Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.

Host range and structural analysis of bat-origin RshSTT182/200 coronavirus binding to human ACE2 and its animal orthologs.

Bat-origin RshSTT182 and RshSTT200 coronaviruses (CoV) from Rhinolophus shameli in Southeast Asia (Cambodia) share 92.6% whole-genome identity with SARS-CoV-2 and show identical receptor-binding domains (RBDs). In this study, we determined the structure of the RshSTT182/200 receptor binding domain (RBD) in complex with human angiotensin-converting enzyme 2 (hACE2) and identified the key residues that influence receptor binding. The binding of the RshSTT182/200 RBD to ACE2 orthologs from 39 animal species, including 18 bat species, was used to evaluate its host range. The RshSTT182/200 RBD broadly recognized 21 of 39 ACE2 orthologs, although its binding affinities for the orthologs were weaker than those of the RBD of SARS-CoV-2. Furthermore, RshSTT182 pseudovirus could utilize human, fox, and Rhinolophus affinis ACE2 receptors for cell entry. Moreover, we found that SARS-CoV-2 induces cross-neutralizing antibodies against RshSTT182 pseudovirus. Taken together, these findings indicate that RshSTT182/200 can potentially infect susceptible animals, but requires further evolution to obtain strong interspecies transmission abilities like SARS-CoV-2.

Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris.

Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.

Structural basis of white-tailed deer, Odocoileus virginianus, ACE2 recognizing all the SARS-CoV-2 variants of concern with high affinity.

SARS-CoV-2 has been expanding its host range, among which the white-tailed deer (WTD), Odocoileus virginianus, became the first wildlife species infected on a large scale and might serve as a host reservoir for variants of concern (VOCs) in case no longer circulating in humans. In this study, we comprehensively assessed the binding of the WTD angiotensin-converting enzyme 2 (ACE2) receptor to the spike (S) receptor-binding domains (RBDs) from the SARS-CoV-2 prototype (PT) strain and multiple variants. We found that WTD ACE2 could be broadly recognized by all of the tested RBDs. We further determined the complex structures of WTD ACE2 with PT, Omicron BA.1, and BA.4/5 S trimer. Detailed structural comparison revealed the important roles of RBD residues on 486, 498, and 501 sites for WTD ACE2 binding. This study deepens our understanding of the interspecies transmission mechanisms of SARS-CoV-2 and further addresses the importance of constant monitoring on SARS-CoV-2 infections in wild animals. IMPORTANCE Even if we manage to eliminate the virus among humans, it will still circulate among wildlife and continuously be transmitted back to humans. A recent study indicated that WTD may serve as reservoir for nearly extinct SARS-CoV-2 strains. Therefore, it is critical to evaluate the binding abilities of SARS-CoV-2 variants to the WTD ACE2 receptor and elucidate the molecular mechanisms of binding of the RBDs to assess the risk of spillback events.

Cross-species recognition of SARS-CoV-2 to bat ACE2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2-related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 from Rhinolophus macrotis (bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

Genome-Wide Association Study Identifies Rice Panicle Blast-Resistant Gene Pb4 Encoding a Wall-Associated Kinase.

Rice blast is one of the most devastating diseases, causing a significant reduction in global rice production. Developing and utilizing resistant varieties has proven to be the most efficient and cost-effective approach to control blasts. However, due to environmental pressure and intense pathogenic selection, resistance has rapidly broken down, and more durable resistance genes are being discovered. In this paper, a novel wall-associated kinase (WAK) gene, Pb4, which confers resistance to rice blast, was identified through a genome-wide association study (GWAS) utilizing 249 rice accessions. Pb4 comprises an N-terminal signal peptide, extracellular GUB domain, EGF domain, EGF-Ca2+ domain, and intracellular Ser/Thr protein kinase domain. The extracellular domain (GUB domain, EGF domain, and EGF-Ca2+ domain) of Pb4 can interact with the extracellular domain of CEBiP. Additionally, its expression is induced by chitin and polygalacturonic acid. Furthermore, transgenic plants overexpressing Pb4 enhance resistance to rice blast. In summary, this study identified a novel rice blast-resistant gene, Pb4, and provides a theoretical basis for understanding the role of WAKs in mediating rice resistance against rice blast disease.

