in the range 0-25.0% with a detection limit of 0.27%. Satisfactory recoveries (94.91±4.09% to 103.23±2.38%) are obtained in spiked ethanol solvent samples, which demonstrate that the MnBr4(TMN)2-based paper sensor is capable of detecting water content in real ethanol solvent samples.

Griseofulvin Inhibits Root Growth by Targeting Microtubule-Associated Proteins Rather Tubulins in Arabidopsis.

Griseofulvin was considered an effective agent for cancer therapy in past decades. Although the negative effects of griseofulvin on microtubule stability are known, the exact target and mechanism of action in plants remain unclear. Here, we used trifluralin, a well-known herbicide targeting microtubules, as a reference and revealed the differences in root tip morphology, reactive oxygen species production (ROS), microtubule dynamics, and transcriptome analysis between Arabidopsis treated with griseofulvin and trifluralin to elucidate the mechanism of root growth inhibition by griseofulvin. Like trifluralin, griseofulvin inhibited root growth and caused significant swelling of the root tip due to cell death induced by ROS. However, the presence of griseofulvin and trifluralin caused cell swelling in the transition zone (TZ) and meristematic zone (MZ) of root tips, respectively. Further observations revealed that griseofulvin first destroyed cortical microtubules in the cells of the TZ and early elongation zone (EZ) and then gradually affected the cells of other zones. The first target of trifluralin is the microtubules in the root MZ cells. Transcriptome analysis showed that griseofulvin mainly affected the expression of microtubule-associated protein (MAP) genes rather than tubulin genes, whereas trifluralin significantly suppressed the expression of αß-tubulin genes. Finally, it was proposed that griseofulvin could first reduce the expression of MAP genes, meanwhile increasing the expression of auxin and ethylene-related genes to disrupt microtubule alignment in root tip TZ and early EZ cells, induce dramatic ROS production, and cause severe cell death, eventually leading to cell swelling in the corresponding zones and inhibition of root growth.

Structural comparisons of host and African swine fever virus dUTPases reveal new clues for inhibitor development.

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal ß-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (Kd = 15.6 µM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors.

E2f4 is required for intestinal and otolith development in zebrafish.

E2f4 is a multifunctional transcription factor that is essential for many cellular processes. Although the role of E2f4 during cell cycle progression has been investigated in great detail, less is known about E2f4 during embryonic development. Here, we investigated the role of E2f4 during zebrafish development. Zebrafish e2f4 mutants displayed ectopic otolith formation due to abnormal ciliary beating in the otic vesicle. The beating defects of motile cilia were caused by abnormal expression of ciliary motility genes. The expression of two genes, lrrc23 and ccdc151, were significantly decreased in the absence of E2f4. In addition to that, e2f4 mutants also displayed growth retardation both in the body length and body weight and mostly died at around 6 months old. Although food intake was normal in the mutants, we found that the microvilli of the intestinal epithelia were significantly affected in the mutants. Finally, the intestinal epithelia of e2f4 mutants also displayed reduced cell proliferation, together with an increased level of cell apoptosis. Our data suggested a tissue-specific role of E2f4 during zebrafish development, which is distinct from the traditional views of this protein as a transcription repressor.

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.

Establishment of Full-Length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant feline coronavirus (FCoV) 79-1146_CA (r79-1146_CA). In animal experiments with 1- to 2-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study pathogenesis, antiviral drugs, and vaccines for FCoV. IMPORTANCE Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

Sperm associated antigen 9 promotes oncogenic KSHV-encoded interferon regulatory factor-induced cellular transformation and angiogenesis by activating the JNK/VEGFA pathway.

