FLECS technology for high-throughput screening of hypercontractile cellular phenotypes in fibrosis: A function-first approach to anti-fibrotic drug discovery.

The pivotal role of myofibroblast contractility in the pathophysiology of fibrosis is widely recognized, yet HTS approaches are not available to quantify this critically important function in drug discovery. We developed, validated, and scaled-up a HTS platform that quantifies contractile function of primary human lung myofibroblasts upon treatment with pro-fibrotic TGF-ß1. With the fully automated assay we screened a library of 40,000 novel small molecules in under 80 h of total assay run-time. We identified 42 hit compounds that inhibited the TGF-ß1-induced contractile phenotype of myofibroblasts, and enriched for 19 that specifically target myofibroblasts but not phenotypically related smooth muscle cells. Selected hits were validated in an ex vivo lung tissue models for their inhibitory effects on fibrotic gene upregulation by TGF-ß1. Our results demonstrate that integrating a functional contraction test into the drug screening process is key to identify compounds with targeted and diverse activity as potential anti-fibrotic agents.

Comparative Analysis of Molecular Pathogenic Mechanisms and Antiviral Development Targeting Old and New World Hantaviruses.

Background: Hantaviruses - dichotomized into New World (i.e. Andes virus, ANDV; Sin Nombre virus, SNV) and Old-World viruses (i.e. Hantaan virus, HTNV) - are zoonotic viruses transmitted from rodents to humans. Currently, no FDA-approved vaccines against hantaviruses exist. Given the recent breakthrough to human-human transmission by the ANDV, an essential step is to establish an effective pandemic preparedness infrastructure to rapidly identify cell tropism, infective potential, and effective therapeutic agents through systematic investigation. Methods: We established human cell model systems in lung (airway and distal lung epithelial cells), heart (pluripotent stem cell-derived (PSC-) cardiomyocytes), and brain (PSC-astrocytes) cell types and subsequently evaluated ANDV, HTNV and SNV tropisms. Transcriptomic, lipidomic and bioinformatic data analyses were performed to identify the molecular pathogenic mechanisms of viruses in different cell types. This cell-based infection system was utilized to establish a drug testing platform and pharmacogenomic comparisons. Results: ANDV showed broad tropism for all cell types assessed. HTNV replication was predominantly observed in heart and brain cells. ANDV efficiently replicated in human and mouse 3D distal lung organoids. Transcriptomic analysis showed that ANDV infection resulted in pronounced inflammatory response and downregulation of cholesterol biosynthesis pathway in lung cells. Lipidomic profiling revealed that ANDV-infected cells showed reduced level of cholesterol esters and triglycerides. Further analysis of pathway-based molecular signatures showed that, compared to SNV and HTNV, ANDV infection caused drastic lung cell injury responses. A selective drug screening identified STING agonists, nucleoside analogues and plant-derived compounds that inhibited ANDV viral infection and rescued cellular metabolism. In line with experimental results, transcriptome data shows that the least number of total and unique differentially expressed genes were identified in urolithin B- and favipiravir-treated cells, confirming the higher efficiency of these two drugs in inhibiting ANDV, resulting in host cell ability to balance gene expression to establish proper cell functioning. Conclusions: Overall, our study describes advanced human PSC-derived model systems and systems-level transcriptomics and lipidomic data to better understand Old and New World hantaviral tropism, as well as drug candidates that can be further assessed for potential rapid deployment in the event of a pandemic.

Adaptation of the Tumor Antigen Presentation Machinery to Ionizing Radiation.

Ionizing radiation (IR) can reprogram proteasome structure and function in cells and tissues. In this article, we show that IR can promote immunoproteasome synthesis with important implications for Ag processing and presentation and tumor immunity. Irradiation of a murine fibrosarcoma (FSA) induced dose-dependent de novo biosynthesis of the immunoproteasome subunits LMP7, LMP2, and Mecl-1, in concert with other changes in the Ag-presentation machinery (APM) essential for CD8+ T cell-mediated immunity, including enhanced expression of MHC class I (MHC-I), ß2-microglobulin, transporters associated with Ag processing molecules, and their key transcriptional activator NOD-like receptor family CARD domain containing 5. In contrast, in another less immunogenic, murine fibrosarcoma (NFSA), LMP7 transcripts and expression of components of the immunoproteasome and the APM were muted after IR, which affected MHC-I expression and CD8+ T lymphocyte infiltration into NFSA tumors in vivo. Introduction of LMP7 into NFSA largely corrected these deficiencies, enhancing MHC-I expression and in vivo tumor immunogenicity. The immune adaptation in response to IR mirrored many aspects of the response to IFN-γ in coordinating the transcriptional MHC-I program, albeit with notable differences. Further investigations showed divergent upstream pathways in that, unlike IFN-γ, IR failed to activate STAT-1 in either FSA or NFSA cells while heavily relying on NF-κB activation. The IR-induced shift toward immunoproteasome production within a tumor indicates that proteasomal reprogramming is part of an integrated and dynamic tumor-host response that is specific to the stressor and the tumor and therefore is of clinical relevance for radiation oncology.