Diphenyl ethers from endophytic fungus Rhexocercosporidium sp. Dzf14 and their antibacterial activity by affecting homeostasis of cell membranes.

BACKGROUND: Phytopathogenic bacteria cause severe losses to crops every year. The management of crop bacterial diseases with chemical agents has been considered as the main strategy. In order to cope with the bactericide resistance made by the pathogens, new antibacterials need to be continuously developed. RESULTS: A chemical investigation from the endophytic fungus Rhexocercosporidium sp. Dzf14 has led to the isolation of 12 diphenyl ethers including two new ones named rhexocerin E (1) and rhexocercosporin G (2), along with two new depsides named rhexocerdepsides A (3) and B (4). The structures and absolute configurations of the new compounds were determined through comprehensive analysis of spectroscopic data and quantum chemical ECD calculations. Diphenyl ethers showed obviously antibacterial activity on Gram-positive bacteria. The structure-activity relationship of diphenyl ethers revealed that prenylation was critical to the antibacterial activity. Among them, rhexocercosporin D (12) possessed the strongest activity against Clavibacter michiganensis and Bacillus subtilis, and was selected for further mechanistic studies. It was found that rhexocercosporin D displayed bactericidal activity by affecting homeostasis of cell membranes. In addition to its rapid bactericidal effects on Gram-positive bacteria, rhexocercosporin D could restore the susceptibility against Gram-negative Agrobacterium tumefaciens by synergistic action with colistin. CONCLUSION: Twelve diphenyl ethers and two depsides were isolated from endophytic fungus Rhexocercosporidium sp. Dzf14. Isopentenyl was critical for diphenyl ethers against Gram-positive bacteria. Rhexocercosporin D could affect homeostasis of bacterial cell membrane to exert rapid bactericidal activity. These findings highlight the antibacterial potential of the diphenyl ethers in crop bacterial disease management. © 2024 Society of Chemical Industry.

Rosellichalasins A-H, cytotoxic cytochalasans from the endophytic fungus Rosellinia sp. Glinf021.

Eight new cytochalasans rosellichalasins A-H (1-8), as well as two new shunt metabolites rosellinins A (9) and B (10) before intramolecular Diels-Alder cycloaddition reaction in cytochalasan biosynthesis, along with nine known cytochalsans (11-19) were isolated from the endophytic fungus Rosellinia sp. Glinf021, which was derived from the medicinal plant Glycyrrhiza inflata. Their structures were characterized by extensive analysis of 1D and 2D NMR as well as HRESIMS spectra and quantum chemical ECD calculations. The cytotoxic activities of these compounds were evaluated against four human cancer cell lines including HCT116, MDA-MB-231, BGC823, and PANC-1 with IC50 values ranging from 0.5 to 58.2 µM.

Phytotoxic Isocassadiene-Type Diterpenoids from Tomato Fusarium Crown and Root Rot Pathogen Fusarium oxysporum f. sp. radicis-lycopersici.

Fusarium crown and root rot (FCRR) has emerged as a highly destructive soil-borne disease, posing a significant threat to the safe cultivation of tomatoes in recent years. The pathogen of tomato FCRR is Fusarium oxysporum f. sp. radicis-lycopersici (Forl). To explore potential phytotoxins from Forl, eight undescribed diterpenoids namely fusariumic acids A-H (1-8) were isolated. Their structures were elucidated by using spectroscopic data analyses, quantum chemical calculations, and X-ray crystallography. Fusariumic acids A (1) and C-H (3-8) were typical isocassadiene-type diterpenoids, while fusariumic acid B (2) contained a cage-like structure with an unusual 7,8-seco-isocassadiene skeleton. A biosynthetic pathway of 2 was proposed. Fusariumic acids A (1) and C-H (3-8) were further assessed for their phytotoxic effects on tomato seedlings at 200 µg/mL. Among them, fusariumic acid F (6) exhibited the strongest inhibition against the hypocotyl and root elongation of tomato seedlings, with inhibitory rates of 61.3 and 45.3%, respectively.

Rosellosides A and B, two phenyloxazole glycosides from Glycyrrhiza inflata-derived fungus Rosellinia sp. Glinf021.