Kaposi's sarcoma (KS), caused by Kaposi's sarcoma-associated herpesvirus (KSHV), is a highly angioproliferative disseminated tumor of endothelial cells commonly found in AIDS patients. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) mediates KSHV-induced cell motility (PLoS Pathog. 2019 Jan 30;15(1):e1007578). However, the role of vIRF1 in KSHV-induced cellular transformation and angiogenesis remains unknown. Here, we show that vIRF1 promotes angiogenesis by upregulating sperm associated antigen 9 (SPAG9) using two in vivo angiogenesis models including the chick chorioallantoic membrane assay (CAM) and the matrigel plug angiogenesis assay in mice. Mechanistically, vIRF1 interacts with transcription factor Lef1 to promote SPAG9 transcription. vIRF1-induced SPAG9 promotes the interaction of mitogen-activated protein kinase kinase 4 (MKK4) with JNK1/2 to increase their phosphorylation, resulting in enhanced VEGFA expression, angiogenesis, cell proliferation and migration. Finally, genetic deletion of ORF-K9 from KSHV genome abolishes KSHV-induced cellular transformation and impairs angiogenesis. Our results reveal that vIRF1 transcriptionally activates SPAG9 expression to promote angiogenesis and tumorigenesis via activating JNK/VEGFA signaling. These novel findings define the mechanism of KSHV induction of the SPAG9/JNK/VEGFA pathway and establish the scientific basis for targeting this pathway for treating KSHV-associated cancers.

Free-Energy Landscape and Isomerization Rates of Au4 Clusters at Finite Temperatures.

In metallic nanoparticles, the geometry of atomic positions controls the particle's electronic band structure, polarizability, and catalytic properties. Analyzing the structural properties is a complex problem; the structure of an assembled cluster changes from moment to moment due to thermal fluctuations. Conventional structural analyses based on spectroscopy or diffraction cannot determine the instantaneous structure exactly and can merely provide an averaged structure. Molecular simulations offer an opportunity to examine the assembly and evolution of metallic clusters, as the preferred assemblies and conformations can easily be visualized and explored. Here, we utilize the adaptive biasing force algorithm applied to first-principles molecular dynamics to demonstrate the exploration of a relatively simple system, which permits a comprehensive study of the small metal cluster Au4 in both neutral and charged configurations. Our simulation work offers a quantitative understanding of these clusters' dynamic structure, which is significant for single-site catalytic reactions on metal clusters and provides a starting point for a detailed quantitative understanding of more complex pure metal and alloy clusters' dynamic properties.

Structural Characterization of the Helicase nsp10 Encoded by Porcine Reproductive and Respiratory Syndrome Virus.

Currently, an effective therapeutic treatment for porcine reproductive and respiratory syndrome virus (PRRSV) remains elusive. PRRSV helicase nsp10 is an important component of the replication transcription complex that plays a crucial role in viral replication, making nsp10 an important target for drug development. Here, we report the first crystal structure of full-length nsp10 from the arterivirus PRRSV, which has multiple domains: an N-terminal zinc-binding domain (ZBD), a 1B domain, and helicase core domains 1A and 2A. Importantly, our structural analyses indicate that the conformation of the 1B domain from arterivirus nsp10 undergoes a dynamic transition. The polynucleotide substrate channel formed by domains 1A and 1B adopts an open state, which may create enough space to accommodate and bind double-stranded RNA (dsRNA) during unwinding. Moreover, we report a unique C-terminal domain structure that participates in stabilizing the overall helicase structure. Our biochemical experiments also showed that deletion of the 1B domain and C-terminal domain significantly reduced the helicase activity of nsp10, indicating that the four domains must cooperate to contribute to helicase function. In addition, our results indicate that nidoviruses contain a conserved helicase core domain and key amino acid sites affecting helicase function, which share a common mechanism of helicase translocation and unwinding activity. These findings will help to further our understanding of the mechanism of helicase function and provide new targets for the development of antiviral drugs.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory disease agent in pigs that causes enormous economic losses to the global swine industry. PRRSV helicase nsp10 is a multifunctional protein with translocation and unwinding activities and plays a vital role in viral RNA synthesis. Here, we report the first structure of full-length nsp10 from the arterivirus PRRSV at 3.0-Å resolution. Our results show that the 1B domain of PRRSV nsp10 adopts a novel open state and has a unique C-terminal domain structure, which plays a crucial role in nsp10 helicase activity. Furthermore, mutagenesis and structural analysis revealed conservation of the helicase catalytic domain across the order Nidovirales (families Arteriviridae and Coronaviridae). Importantly, our results will provide a structural basis for further understanding the function of helicases in the order Nidovirales.

