Fluorogenic reporter enables identification of compounds that inhibit SARS-CoV-2.

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.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

A defective viral genome strategy elicits broad protective immunity against respiratory viruses.

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.

Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers.

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-ß1 levels between smokers and non-smokers.

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles.

The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 µM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.

SARS-CoV-2 infects human pancreatic ß cells and elicits ß cell impairment.

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic ß cells can be infected by SARS-CoV-2 and cause ß cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in ß cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic ß cells in patients who succumbed to COVID-19 and selectively infects human islet ß cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces ß cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic ß cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce ß cell killing.

Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models.

SARS-CoV-2 is the coronavirus that causes the respiratory disease COVID-19, which is now the third-leading cause of death in the United States. The FDA has recently approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though recent data from the WHO shows little to no benefit with use of this anti-viral agent. Here we report the discovery of ethacridine, a safe antiseptic use in humans, as a potent drug for use against SARS-CoV-2 (EC50 ~ 0.08 µM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Indeed, ethacridine is effective in various cell types, including primary human nasal epithelial cells. Taken together, these data identify a promising, potent, and new use of the old drug possessing a distinct mode of action for inhibiting SARS-CoV-2.