Two new chlorinated phenyloxazole glycosides, named rosellosides A (1) and B (2), were isolated from the endophytic fungus Rosellinia sp. Glinf021, which was derived from the medicinal plant Glycyrrhiza inflata (Leguminosae). Both compounds were rare chlorinated polyketide glycosides bearing an oxazole moiety. Their structures were elucidated by analysis of the NMR and HRESIMS data, and their absolute configurations were determined by quantum chemical ECD calculations and X-ray crystallography.

Activation of Ustilaginoidin Biosynthesis Gene uvpks1 in Villosiclava virens Albino Strain LN02 Influences Development, Stress Responses, and Inhibition of Rice Seed Germination.

Villosiclava virens (anamorph: Ustilaginoidea virens) is the pathogen of rice false smut (RFS), which is a destructive rice fungal disease. The albino strain LN02 is a natural white-phenotype mutant of V. virens due to its incapability to produce toxic ustilaginoidins. In this study, three strains including the normal strain P1, albino strain LN02, and complemented strain uvpks1C-1 of the LN02 strain were employed to investigate the activation of the ustilaginoidin biosynthesis gene uvpks1 in the albino strain LN02 to influence sporulation, conidia germination, pigment production, stress responses, and the inhibition of rice seed germination. The activation of the ustilaginoidin biosynthesis gene uvpks1 increased fungal tolerances to NaCl-induced osmotic stress, Congo-red-induced cell wall stress, SDS-induced cell membrane stress, and H2O2-induced oxidative stress. The activation of uvpks1 also increased sporulation, conidia germination, pigment production, and the inhibition of rice seed germination. In addition, the activation of uvpks1 was able to increase the mycelial growth of the V. virens albino strain LN02 at 23 °C and a pH from 5.5 to 7.5. The findings help in understanding the effects of the activation of uvpks1 in albino strain LN02 on development, pigment production, stress responses, and the inhibition of rice seed germination by controlling ustilaginoidin biosynthesis.

Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the recent pandemic COVID-19, is reported to have originated from bats, with its intermediate host unknown to date. Here, we screened 26 animal counterparts of the human ACE2 (hACE2), the receptor for SARS-CoV-2 and SARS-CoV, and found that the ACE2s from various species, including pets, domestic animals and multiple wild animals, could bind to SARS-CoV-2 receptor binding domain (RBD) and facilitate the transduction of SARS-CoV-2 pseudovirus. Comparing to SARS-CoV-2, SARS-CoV seems to have a slightly wider range in choosing its receptor. We further resolved the cryo-electron microscopy (cryo-EM) structure of the cat ACE2 (cACE2) in complex with the SARS-CoV-2 RBD at a resolution of 3 Å, revealing similar binding mode as hACE2 to the SARS-CoV-2 RBD. These results shed light on pursuing the intermediate host of SARS-CoV-2 and highlight the necessity of monitoring susceptible hosts to prevent further outbreaks.

Enhanced effect of folated pluronic F87-PLA/TPGS mixed micelles on targeted delivery of paclitaxel.

Targeted drug delivery systems have great potential to overcome the side effect and improve the bioavailability of conventional anticancer drugs. In order to further improve the antitumor efficacy of paclitaxel (PTX) loaded in folated Pluronic F87/poly(lactic acid) (FA-F87-PLA) micelles, D-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS or Vitamin E TPGS) were added into FA-F87-PLA to form FA-F87-PLA/TPGS mixed micelles. The LE of PTX-loaded mixed micelles (13.5%) was highest in the mass ratio 5 to 3 of FA-F87-PLA to TPGS. The in vitro cytotoxicity assays indicated that the IC50 values for free PTX injections, PTX-loaded FA-F87-PLA micelles and PTX-loaded FA-F87-PLA/TPGS mixed micelles after 72h of incubation were 1.52, 0.42 and 0.037mg/L, respectively. The quantitative cellular uptake of coumarin 6-loaded FA-F87-PLA/TPGS and FA-F87-PLA micelles showed that the cellular uptake efficiency of mixed micelles was higher for 2 and 4h incubation, respectively. In vivo pharmacokinetic studies found that the AUC of PTX-loaded FA-F87-PLA/TPGS mixed micelles is almost 1.4 times of that of PTX-loaded FA-F87-PLA micelles. The decreased particle size and inhibition of P-glycoprotein effect induced by the addition of TPGS could result in enhancing the cellular uptake and improving the antitumor efficiency of PTX-loaded FA-F87-PLA/TPGS mixed micelles.