SARS Unique Domain (SUD) of Severe Acute Respiratory Syndrome Coronavirus Induces NLRP3 Inflammasome-Dependent CXCL10-Mediated Pulmonary Inflammation.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) initiates the cytokine/chemokine storm-mediated lung injury. The SARS-CoV unique domain (SUD) with three macrodomains (N, M, and C), showing the G-quadruplex binding activity, was examined the possible role in SARS pathogenesis in this study. The chemokine profile analysis indicated that SARS-CoV SUD significantly up-regulated the expression of CXCL10, CCL5 and interleukin (IL)-1ß in human lung epithelial cells and in the lung tissues of the mice intratracheally instilled with the recombinant plasmids. Among the SUD subdomains, SUD-MC substantially activated AP-1-mediated CXCL10 expression in vitro. In the wild type mice, SARS-CoV SUD-MC triggered the pulmonary infiltration of macrophages and monocytes, inducing CXCL10-mediated inflammatory responses and severe diffuse alveolar damage symptoms. Moreover, SUD-MC actuated NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome-dependent pulmonary inflammation, as confirmed by the NLRP3 inflammasome inhibitor and the NLRP3-/- mouse model. This study demonstrated that SARS-CoV SUD modulated NLRP3 inflammasome-dependent CXCL10-mediated pulmonary inflammation, providing the potential therapeutic targets for developing the antiviral agents.

Functional assessments of SARS-CoV-2 single-round infectious particles with variant-specific spike proteins on infectivity, drug sensitivity, and antibody neutralization.

Working with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is restricted to biosafety level III (BSL-3) laboratory. The study used a trans-complementation system consisting of virus-like particles (VLPs) and DNA-launched replicons to generate SARS-CoV-2 single-round infectious particles (SRIPs) with variant-specific spike (S) proteins. S gene of Wuhan-Hu-1 strain (SWH1) or Omicron BA.1 variant (SBA.1), along with the envelope (E) and membrane (M) genes, were cloned into a tricistronic vector, co-expressed in the cells to produce variant-specific S-VLPs. Additionally, the replicon of the WH1-like strain without S, E, M and accessory genes, was engineered under the control by a CMV promoter to produce self-replicating RNAs within VLP-producing cells, led to create SWH1- and SBA.1-based SARS-CoV-2 SRIPs. The SBA.1-based SRIP showed lower virus yield, replication, N protein expression, fusogenicity, and infectivity compared to SWH1-based SRIPs. SBA.1-based SRIP also exhibited intermediate resistance to neutralizing antibodies produced by SWH1-based vaccines, but were effective at infecting cells with low ACE2 expression. Importantly, both S-based SRIPs responded similarly to remdesivir and GC376, with EC50 values ranging from 0.17 to 1.46 µM, respectively. The study demonstrated that this trans-complementation system is a reliable and efficient tool for generating SARS-CoV-2 SRIPs with variant-specific S proteins. SARS-CoV-2 SRIPs, mimicking authentic live viruses, facilitate comprehensive analysis of variant-specific virological characteristics, including antibody neutralization, and drug sensitivity in non-BSL-3 laboratories.

Development of Fluorescence-Tagged SARS-CoV-2 Virus-like Particles by a Tri-Cistronic Vector Expression System for Investigating the Cellular Entry of SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has caused the pandemic that began late December 2019. The co-expression of SARS-CoV-2 structural proteins in cells could assemble into several types of virus-like particles (VLPs) without a viral RNA genome. VLPs containing S proteins with the structural and functional properties of authentic virions are safe materials to exploit for virus-cell entry and vaccine development. In this study, to generate SARS-CoV-2 VLPs (SCoV2-SEM VLPs) composed of three structural proteins including spike (S), envelop (E) protein and membrane (M) protein, a tri-cistronic vector expression system was established in a cell line co-expressing SARS-CoV-2 S, E and M proteins. The SCoV2-SEM VLPs were harvested from the cultured medium, and three structure proteins were confirmed by Western blot assay. A negative-stain TEM assay demonstrated the size of the SCoV2-SEM VLPs with a diameter of about 90 nm. To further characterize the infectious properties of SCoV2-SEM VLPs, the VLPs (atto647N-SCoV2-SEM VLPs) were fluorescence-labeled by conjugation with atto-647N and visualized under confocal microscopy at a single-particle resolution. The results of the infection assay revealed that atto647N-SCoV2-SEM VLPs attached to the surface of the HEK293T cells at the pre-binding phase in a ACE2-dependent manner. At the post-infection phase, atto647N-SCoV2-SEM VLPs either fused with the cellular membrane or internalized into the cytoplasm with mCherry-rab5-positive early endosomes. Moreover, fusion with the cellular membrane and the internalization with early endosomes could be inhibited by the treatment of camostat (a pharmacological inhibitor of TMPRSS2) and chlorpromazine (an endocytosis inhibitor), respectively. These results elucidated that SCoV2-SEM VLPs behave similarly to the authentic live SARS-CoV-2 virus, suggesting that the development of SCoV2-SEM VLPs provide a realistic and safe experimental model for studying the infectious mechanism of SARS-CoV-2.

