Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

Innate immune pathway modulator screen identifies STING pathway activation as a strategy to inhibit multiple families of arbo and respiratory viruses.

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

Longitudinal Evaluation of Antibody Persistence in Mother-Infant Dyads Following SARS-CoV-2 Infection in Pregnancy.

BACKGROUND: There are limited data on how COVID-19 severity, timing of infection, and subsequent vaccination impact transplacental transfer and persistence of maternal and infant antibodies. METHODS: In a longitudinal cohort of pregnant women with PCR-confirmed SARS-CoV-2 infection, maternal/infant sera were collected at enrollment, delivery/birth, and 6 months. Anti-SARS-CoV-2 spike IgG, IgM and IgA were measured by ELISA. RESULTS: 256 pregnant women and 135 infants were enrolled; 148 maternal and 122 neonatal specimens were collected at delivery/birth; 45 maternal and 48 infant specimens were collected at 6 months. Sixty-eight percent of women produced all anti-SARS-CoV-2 isotypes at delivery (IgG, IgM, IgA); 96% had at least one isotype. Symptomatic disease, and vaccination prior to delivery, were associated with higher maternal IgG at L&D. Detectable IgG in infants dropped from 78% at birth to 52% at 6 months. In the multivariate analysis evaluating factors associated with detectable IgG in infants at delivery, significant predictors were 3rd trimester infection (OR 4.0), mild/moderate disease (OR 4.8), severe/critical disease (OR 6.3), and maternal vaccination prior to delivery (OR 18.8). No factors were significant in the multivariate analysis at 6 months postpartum. CONCLUSIONS: Vaccination in pregnancy post-COVID-19 recovery is a strategy for boosting antibodies in mother-infant dyads.

Hippo signaling pathway activation during SARS-CoV-2 infection contributes to host antiviral response.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19-associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin, a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

In Silico Genome Analysis Reveals the Evolution and Potential Impact of SARS-CoV-2 Omicron Structural Changes on Host Immune Evasion and Antiviral Therapeutics.

New variants of SARS-CoV-2 continue to evolve. The novel SARS-CoV-2 variant of concern (VOC) B.1.1.529 (Omicron) was particularly menacing due to the presence of numerous consequential mutations. In this study, we reviewed about 12 million SARS-CoV-2 genomic and associated metadata using extensive bioinformatic approaches to understand how evolutionary and mutational changes affect Omicron variant properties. Subsampled global data based analysis of molecular clock in the phylogenetic tree showed 29.56 substitutions per year as the evolutionary rate of five VOCs. We observed extensive mutational changes in the spike structural protein of the Omicron variant. A total of 20% of 7230 amino acid and structural changes exclusive to Omicron's spike protein were detected in the receptor binding domain (RBD), suggesting differential selection pressures exerted during evolution. Analyzing key drug targets revealed mutation-derived differential binding affinities between Delta and Omicron variants. Nine single-RBD substitutions were detected within the binding site of approved therapeutic monoclonal antibodies. T-cell epitope prediction revealed eight immunologically important functional hotspots in three conserved non-structural proteins. A universal vaccine based on these regions may likely protect against all these SARS-CoV-2 variants. We observed key structural changes in the spike protein, which decreased binding affinities, indicating that these changes may help the virus escape host cellular immunity. These findings emphasize the need for continuous genomic surveillance of SARS-CoV-2 to better understand how novel mutations may impact viral spread and disease outcome.

Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development.

Development of off-the-shelf hematopoietic stem cell-engineered invariant natural killer T cells for COVID-19 therapeutic intervention.