NSC95397 is a Novel HIV Latency Reversing Agent.

The latent viral reservoir represents one of the major barriers of curing HIV. Focus on the "kick and kill" approach, in which virus expression is reactivated then cells producing virus are selectively depleted, has led to the discovery of many latency reversing agents (LRAs) that can reactivate latently integrated virus and further our understanding of the mechanisms driving HIV latency and latency reversal. Thus far, individual compounds have yet to be robust enough to work as a therapy, highlighting the importance of identifying new compounds that can act in novel pathways and synergize with known LRAs. In this study, we identified a promising LRA, NSC95397, from a screen of ~4250 compounds in J-Lat cell lines. We validated that NSC95397 reactivates latent viral transcription and protein expression from cells with unique integration events. Cotreating cells with NSC95397 and known LRAs demonstrated that NSC95397 has the potential to synergize with different drugs, such prostratin, a PKC agonist, and SAHA, an HDAC inhibitor. By looking at multiple common markers of open chromatin, we show that NSC95397 does not increase open chromatin globally. Bulk RNA sequencing revealed that NSC95397 does not greatly change cellular transcription. Instead, NSC95397 downregulates many pathways key to metabolism, cell growth, and DNA repair - highlighting the potential of these pathways in regulating HIV latency. Overall, we identified NSC95397 as a novel LRA that does not alter global transcription, that shows potential for synergy with known LRAs, and that may act through novel pathways not previously recognized for their ability to modulate HIV latency.

Osteopontin as a Biomarker in Chronic Kidney Disease.

Osteopontin (OPN) is a ubiquitously expressed protein with a wide range of physiological functions, including roles in bone mineralization, immune regulation, and wound healing. OPN has been implicated in the pathogenesis of several forms of chronic kidney disease (CKD) where it promotes inflammation and fibrosis and regulates calcium and phosphate metabolism. OPN expression is increased in the kidneys, blood, and urine of patients with CKD, particularly in those with diabetic kidney disease and glomerulonephritis. The full-length OPN protein is cleaved by various proteases, including thrombin, matrix metalloproteinase (MMP)-3, MMP-7, cathepsin-D, and plasmin, producing N-terminal OPN (ntOPN), which may have more detrimental effects in CKD. Studies suggest that OPN may serve as a biomarker in CKD, and while more research is needed to fully evaluate and validate OPN and ntOPN as CKD biomarkers, the available evidence suggests that they are promising candidates for further investigation. Targeting OPN may be a potential treatment strategy. Several studies show that inhibition of OPN expression or activity can attenuate kidney injury and improve kidney function. In addition to its effects on kidney function, OPN has been linked to cardiovascular disease, which is a major cause of morbidity and mortality in patients with CKD.

MEK Inhibition Sensitizes Pancreatic Cancer to STING Agonism by Tumor Cell-intrinsic Amplification of Type I IFN Signaling.

PURPOSE: Stimulator of interferon genes (STING) agonists are currently in development for treatment of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Response rates to STING agonists alone have been promising yet modest, and combination therapies will likely be required to elicit their full potency. We sought to identify combination therapies and mechanisms that augment the tumor cell-intrinsic effect of therapeutically relevant STING agonists apart from their known effects on tumor immunity. EXPERIMENTAL DESIGN: We screened 430 kinase inhibitors to identify synergistic effectors of tumor cell death with diABZI, an intravenously administered and systemically available STING agonist. We deciphered the mechanisms of synergy with STING agonism that cause tumor cell death in vitro and tumor regression in vivo. RESULTS: We found that MEK inhibitors caused the greatest synergy with diABZI and that this effect was most pronounced in cells with high STING expression. MEK inhibition enhanced the ability of STING agonism to induce type I IFN-dependent cell death in vitro and tumor regression in vivo. We parsed NFκB-dependent and NFκB-independent mechanisms that mediate STING-driven type I IFN production and show that MEK signaling inhibits this effect by suppressing NFκB activation. CONCLUSIONS: Our results highlight the cytotoxic effects of STING agonism on PDAC cells that are independent of tumor immunity and that these therapeutic benefits of STING agonism can be synergistically enhanced by MEK inhibition.

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.

Pacritinib inhibits glucose consumption in squamous cell lung cancer cells by targeting FLT3.

