Characterization of a pangolin SARS-CoV-2-related virus isolate that uses the human ACE2 receptor.

Various SARS-CoV-2-related coronaviruses have been increasingly identified in pangolins, showing a potential threat to humans. Here we report the infectivity and pathogenicity of the SARS-CoV-2-related virus, PCoV-GX/P2V, which was isolated from a Malayan pangolin (Manis javanica). PCoV-GX/P2V could grow in human hepatoma, colorectal adenocarcinoma cells, and human primary nasal epithelial cells. It replicated more efficiently in cells expressing human angiotensin-converting enzyme 2 (hACE2) as SARS-CoV-2 did. After intranasal inoculation to the hACE2-transgenic mice, PCoV-GX/P2V not only replicated in nasal turbinate and lungs, but also caused interstitial pneumonia, characterized by infiltration of mixed inflammatory cells and multifocal alveolar hemorrhage. Existing population immunity established by SARS-CoV-2 infection and vaccination may not protect people from PCoV-GX/P2V infection. These findings further verify the hACE2 utility of PCoV-GX/P2V by in vivo experiments using authentic viruses and highlight the importance for intensive surveillance to prevent possible cross-species transmission.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Disulfiram ameliorates STING/MITA-dependent inflammation and autoimmunity by targeting RNF115.

STING (also known as MITA) is an adaptor protein that mediates cytoplasmic DNA-triggered signaling, and aberrant activation of STING/MITA by cytosolic self-DNA or gain-of-function mutations causes severe inflammation. Here, we show that STING-mediated inflammation and autoimmunity are promoted by RNF115 and alleviated by the RNF115 inhibitor disulfiram (DSF). Knockout of RNF115 or treatment with DSF significantly inhibit systemic inflammation and autoimmune lethality and restore immune cell development in Trex1-/- mice and STINGN153S/WT bone marrow chimeric mice. In addition, knockdown or pharmacological inhibition of RNF115 substantially downregulate the expression of IFN-α, IFN-γ and proinflammatory cytokines in PBMCs from patients with systemic lupus erythematosus (SLE) who exhibit high concentrations of dsDNA in peripheral blood. Mechanistically, knockout or inhibition of RNF115 impair the oligomerization and Golgi localization of STING in various types of cells transfected with cGAMP and in organs and cells from Trex1-/- mice. Interestingly, knockout of RNF115 inhibits the activation and Golgi localization of STINGN153S as well as the expression of proinflammatory cytokines in myeloid cells but not in endothelial cells or fibroblasts. Taken together, these findings highlight the RNF115-mediated cell type-specific regulation of STING and STINGN153S and provide potential targeted intervention strategies for STING-related autoimmune diseases.

To Investigate the Potential Mechanism of Huanglian Jiangtang Formula Lowering Blood Sugar in View of Network Pharmacology and Molecular Docking Technology.

Objective: In view of network pharmacology and molecular docking technology, to explore the targets as well as effect mechanism of the Huanglian Jiangtang formula (including Coptis chinensis, Anemarrhena asphodeloides, rhubarb wine, Cortex Moutan, Rehmannia glutinosa, and dried ginger) in the type II diabetes therapy. Methods: TCMSP and Batman database (DB) were used to retrieve the chemical components and action targets of drugs; GeneCards, OMIM, TTD, DrugBank, and other databases were applied to screen the disease targets. We used the UniProt DB to annotate the targets before building the drug-compound-target network with Cytoscape 3.9.1. We also exploited the String DB to construct the protein-protein interaction (PPI) network. In addition, the targets for the treatment of type II diabetes were searched in the DrugBank, OMIM, GeneCards, and TTD database; then, we utilized Venn to intersect the key targets for the therapy of type II diabetes and active ingredient targets to obtain common targets. Furthermore, we exploited the common targets using GO and KEGG enrichment analysis method. The common targets and core components were analyzed by molecular docking using the AutoDock software. Results: A total of 61 effective components of this compound were screened out; drugs and type II diabetes have 278 common targets; the PPI network screened core target proteins such as CDKN1A, CDK2, and E2F1 with the help of molecular docking technology; the three main compounds including quercetin, kaempferol, and gamma-aminobutyric acid were obtained. Besides, the key target proteins had excellent binding properties with the main components. The signal pathways of six compound interventions in type II diabetes were mostly related to cancer, cocaine addiction, aminoacyl-tRNA biosynthesis, glycine, serine, threonine metabolism, platinum drug resistance, and other pathways, according to the KEGG enrichment analysis method. Conclusion: In the treatment of diabetes, the Huanglian Jiangtang formula has sorts of properties especially in the aspects of composition, target, and pathway. Its molecular target and mechanism of action may be related to pathways in cancer, cocaine addiction, aminoacyl-tRNA biosynthesis, glycine, serine, threonine metabolism, platinum drug resistance, and other pathways. This conclusion can provide theoretical support and science for further research.

The E3 ubiquitin ligase ARIH1 promotes antiviral immunity and autoimmunity by inducing mono-ISGylation and oligomerization of cGAS.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) plays a critical role in antiviral immunity and autoimmunity. The activity and stability of cGAS are fine-tuned by post-translational modifications. Here, we show that ariadne RBR E3 ubiquitin protein ligase 1 (ARIH1) catalyzes the mono-ISGylation and induces the oligomerization of cGAS, thereby promoting antiviral immunity and autoimmunity. Knockdown or knockout of ARIH1 significantly inhibits herpes simplex virus 1 (HSV-1)- or cytoplasmic DNA-induced expression of type I interferons (IFNs) and proinflammatory cytokines. Consistently, tamoxifen-treated ER-Cre;Arih1fl/fl mice and Lyz2-Cre; Arih1fl/fl mice are hypersensitive to HSV-1 infection compared with the controls. In addition, deletion of ARIH1 in myeloid cells alleviates the autoimmune phenotypes and completely rescues the autoimmune lethality caused by TREX1 deficiency. Mechanistically, HSV-1- or cytosolic DNA-induced oligomerization and activation of cGAS are potentiated by ISGylation at its K187 residue, which is catalyzed by ARIH1. Our findings thus reveal an important role of ARIH1 in innate antiviral and autoimmune responses and provide insight into the post-translational regulation of cGAS.

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates.

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.