Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection.

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.

Functional studies of HLA and its role in SARS-CoV-2: Stimulating T cell response and vaccine development.

In the biological immune process, the major histocompatibility complex (MHC) plays an indispensable role in the expression of HLA molecules in the human body when viral infection activates the T-cell response to remove the virus. Since the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2019, how to address and prevent SARS-CoV-2 has become a common problem facing all mankind. The T-cell immune response activated by MHC peptides is a way to construct a defense line and reduce the transmission and harm of the virus. Presentation of SARS-CoV-2 antigen is associated with different types of HLA phenotypes, and different HLA phenotypes induce different immune responses. The prediction of SARS-CoV-2 mutation information and the design of vaccines based on HLAs can effectively activate autoimmunity and cope with virus mutations, which can provide some references for the prevention and treatment of SARS-CoV-2.

Nucleocapsid mutations R203K/G204R increase the infectivity, fitness, and virulence of SARS-CoV-2.

Previous work found that the co-occurring mutations R203K/G204R on the SARS-CoV-2 nucleocapsid (N) protein are increasing in frequency among emerging variants of concern or interest. Through a combination of in silico analyses, this study demonstrates that R203K/G204R are adaptive, while large-scale phylogenetic analyses indicate that R203K/G204R associate with the emergence of the high-transmissibility SARS-CoV-2 lineage B.1.1.7. Competition experiments suggest that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly. Moreover, the 203K/204R virus shows increased infectivity in human lung cells and hamsters. Accordingly, we observe a positive association between increased COVID-19 severity and sample frequency of 203K/204R. Our work suggests that the 203K/204R mutations contribute to the increased transmission and virulence of select SARS-CoV-2 variants. In addition to mutations in the spike protein, mutations in the nucleocapsid protein are important for viral spreading during the pandemic.

Structure-Based Discovery of Novel Nonpeptide Inhibitors Targeting SARS-CoV-2 Mpro.

The continual spread of novel coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), posing a severe threat to the health worldwide. The main protease (Mpro, alias 3CLpro) of SARS-CoV-2 is a crucial enzyme for the maturation of viral particles and is a very attractive target for designing drugs to treat COVID-19. Here, we propose a multiple conformation-based virtual screening strategy to discover inhibitors that can target SARS-CoV-2 Mpro. Based on this strategy, nine Mpro structures and a protein mimetics library with 8960 commercially available compounds were prepared to carry out ensemble docking for the first time. Five of the nine structures are apo forms presented in different conformations, whereas the other four structures are holo forms complexed with different ligands. The surface plasmon resonance assay revealed that 6 out of 49 compounds had the ability to bind to SARS-CoV-2 Mpro. The fluorescence resonance energy transfer experiment showed that the biochemical half-maximal inhibitory concentration (IC50) values of the six compounds could hamper Mpro activities ranged from 0.69 ± 0.05 to 2.05 ± 0.92 µM. Evaluation of antiviral activity using the cell-based assay indicated that two compounds (Z1244904919 and Z1759961356) could strongly inhibit the cytopathic effect and reduce replication of the living virus in Vero E6 cells with the half-maximal effective concentrations (EC50) of 4.98 ± 1.83 and 8.52 ± 0.92 µM, respectively. The mechanism of the action for the two inhibitors were further elucidated at the molecular level by molecular dynamics simulation and subsequent binding free energy analysis. As a result, the discovered noncovalent reversible inhibitors with novel scaffolds are promising antiviral drug candidates, which may be used to develop the treatment of COVID-19.

ORF8 contributes to cytokine storm during SARS-CoV-2 infection by activating IL-17 pathway.

Recently, COVID-19 caused by the novel coronavirus SARS-CoV-2 has brought great challenges to the world. More and more studies have shown that patients with severe COVID-19 may suffer from cytokine storm syndrome; however, there are few studies on its pathogenesis. Here we demonstrated that SARS-CoV-2 coding protein open reading frame 8 (ORF8) acted as a contributing factor to cytokine storm during COVID-19 infection. ORF8 could activate IL-17 signaling pathway and promote the expression of pro-inflammatory factors. Moreover, we demonstrated that treatment of IL17RA antibody protected mice from ORF8-induced inflammation. Our findings are helpful to understand the pathogenesis of cytokine storm caused by SARS-CoV-2 and provide a potential target for the development of COVID-19 therapeutic drugs.

Network pharmacological analysis and mechanism prediction of Xingnaojing Injection in treatment of neurological damage caused by SARS-CoV-2

Objective: To explore the active compounds, targets and signaling pathways of Xingnaojing Injection (XNJI) for the treatment of neurological damage caused by SARS-CoV-2, so as to explore its mechanism. Methods: Using TCMSP, BATMAN, Swiss Target Prediction, and other databases, the chemical compounds and targets of XNJI were retrieved. Cytoscape software was used to construct XNJI efficacy network of "drug-compounds-targets" for coronavirus and neuroprotection, and the action mechanism was predicted by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Then core compounds were verified by molecular docking with 3CL Mpro, ACE2, and 2019-nCoV RBD/ACE2-B0AT1 complex. Results: A total of 105 active compounds of XNJI, 928 drug targets, 741 targets of coronavirus, 611 targets of neuroprotection, 83 drug-disease common targets, 12 core compounds, and seven key targets were obtained. The function enrichment analysis of GO yielded 204 entries, KEGG pathway enrichment screened 120 signaling pathways, which included Hepatitis B, pathways in cancer, TNF, HIF-1, and VEGF signaling pathway, and so on. The results of molecular docking showed that core compounds of XNJI had a good bonding activity with 3CL Mpro, ACE2 and complex. The chlorogenin and kaempferol had the lowest binding energy with three proteins and might play an important role in treatment. Conclusion: The core compounds in XNJI including chlorogenin, kaempferol, 5-hydroxy- 6,7,3',4',5'-pentamethoxyflavone, 3-methylkempferol, morin, gardenin, quercetin, artemisetin, genistein, dryobalanone, curcumin, and elemicin, which might interfere with various signaling pathways by acting on key targets like PARP1, PTGS2, MMP9, CDK2, ADORA2A, ALOX5, GSK3B, and regulate the inflammatory response, apoptosis, oxidative stress, angiogenesis, and other processes to improve the neurological damage caused by SARS-CoV-2, and inhibit virus replication and prevent infection of the host cell by binding with 3CL Mpro, ACE2 and complex, which suggest that XNJI may have a positive therapeutic effect on the neurological damage caused by SARS-CoV-2.