Squamous cell lung cancer maintains its growth through elevated glucose consumption, but selective glucose consumption inhibitors are lacking. Here, we discovered using a high-throughput screen new compounds that block glucose consumption in three squamous cell lung cancer cell lines and identified 79 compounds that block glucose consumption in one or more of these cell lines. Based on its ability to block glucose consumption in all three cell lines, pacritinib, an inhibitor of FMS Related Receptor Tyrosine Kinase 3 (FLT3) and Janus Kinase 2 (JAK2), was further studied. Pacritinib decreased glucose consumption in squamous cell lung cancer cells in cell culture and in vivo without affecting glucose consumption in healthy tissues. Pacritinib blocked hexokinase activity, and Hexokinase 1 and 2 mRNA and protein expression. Overexpression of Hexokinase 1 blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Overexpression of FLT3 but not JAK2 significantly increased glucose consumption and blocked the ability of pacritinib to inhibit glucose consumption in squamous cell lung cancer cells. Additional FLT3 inhibitors blocked glucose consumption in squamous cell lung cancer cells. Our study identifies FLT3 inhibitors as a new class of inhibitors that can block glucose consumption in squamous cell lung cancer.

Quantitative In Vivo Imaging of the Androgen Receptor Axis Reveals Degree of Prostate Cancer Radiotherapy Response.

Noninvasive biomarkers for androgen receptor (AR) pathway activation are urgently needed to better monitor patient response to prostate cancer therapies. AR is a critical driver and mediator of resistance of prostate cancer but currently available noninvasive prostate cancer biomarkers to monitor AR activity are discordant with downstream AR pathway activity. External beam radiotherapy (EBRT) remains a common treatment for all stages of prostate cancer, and DNA damage induced by EBRT upregulates AR pathway activity to promote therapeutic resistance. [89Zr]11B6-PET is a novel modality targeting prostate-specific protein human kallikrein 2 (hK2), which is a surrogate biomarker for AR activity. Here, we studied whether [89Zr]11B6-PET can accurately assess EBRT-induced AR activity.Genetic and human prostate cancer mouse models received EBRT (2-50 Gy) and treatment response was monitored by [89Zr]11B6-PET/CT. Radiotracer uptake and expression of AR and AR target genes was quantified in resected tissue.EBRT increased AR pathway activity and [89Zr]11B6 uptake in LNCaP-AR and 22RV1 tumors. EBRT increased prostate-specific [89Zr]11B6 uptake in prostate cancer-bearing mice (Hi-Myc x Pb_KLK2) with no significant changes in uptake in healthy (Pb_KLK2) mice, and this correlated with hK2 protein levels. IMPLICATIONS: hK2 expression in prostate cancer tissue is a proxy of EBRT-induced AR activity that can noninvasively be detected using [89Zr]11B6-PET; further clinical evaluation of hK2-PET for monitoring response and development of resistance to EBRT in real time is warranted.

Early Reduction of Glucose Consumption Is a Biomarker of Kinase Inhibitor Efficacy Which Can Be Reversed with GLUT1 Overexpression in Lung Cancer Cells.

PURPOSE: Small molecule inhibitors that target oncogenic driver kinases are an important class of therapies for non-small cell lung cancer (NSCLC) and other malignancies. However, these therapies are not without their challenges. Each inhibitor works on only a subset of patients, the pharmacokinetics of these inhibitors is variable, and these inhibitors are associated with significant side effects. Many of these inhibitors lack non-invasive biomarkers to confirm pharmacodynamic efficacy, and our understanding of how these inhibitors block cancer cell growth remains incomplete. Limited clinical studies suggest that early (< 2 weeks after start of therapy) changes in tumor glucose consumption, measured by [18F]FDG PET imaging, can predict therapeutic efficacy, but the scope of this strategy and functional relevance of this inhibition of glucose consumption remains understudied. Here we demonstrate that early inhibition of glucose consumption as can be measured clinically with [18F]FDG PET is a consistent phenotype of efficacious targeted kinase inhibitors and is necessary for the subsequent inhibition of growth across models of NSCLC. METHODS: We tested nine NSCLC cell lines (A549, H1129, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells) and ten targeted therapies (afatinib, buparlisib, ceritinib, cabozantinib, crizotinib, dovitinib, erlotinib, ponatinib, trametinib, and vemurafenib) across concentrations ranging from 1.6 nM to 5 µM to evaluate whether these inhibitors block glucose consumption at 24-h post-drug treatment and cell growth at 72-h post-drug treatment. We overexpressed the facilitative glucose transporter SLC2A1 (GLUT1) to test the functional connection between blocked glucose consumption and cell growth after treatment with a kinase inhibitor. A subset of these inhibitors and cell lines were studied in vivo. RESULTS: Across the nine NSCLC cell lines, ten targeted therapies, and a range of inhibitor concentrations, whether a kinase inhibitor blocked glucose consumption at 24-h post-drug treatment strongly correlated with whether that inhibitor blocked cell growth at 72-h post-drug treatment in cell culture. These results were confirmed in vivo with [18F]FDG PET imaging. GLUT1 overexpression blocked the kinase inhibitors from limiting glucose consumption and cell growth. CONCLUSIONS: Our results demonstrate that the early inhibition of lung cancer glucose consumption in response to a kinase inhibitor is a strong biomarker of and is often required for the subsequent inhibition of cell growth. Early inhibition of glucose consumption may provide complementary information to other biomarkers in determining whether a drug will effectively limit tumor growth.

