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1.
Nature Microbiology ; 8(1):121-134, 2023.
Article in English | MEDLINE | ID: covidwho-2185891

ABSTRACT

The coronavirus SARS-CoV-2 causes the severe disease COVID-19. SARS-CoV-2 infection is initiated by interaction of the viral spike protein and host receptor angiotensin-converting enzyme 2 (ACE2). We report an improved bright and reversible fluorogenic reporter, named SURF (split UnaG-based reversible and fluorogenic protein-protein interaction reporter), that we apply to monitor real-time interactions between spike and ACE2 in living cells. SURF has a large dynamic range with a dark-to-bright fluorescence signal that requires no exogenous cofactors. Utilizing this reporter, we carried out a high-throughput screening of small-molecule libraries. We identified three natural compounds that block replication of SARS-CoV-2 in both Vero cells and human primary nasal and bronchial epithelial cells. Cell biological and biochemical experiments validated all three compounds and showed that they block the early stages of viral infection. Two of the inhibitors, bruceine A and gamabufotalin, were also found to block replication of the Delta and Omicron variants of SARS-CoV-2. Both bruceine A and gamabufotalin exhibited potent antiviral activity in K18-hACE2 and wild-type C57BL6/J mice, as evidenced by reduced viral titres in the lung and brain, and protection from alveolar and peribronchial inflammation in the lung, thereby limiting disease progression. We propose that our fluorescent assay can be applied to identify antiviral compounds with potential as therapeutic treatment for COVID-19 and other respiratory diseases.

2.
American Journal of Respiratory and Critical Care Medicine ; 203(9):1, 2021.
Article in English | Web of Science | ID: covidwho-1407044
3.
American Journal of Respiratory and Critical Care Medicine ; 203(9), 2021.
Article in English | EMBASE | ID: covidwho-1277755

ABSTRACT

Rationale: SARS-CoV-2, the virus that causes COVID-19, exhibits an ACE2-dependent airway epithelial tropism, and exploits host cell proteins to replicate and evade detection. The impact of asthma on COVID-19 susceptibility and severity is unclear. We sought to discover how genes encoding SARS-CoV-2-associated host proteins are expressed in primary human bronchial epithelial cells (HBECs), and how these genes are regulated by cytokines important in asthma. Methods: We compiled a list of 342 SARS-CoV-2-associated genes. We cultured primary HBECs at air-liquid interface in the absence of cytokine, or with interleukin (IL)-13, IL-17, interferon (IFN)-α, or IFN-γ. We used bulk RNA-seq and single cell RNA-sequencing to identify changes in gene expression. We correlated cytokine-regulated changes in SARS-CoV-2-associated transcripts on cytokine exposure in vitro with gene expression changes in transcriptomic profiling datasets derived from individuals with mild-to-moderate asthma and chronic obstructive pulmonary disease (COPD). Results: Transcripts encoding 332 of 342 (97%) SARS-CoV-2-associated proteins were detected in HBECs (≥1 RPM in 50% samples);85 (26%) were regulated by at least one cytokine (>1.5-fold change, FDR < 0.05). 21 and 19 of the 41 IL-13 responsive, SARS-CoV-2-associated genes identified in HBECs correlated with type 2 inflammatory gene signature scores in transcriptomic profiling datasets derived from individuals with mild-to-moderate asthma and COPD (p < 0.05);few IL-17 or interferon-responsive genes were correlated with their respective signatures in either dataset. Single cell RNA-sequencing revealed that 143 of the 332 (43%) SARS-CoV-2-associated transcripts detected in HBECs were differentially expressed between cell types (FDR < 0.05). 11 SARS-CoV-2-associated genes were modulated by IL-13 in a cell type-specific manner (>1.25-fold change, FDR < 0.05). Conclusions: Many genes encoding proteins associated with SARS-CoV-2 infection are expressed in HBECs, with substantial differences among cell subsets. IL-13 induces extensive changes in the expression of SARS-CoV-2-related genes that correlated with a measure of type-2 inflammation in vivo, providing a plausible basis for differences in outcome of COVID-19 in individuals with asthma.

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