BACKGROUND: New COVID-19 treatments are desperately needed as case numbers continue to rise and emergent strains threaten vaccine efficacy. Cell therapy has revolutionized cancer treatment and holds much promise in combatting infectious disease, including COVID-19. Invariant natural killer T (iNKT) cells are a rare subset of T cells with potent antiviral and immunoregulatory functions and an excellent safety profile. Current iNKT cell strategies are hindered by the extremely low presence of iNKT cells, and we have developed a platform to overcome this critical limitation. METHODS: We produced allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells through TCR engineering of human cord blood CD34+ hematopoietic stem cells (HSCs) and differentiation of these HSCs into iNKT cells in an Ex Vivo HSC-Derived iNKT Cell Culture. We then established in vitro SARS-CoV-2 infection assays to assess AlloHSC-iNKT cell antiviral and anti-hyperinflammation functions. Lastly, using in vitro and in vivo preclinical models, we evaluated AlloHSC-iNKT cell safety and immunogenicity for off-the-shelf application. RESULTS: We reliably generated AlloHSC-iNKT cells at high-yield and of high-purity; these resulting cells closely resembled endogenous human iNKT cells in phenotypes and functionalities. In cell culture, AlloHSC-iNKT cells directly killed SARS-CoV-2 infected cells and also selectively eliminated SARS-CoV-2 infection-stimulated inflammatory monocytes. In an in vitro mixed lymphocyte reaction (MLR) assay and an NSG mouse xenograft model, AlloHSC-iNKT cells were resistant to T cell-mediated alloreaction and did not cause GvHD. CONCLUSIONS: Here, we report a method to robustly produce therapeutic levels of AlloHSC-iNKT cells. Preclinical studies showed that these AlloHSC-iNKT cells closely resembled endogenous human iNKT cells, could reduce SARS-CoV-2 virus infection load and mitigate virus infection-induced hyperinflammation, and meanwhile were free of GvHD-risk and resistant to T cell-mediated allorejection. These results support the development of AlloHSC-iNKT cells as a promising off-the-shelf cell product for treating COVID-19; such a cell product has the potential to target the new emerging SARS-CoV-2 variants as well as the future new emerging viruses.

Compound screen identifies the small molecule Q34 as an inhibitor of SARS-CoV-2 infection.

The COVID-19 outbreak poses a serious threat to global public health. Effective countermeasures and approved therapeutics are desperately needed. In this study, we screened a small molecule library containing the NCI-DTP compounds to identify molecules that can prevent SARS-CoV-2 cellular entry. By applying a luciferase assay-based screening using a pseudotyped SARS-CoV-2-mediated cell entry assay, we identified a small molecule compound Q34 that can efficiently block cellular entry of the pseudotyped SARS-CoV-2 into human ACE2-expressing HEK293T cells, and inhibit the infection of the authentic SARS-CoV-2 in human ACE2-expressing HEK293T cells, human iPSC-derived neurons and astrocytes, and human lung Calu-3 cells. Importantly, the safety profile of the compound is favorable. There is no obvious toxicity observed in uninfected cells treated with the compound. Thus, this compound holds great potential as both prophylactics and therapeutics for COVID-19 and future pandemics by blocking the entry of SARS-CoV-2 and related viruses into human cells.

ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.

The systemic inflammatory landscape of COVID-19 in pregnancy: Extensive serum proteomic profiling of mother-infant dyads with in utero SARS-CoV-2.

While pregnancy increases the risk for severe COVID-19, the clinical and immunological implications of COVID-19 on maternal-fetal health remain unknown. Here, we present the clinical and immunological landscapes of 93 COVID-19 mothers and 45 of their SARS-CoV-2-exposed infants through comprehensive serum proteomics profiling for >1,400 cytokines of their peripheral and cord blood specimens. Prenatal SARS-CoV-2 infection triggers NF-κB-dependent proinflammatory immune activation. Pregnant women with severe COVID-19 show increased inflammation and unique IFN-λ antiviral signaling, with elevated levels of IFNL1 and IFNLR1. Furthermore, SARS-CoV-2 infection re-shapes maternal immunity at delivery, altering the expression of pregnancy complication-associated cytokines, inducing MMP7, MDK, and ESM1 and reducing BGN and CD209. Finally, COVID-19-exposed infants exhibit induction of T cell-associated cytokines (IL33, NFATC3, and CCL21), while some undergo IL-1ß/IL-18/CASP1 axis-driven neonatal respiratory distress despite birth at term. Our findings demonstrate COVID-19-induced immune rewiring in both mothers and neonates, warranting long-term clinical follow-up to mitigate potential health risks.

Neurological pathophysiology of SARS-CoV-2 and pandemic potential RNA viruses: a comparative analysis.