Antiviral activity of glycyrrhizic acid conjugates with amino acid esters against Zika virus.

Zika virus (ZIKV) is a new pathogenic flavivirus transmitted by mosquitoes Aedes spp. ZIKV infection is accompanied by serious neurological complications and is especially dangerous for pregnant women, in which it can lead to congenital malformations of the fetus and microcephaly in neonates. Currently, there are no licensed vaccines or specific post-infectious therapies for ZIKV infection. This report is devoted to the study of glycyrrhizic acid (GL) derivatives as ZIKV inhibitors. The inhibitory assays on the cytopathic effect (CPE) and viral infectivity of ZIKV in three different human cell lines revealed that the conjugation of GL with amino acids and their esters (methyl, ethyl) is influenced by the antiviral activity of the compounds. GL conjugates with Glu(OMe)-OMe 11, Glu(OH)-OMe 12, Asp(OMe)-OMe 13, TyrOMe 14, LeuOEt 15, and PheOEt 16 with free COOH groups in the triterpene moiety were active against ZIKV. The most active compounds 13 and 14 have IC50 values of 0.23 µM and 0.09 µM against low doses (MOI = 0.05) of ZIKV strain PRVABC59, 1.20 µM and 0.74 µM against high doses (MOI = 10) of ZIKV strain Natal RGN single-round infectious particles, respectively. The lead compound was 14 with a high selectivity index (SI < 500). Compound 13 showed a higher inhibitory effect on the early stage (entry) of ZIKV replication than compound 14, and was less potent than compound 14 at the post-entry stage, consistent with the docking models. Compounds 13 and 14 also had a strong interaction with the active site pocket of NS5 MTase. Compounds 13 and 14 are recommended for expanded antiviral studies against ZIKV infection.

Antiviral Action of Tryptanthrin Isolated from Strobilanthes cusia Leaf against Human Coronavirus NL63.

Strobilanthes cusia (Nees) Kuntze is a Chinese herbal medicine used in the treatment of respiratory virus infections. The methanol extract of S. cusia leaf contains chemical components such as ß-sitosterol, indirubin, tryptanthrin, betulin, indigodole A, and indigodole B that have diverse biological activities. However, the antiviral action of S. cusia leaf and its components against human coronavirus remains to be elucidated. Human coronavirus NL63 infection is frequent among immunocompromised individuals, young children, and in the elderly. This study investigated the anti-Human coronavirus NL63 (HCoV-NL63) activity of the methanol extract of S. cusia leaf and its major components. The methanol extract of S. cusia leaf effectively inhibited the cytopathic effect (CPE) and virus yield (IC50 = 0.64 µg/mL) in HCoV-NL63-infected cells. Moreover, this extract potently inhibited the HCoV-NL63 infection in a concentration-dependent manner. Among the six components identified in the methanol extract of S. cusia leaf, tryptanthrin and indigodole B (5aR-ethyltryptanthrin) exhibited potent antiviral activity in reducing the CPE and progeny virus production. The IC50 values against virus yield were 1.52 µM and 2.60 µM for tryptanthrin and indigodole B, respectively. Different modes of time-of-addition/removal assay indicated that tryptanthrin prevented the early and late stages of HCoV-NL63 replication, particularly by blocking viral RNA genome synthesis and papain-like protease 2 activity. Notably, tryptanthrin (IC50 = 0.06 µM) and indigodole B (IC50 = 2.09 µM) exhibited strong virucidal activity as well. This study identified tryptanthrin as the key active component of S. cusia leaf methanol extract that acted against HCoV-NL63 in a cell-type independent manner. The results specify that tryptanthrin possesses antiviral potential against HCoV-NL63 infection.

The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles.

Zika virus (ZIKV) is transmitted by Aedes mosquitoes and exhibits genetic variation with African and Asian lineages. ZIKV Natal RGN strain, an Asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. However, ZIKV Natal RGN strain has not been isolated; its biological features are not yet illustrated. This study rescued and characterized recombinant, single-round infectious particles (SRIPs) of the ZIKV Natal RGN strain using reverse genetic and synthetic biology techniques. The DNA-launched replicon of ZIKV Natal RGN was constructed and contains the EGFP reporter, lacks prM-E genes, and replicates under CMV promoter control. The peak in the ZIKV Natal RGN SRIP titer reached 6.25 × 106 TCID50/mL in the supernatant of prM-E-expressing packaging cells 72 h post-transfection with a ZIKV Natal RGN replicon. The infectivity of ZIKV Natal RGN SRIPs has been demonstrated to correlate with the green florescence intensity of the EGFP reporter, the SRIP-induced cytopathic effect, and ZIKV's non-structural protein expression. Moreover, ZIKV Natal RGN SRIPs effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. Therefore, the recombinant ZIKV Natal RGN strain was rescued as SRIPs that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates.