SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

Infection with coronavirus rearranges the host cell membrane to assemble a replication/transcription complex in which replication of the viral genome and transcription of viral mRNA occur. Although coexistence of nsp3 and nsp4 is known to cause membrane rearrangement, the mechanisms underlying the interaction of these two proteins remain unclear. We demonstrated that binding of nsp4 with nsp3 is essential for membrane rearrangement and identified amino acid residues in nsp4 responsible for the interaction with nsp3. In addition, we revealed that the nsp3-nsp4 interaction is not sufficient to induce membrane rearrangement, suggesting the participation of other factors such as host proteins. Finally, we showed that loss of the nsp3-nsp4 interaction eliminated viral replication by using an infectious cDNA clone and replicon system of SARS-CoV. These findings provide clues to the mechanism of the replication/transcription complex assembly of SARS-CoV and could reveal an antiviral target for the treatment of betacoronavirus infection.

54 55 Abbreviations 56 2D two-dimensional 57 ACE2 angiotensin-converting enzyme 2 58 CC10 club cell protein 10 59 FGF fibroblast growth factor 60 hBEpC human bronchial epithelial cells 61 hBO human bronchial organoids 62 IFN-I type I interferon 63 IHC immunohistochemistry 64 KRT5 keratin 5 65 LDH lactate dehydrogenase 66 PSC pluripotent stem cell 67 RdRp RNA-dependent RNA polymerase 68 RNA seq RNA sequencing 69 SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 70 TMPRSS2 transmembrane serine proteinase 2 Abstract 74 Coronavirus disease 2019 (COVID-19) is a disease that causes fatal disorders 75 including severe pneumonia. To develop a therapeutic drug for COVID-19, a model that 76 can reproduce the viral life cycle and evaluate the drug efficacy of anti-viral drugs is 77 essential. In this study, we established a method to generate human bronchial organoids 78 (hBO) from commercially available cryopreserved human bronchial epithelial cells and 79 examined whether they could be used as a model for severe acute respiratory syndrome 80 coronavirus 2 (SARS-CoV-2) research. Our hBO contain basal, club, ciliated, and 81 goblet cells. Angiotensin-converting enzyme 2 (ACE2), which is a receptor for 82 SARS-CoV-2, and transmembrane serine proteinase 2 (TMPRSS2), which is an 83 essential serine protease for priming spike (S) protein of SARS-CoV-2, were highly 84 expressed. After SARS-CoV-2 infection, not only the intracellular viral genome, but 85 also progeny virus, cytotoxicity, pyknotic cells, and moderate increases of the type I 86 interferon signal could be observed. Treatment with camostat, an inhibitor of TMPRSS2, 87 reduced the viral copy number to 2% of the control group. Furthermore, the gene 88 expression profile in SARS-CoV-2-infected hBO was obtained by performing RNA-seq 89 analysis. In conclusion, we succeeded in generating hBO that can be used for 90 SARS-CoV-2 research and COVID-19 drug discovery. 91 92 93 5 Graphical abstract 94 95 96 Key words 97 SARS-CoV-2, bronchial organoids, COVID-19, camostat 98 99 6 100 101 The "2019-new coronavirus disease (COVID-19) was first reported in China in 102 December 2019 1 and declared a pandemic by the WHO in March 2020 2 . Severe 103 pneumonia is most frequently observed in COVID-19 patients, and the number of 104 COVID-19 patients and deaths are still increasing. These conditions have made it 105 difficult for research on severe acute respiratory syndrome coronavirus 2 106 (SARS-CoV-2), which is the causative virus of COVID-19, to keep pace. SARS-CoV-2 107 is composed of four proteins: S (spike), E (envelope), M (membrane), and N 108 (nucleocapsid) proteins. It is known that angiotensin-converting enzyme 2 (ACE2) is a 109 SARS-CoV-2 receptor, and transmembrane serine proteinase 2 (TMPRSS2) is essential 110 for priming S protein 3 . Thus, to accelerate SARS-CoV-2 research, a novel lung model 111 that reproduces the viral life cycle with intact expression of these host factors is 112 indispensable. 113 A number of animal and cell models that can be used for SA...

Inhibition of programmed death 1 (PD-1), expressed on activated T cells, can break through immune resistance and elicit durable responses in human melanoma as well as other types of cancers. Canine oral malignant melanoma is one of the most aggressive tumors bearing poor prognosis due to its high metastatic potency. However, there are few effective treatments for the advanced stages of melanoma in veterinary medicine. Only one previous study indicated the potential of the immune checkpoint inhibitor, anti-canine PD-L1 therapeutic antibody in dogs, and no anti-canine PD-1 therapeutic antibodies are currently available. Here, we developed two therapeutic antibodies, rat-dog chimeric and caninized anti-canine PD-1 monoclonal antibodies and evaluated in vitro functionality for these antibodies. Moreover, we conducted a pilot study to determine their safety profiles and clinical efficacy in spontaneously occurring canine cancers. In conclusion, the anti-canine PD-1 monoclonal antibody was relatively safe and effective in dogs with advanced oral malignant melanoma and other cancers. Thus, our study suggests that PD-1 blockade may be an attractive treatment option in canine cancers.

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide. Chemotherapies against gastric cancer often fail, with cancer recurrence due potentially to the persistence of cancer stem cells. This unique subpopulation of cells in tumors possesses the ability to self-renew and dedifferentiate. These cancer stem cells are critical for initiation, maintenance, metastasis, and relapse of cancers; however, the molecular mechanisms supporting cancer stemness remain largely unknown. Increased kinase and decreased phosphatase activity are hallmarks of oncogenic signaling. Protein phosphatase 2A (PP2A) functions as a tumor-suppressor enzyme, and elevated levels of SET/I2PP2A, an endogenous PP2A protein inhibitor, are correlated with poor prognosis of several human cancers. Here, it was determined that SET expression was elevated in tumor tissue in a gastric cancer mouse model system, and SET expression was positively correlated with poor survival of human gastric cancer patients. Mechanistically, SET knockdown decreased E2F1 levels and suppressed the stemness of cancer cell lines. Immunoprecipitations show SET associated with the PP2A-B56 complex, and the B56 subunit interacted with the E2F1 transcription factor. Treatment of gastric cancer cells with the SET-targeting drug OP449 increased PP2A activity, decreased E2F1 protein levels, and suppressed stemness of cancer cells. These data indicate that a SET/PP2A/E2F1 axis regulates cancer cell stemness and is a potential target for gastric cancer therapy. This study highlights the oncogenic role of SET/I2PP2A in gastric cancer and suggests that SET maintains cancer cell stemness by suppressing PP2A activity and stabilizing E2F1. .

Fusogenic reoviruses encode fusion-associated small transmembrane (FAST) protein, which induces cell–cell fusion. FAST protein is the only known fusogenic protein in non-enveloped viruses, and its role in virus replication is not yet known. We generated replication-competent, FAST protein-deficient pteropine orthoreovirus and demonstrated that FAST protein was not essential for viral replication, but enhanced viral replication in the early phase of infection. Addition of recombinant FAST protein enhanced replication of FAST-deficient virus and other non-fusogenic viruses in a fusion-dependent and FAST-species-independent manner. In a mouse model, replication and pathogenicity of FAST-deficient virus were severely impaired relative to wild-type virus, indicating that FAST protein is a major determinant of the high pathogenicity of fusogenic reovirus. FAST-deficient virus also conferred effective protection against challenge with lethal homologous virus strains in mice. Our results demonstrate a novel role of a viral fusogenic protein and the existence of a cell–cell fusion-dependent replication system in non-enveloped viruses.