SARS-CoV-2 has infected hundreds of millions of people with over four million dead, resulting in one of the worst global pandemics in recent history. Neurological symptoms associated with COVID-19 include anosmia, ageusia, headaches, confusion, delirium, and strokes. These may manifest due to viral entry into the central nervous system (CNS) through the blood-brain barrier (BBB) by means of ill-defined mechanisms. Here, we summarize the abilities of SARS-CoV-2 and other neurotropic RNA viruses, including Zika virus and Nipah virus, to cross the BBB into the CNS, highlighting the role of magnetic resonance imaging (MRI) in assessing presence and severity of brain structural changes in COVID-19 patients. We present new insight into key mutations in SARS-CoV-2 variants B.1.1.7 (P681H) and B.1.617.2 (P681R), which may impact on neuropilin 1 (NRP1) binding and CNS invasion. We postulate that SARS-CoV-2 may infect both peripheral cells capable of crossing the BBB and brain endothelial cells to traverse the BBB and spread into the brain. COVID-19 patients can be followed up with MRI modalities to better understand the long-term effects of COVID-19 on the brain.

SARS-CoV-2 and its beta variant of concern infect human conjunctival epithelial cells and induce differential antiviral innate immune response.

PURPOSE: SARS-CoV-2 RNA has been detected in ocular tissues, but their susceptibility to SARS-CoV-2 infection is unclear. Here, we tested whether SARS-CoV-2 can infect human conjunctival epithelial cells (hCECs) and induce innate immune response. METHODS: Conjunctival tissue from COVID-19 donors was used to detect SARS-CoV-2 spike and envelope proteins. Primary hCECs isolated from cadaver eyes were infected with the parental SARS-CoV-2 and its beta variant of concern (VOC). Viral genome copy number, and expression of viral entry receptors, TLRs, interferons, and innate immune response genes were determined by qPCR. Viral entry receptors were examined in hCECs and tissue sections by immunostaining. Spike protein was detected in the cell culture supernatant by dot blot. RESULTS: Spike and envelope proteins were found in conjunctiva from COVID-19 patients. SARS-CoV-2 infected hCECs showed high viral copy numbers at 24-72h post-infection; spike protein levels were the highest at 24hpi. Viral entry receptors ACE2, TMPRSS2, CD147, Axl, and NRP1 were detected in conjunctival tissue and hCECs. SARS-CoV-2 infection-induced receptor gene expression peaked at early time points post-infection, but gene expression of most TLRs peaked at 48 or 72hpi. SARS-CoV-2 infected hCECs showed higher expression of genes regulating antiviral response, RIG-I, interferons (α, ß, & λ), ISG15 & OAS2, cytokines (IL6, IL1ß, TNFα), and chemokines (CXCL10, CCL5). Compared to the parental strain, beta VOC induced increased viral copy number and innate response in hCECs. CONCLUSIONS: Conjunctival epithelial cells are susceptible to SARS-CoV-2 infection. Beta VOC is more infectious than the parental strain and evokes a higher antiviral and inflammatory response.

Targeting the coronavirus nucleocapsid protein through GSK-3 inhibition.

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

Comparative transcriptomic analysis of SARS-CoV-2 infected cell model systems reveals differential innate immune responses.

The transcriptome of SARS-CoV-2-infected cells that reflects the interplay between host and virus has provided valuable insights into mechanisms underlying SARS-CoV-2 infection and COVID-19 disease progression. In this study, we show that SARS-CoV-2 can establish a robust infection in HEK293T cells that overexpress human angiotensin-converting enzyme 2 (hACE2) without triggering significant host immune response. Instead, endoplasmic reticulum stress and unfolded protein response-related pathways are predominantly activated. By comparing our data with published transcriptome of SARS-CoV-2 infection in other cell lines, we found that the expression level of hACE2 directly correlates with the viral load in infected cells but not with the scale of immune responses. Only cells that express high level of endogenous hACE2 exhibit an extensive immune attack even with a low viral load. Therefore, the infection route may be critical for the extent of the immune response, thus the severity of COVID-19 disease status.

A systems-level study reveals host-targeted repurposable drugs against SARS-CoV-2 infection.