A quantitative high-throughput screen identifies compounds that lower expression of the SCA2-and ALS-associated gene ATXN2.

CAG repeat expansions in the ATXN2 (ataxin-2) gene can cause the autosomal dominant disorder spinocerebellar ataxia type 2 (SCA2) as well as increase the risk of ALS. Abnormal molecular, motor, and neurophysiological phenotypes in SCA2 mouse models are normalized by lowering ATXN2 transcription, and reduction of nonmutant Atxn2 expression has been shown to increase the life span of mice overexpressing the TDP-43 (transactive response DNA-binding protein 43 kDa) ALS protein, demonstrating the potential benefits of targeting ATXN2 transcription in humans. Here, we describe a quantitative high-throughput screen to identify compounds that lower ATXN2 transcription. We screened 428,759 compounds in a multiplexed assay using an ATXN2-luciferase reporter in human embryonic kidney 293 (HEK-293) cells and identified a diverse set of compounds capable of lowering ATXN2 transcription. We observed dose-dependent reductions of endogenous ATXN2 in HEK-293 cells treated with procillaridin A, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), and heat shock protein 990 (HSP990), known inhibitors of HSP90 and Na+/K+-ATPases. Furthermore, HEK-293 cells expressing polyglutamine-expanded ATXN2-Q58 treated with 17-DMAG had minimally detectable ATXN2, as well as normalized markers of autophagy and endoplasmic reticulum stress, including STAU1 (Staufen 1), molecular target of rapamycin, p62, LC3-II (microtubule-associated protein 1A/1B-light chain 3II), CHOP (C/EBP homologous protein), and phospho-eIF2α (eukaryotic initiation factor 2α). Finally, bacterial artificial chromosome ATXN2-Q22 mice treated with 17-DMAG or HSP990 exhibited highly reduced ATXN2 protein abundance in the cerebellum. Taken together, our study demonstrates inhibition of HSP90 or Na+/K+-ATPases as potentially effective therapeutic strategies for treating SCA2 and ALS.

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers.

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

Suspendable Hydrogel Nanovials for Massively Parallel Single-Cell Functional Analysis and Sorting.

Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology as well as accelerate the development of next-generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein, we describe methods to fabricate hydrogel-based chemically functionalized microcontainers, which we call nanovials, and demonstrate their use for sorting single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. These nanovials act as solid supports that facilitate attachment of a variety of adherent and suspension cell types, partition uniform aqueous compartments, and capture secreted proteins. Solutions can be exchanged around nanovials to perform fluorescence immunoassays on secreted proteins. Using this platform and commercial flow sorters, we demonstrate high-throughput screening of stably and transiently transfected producer cells based on relative IgG production. Chinese hamster ovary cells sorted based on IgG production regrew and maintained a high secretion phenotype over at least a week, yielding >40% increase in bulk IgG production rates. We also sorted hybridomas and B lymphocytes based on antigen-specific antibody production. Hybridoma cells secreting an antihen egg lysozyme antibody were recovered from background cells, enriching a population of ∼4% prevalence to >90% following sorting. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1 million events in <1 h. IgG secreting mouse B cells were also sorted and enriched based on antigen-specific binding. Successful sorting of antibody-secreting B cells combined with the ability to perform single-cell RT-PCR to recover sequence information suggests the potential to perform antibody discovery workflows. The reported nanovials can be easily stored and distributed among researchers, democratizing access to high-throughput functional cell screening.

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.

We determine that type I interferon (IFN) response biomarkers are enriched in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors; however, actionable vulnerabilities associated with IFN signaling have not been systematically defined. Integration of a phosphoproteomic analysis and a chemical genomics synergy screen reveals that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) in PDAC cells and sensitizes them to ATR inhibitors. IFN triggers cell-cycle arrest in S-phase, which is accompanied by nucleotide pool insufficiency and nucleoside efflux. In combination with IFN, ATR inhibitors induce lethal DNA damage and downregulate nucleotide biosynthesis. ATR inhibition limits the growth of PDAC tumors in which IFN signaling is driven by stimulator of interferon genes (STING). These results identify a cross talk between IFN, DNA replication stress response networks, and nucleotide metabolism while providing the rationale for targeted therapeutic interventions that leverage IFN signaling in tumors.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

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.

Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples.

Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.