An identified PfHMGB1 promotes microcystin-LR-induced liver injury of yellow catfish (Pelteobagrus fulvidraco).

Microcystin-LR (MC-LR) is a potent hepatotoxin that can cause liver inflammation and injury. However, the mode of action of related inflammatory factors is not fully understood. PfHMGB1 is an inflammatory factor induced at the mRNA level in the liver of juvenile yellow catfish (Pelteobagrus fulvidraco) that were intraperitoneally injected with 50 µg/kg MC-LR. The PfHMGB1 mRNA level was highest in the liver and muscle among 11 tissues examined. The full-length cDNA sequence of PfHMGB1 was cloned and overexpressed in E. coli, and the purified protein rPfHMGB1 demonstrated DNA binding affinity. Endotoxin-free rPfHMGB1 (6-150 µg/mL) also showed dose-dependent hepatotoxicity and induced inflammatory gene expression of primary hepatocytes. PfHMGB1 antibody (anti-PfHMGB1) in vitro reduced MC-LR (30 and 50 µmol/L)-induced hepatotoxicity, suggesting PfHMGB1 is important in the toxic effects of MC-LR. In vivo study showed that MC-LR upregulated PfHMGB1 protein in the liver. The anti-PfHMGB1 blocked its counterpart and reduced ALT/AST activities after MC-LR exposure. Anti-PfHMGB1 partly neutralized MC-LR-induced hepatocyte disorganization, nucleus shrinkage, mitochondria, and rough endoplasmic reticula destruction. These findings suggest that PfHMGB1 promotes MC-LR-induced liver damage in the yellow catfish. HMGB1 may help protect catfish against widespread microcystin pollution.

A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence.

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.

Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine Deltacoronavirus functions as a dimer.

Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses.

Novel elastic response in twist-bend nematic models.

Bent-shaped liquid crystals have attracted significant attention recently due to their novel mesostructure and the intriguing behavior of their elastic constants, which are strongly anisotropic and have an unusual temperature dependence. Though theories explain the onset of the twist-bend nematic phase (NTB) through spontaneous symmetry breaking concomitant with transition to a negative bend (K3) elastic constant, this has not been observed as yet in experiments. There, the small bend elastic constant has a strongly non-monotonic temperature dependence, which first increases after crossing the isotropic (I)-nematic (N) transition, then dips near the nematic (N)-twist-bend (NTB) transition before it increases again as the transition is crossed. The molecular mechanisms responsible for this exotic behavior are unclear. Here, we utilize density of states algorithms in Monte Carlo simulation applied to a variant of the Lebwohl-Lasher model which includes bent-shaped-like interactions to analyze the mechanism behind elastic response in this novel mesostructure and understand the temperature dependence of its Frank-Oseen elastic constants.

Metal-Free Dehydrogenation of N-Heterocycles by Ternary h-BCN Nanosheets with Visible Light.

An efficient metal-free catalytic system has been developed based on hexagonal boron carbon nitride (h-BCN) nanosheets for the dehydrogenation of N-heterocycles with visible light; hydrogen gas is released in the process, and thus no proton acceptor is needed. This acceptorless dehydrogenation of hydroquinolines, hydroisoquinolines, and indolines to the corresponding aromatic N-heterocycles occurred in excellent yield under visible-light irradiation at ambient temperature. With h-BCN as the photocatalyst and water as the solvent, this environmentally benign protocol shows broad substitution tolerance and high efficiency.