Understanding the mechanism of SARS-CoV-2 infection and identifying potential therapeutics are global imperatives. Using a quantitative systems pharmacology approach, we identified a set of repurposable and investigational drugs as potential therapeutics against COVID-19. These were deduced from the gene expression signature of SARS-CoV-2-infected A549 cells screened against Connectivity Map and prioritized by network proximity analysis with respect to disease modules in the viral-host interactome. We also identified immuno-modulating compounds aiming at suppressing hyperinflammatory responses in severe COVID-19 patients, based on the transcriptome of ACE2-overexpressing A549 cells. Experiments with Vero-E6 cells infected by SARS-CoV-2, as well as independent syncytia formation assays for probing ACE2/SARS-CoV-2 spike protein-mediated cell fusion using HEK293T and Calu-3 cells, showed that several predicted compounds had inhibitory activities. Among them, salmeterol, rottlerin, and mTOR inhibitors exhibited antiviral activities in Vero-E6 cells; imipramine, linsitinib, hexylresorcinol, ezetimibe, and brompheniramine impaired viral entry. These novel findings provide new paths for broadening the repertoire of compounds pursued as therapeutics against COVID-19.

Deleterious Effects of SARS-CoV-2 Infection on Human Pancreatic Cells.

COVID-19 pandemic has infected more than 154 million people worldwide and caused more than 3.2 million deaths. It is transmitted by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and affects the respiratory tract as well as extra-pulmonary systems, including the pancreas, that express the virus entry receptor, Angiotensin-Converting Enzyme 2 (ACE2) receptor. Importantly, the endocrine and exocrine pancreas, the latter composed of ductal and acinar cells, express high levels of ACE2, which correlates to impaired functionality characterized as acute pancreatitis observed in some cases presenting with COVID-19. Since acute pancreatitis is already one of the most frequent gastrointestinal causes of hospitalization in the U.S. and the majority of studies investigating the effects of SARS-CoV-2 on the pancreas are clinical and observational, we utilized human iPSC technology to investigate the potential deleterious effects of SARS-CoV-2 infection on iPSC-derived pancreatic cultures containing endocrine and exocrine cells. Interestingly, iPSC-derived pancreatic cultures allow SARS-CoV-2 entry and establish infection, thus perturbing their normal molecular and cellular phenotypes. The infection increased a key cytokine, CXCL12, known to be involved in inflammatory responses in the pancreas. Transcriptome analysis of infected pancreatic cultures confirmed that SARS-CoV-2 hijacks the ribosomal machinery in these cells. Notably, the SARS-CoV-2 infectivity of the pancreas was confirmed in post-mortem tissues from COVID-19 patients, which showed co-localization of SARS-CoV-2 in pancreatic endocrine and exocrine cells and increased the expression of some pancreatic ductal stress response genes. Thus, we demonstrate that SARS-CoV-2 can directly infect human iPSC-derived pancreatic cells with strong supporting evidence of presence of the virus in post-mortem pancreatic tissue of confirmed COVID-19 human cases. This novel model of iPSC-derived pancreatic cultures will open new avenues for the comprehension of the SARS-CoV-2 infection and potentially establish a platform for endocrine and exocrine pancreas-specific antiviral drug screening.

A monoclonal antibody against staphylococcal enterotoxin B superantigen inhibits SARS-CoV-2 entry in vitro.

We recently discovered a superantigen-like motif sequentially and structurally similar to a staphylococcal enterotoxin B (SEB) segment, near the S1/S2 cleavage site of the SARS-CoV-2 spike protein, which might explain the multisystem inflammatory syndrome (MIS-C) observed in children and the cytokine storm in severe COVID-19 patients. We show here that an anti-SEB monoclonal antibody (mAb), 6D3, can bind this viral motif at its polybasic (PRRA) insert to inhibit infection in live virus assays. The overlap between the superantigenic site of the spike and its proteolytic cleavage site suggests that the mAb prevents viral entry by interfering with the proteolytic activity of cell proteases (furin and TMPRSS2). The high affinity of 6D3 for this site originates from a polyacidic segment at its heavy chain CDR2. The study points to the potential utility of 6D3 for possibly treating COVID-19, MIS-C, or common colds caused by human coronaviruses that also possess a furin-like cleavage site.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.