VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney.

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased ß-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.

Surfaces with instant and persistent antimicrobial efficacy against bacteria and SARS-CoV-2.

Surfaces contaminated with bacteria and viruses contribute to the transmission of infectious diseases and pose a significant threat to global public health. Modern day disinfection either relies on fast-acting (>3-log reduction within a few minutes), yet impermanent, liquid-, vapor-, or radiation-based disinfection techniques, or long-lasting, but slower-acting, passive antimicrobial surfaces based on heavy metal surfaces, or metallic nanoparticles. There is currently no surface that provides instant and persistent antimicrobial efficacy against a broad spectrum of bacteria and viruses. In this work, we describe a class of extremely durable antimicrobial surfaces incorporating different plant secondary metabolites that are capable of rapid disinfection (>4-log reduction) of current and emerging pathogens within minutes, while maintaining persistent efficacy over several months and under significant environmental duress. We also show that these surfaces can be readily applied onto a variety of desired substrates or devices via simple application techniques such as spray, flow, or brush coating.

In Vitro Evaluation and Mitigation of Niclosamide's Liabilities as a COVID-19 Treatment.

Niclosamide, an FDA-approved oral anthelmintic drug, has broad biological activity including anticancer, antibacterial, and antiviral properties. Niclosamide has also been identified as a potent inhibitor of SARS-CoV-2 infection in vitro, generating interest in its use for the treatment or prevention of COVID-19. Unfortunately, there are several potential issues with using niclosamide for COVID-19, including low bioavailability, significant polypharmacology, high cellular toxicity, and unknown efficacy against emerging SARS-CoV-2 variants of concern. In this study, we used high-content imaging-based immunofluorescence assays in two different cell models to assess these limitations and evaluate the potential for using niclosamide as a COVID-19 antiviral. We show that despite promising preliminary reports, the antiviral efficacy of niclosamide overlaps with its cytotoxicity giving it a poor in vitro selectivity index for anti-SARS-CoV-2 inhibition. We also show that niclosamide has significantly variable potency against the different SARS-CoV-2 variants of concern and is most potent against variants with enhanced cell-to-cell spread including the B.1.1.7 (alpha) variant. Finally, we report the activity of 33 niclosamide analogs, several of which have reduced cytotoxicity and increased potency relative to niclosamide. A preliminary structure-activity relationship analysis reveals dependence on a protonophore for antiviral efficacy, which implicates nonspecific endolysosomal neutralization as a dominant mechanism of action. Further single-cell morphological profiling suggests niclosamide also inhibits viral entry and cell-to-cell spread by syncytia. Altogether, our results suggest that niclosamide is not an ideal candidate for the treatment of COVID-19, but that there is potential for developing improved analogs with higher clinical translational potential in the future.

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19.

The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site (i.e., RNA synthesis) of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise.

Identification of cell type specific ACE2 modifiers by CRISPR screening.

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Host-Virus Chimeric Events in SARS-CoV-2-Infected Cells Are Infrequent and Artifactual.

The pathogenic mechanisms underlying severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection remain largely unelucidated. High-throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen- and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in transcriptome sequencing (RNA-seq) data from SARS-CoV-2-infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV-2 is a positive-sense RNA virus that replicates in the cytoplasm, it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events like mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with coronavirus disease 2019 (COVID-19) and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spiked-in RNA from an unrelated species, such as the fruit fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV-2 RNA-seq, we found that the frequency of HVC events was, in fact, not greater than this background "noise." Finally, we developed a novel experimental approach to enrich SARS-CoV-2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV-2-infected cells are extremely rare and are likely artifacts arising from random template switching of reverse transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV-2 fusion to cellular genes and/or integration into human genomes. IMPORTANCE The pathogenic mechanisms underlying SARS-CoV-2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known about the reasons some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV-2-infected cells and suggested that HVC events support potential "human genome invasion" and "integration" by SARS-CoV-2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here, we provide several lines of evidence suggesting that the observed HVC events are likely artifactual.

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 Replication in an Immunocompromised Patient.

We describe a case of chronic coronavirus disease 2019 (COVID-19) in a patient with lymphoma and associated B-cell immunodeficiency. Viral cultures and sequence analysis demonstrate ongoing replication of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for at least 119 days. The patient had 3 admissions related to COVID-19 over a 4-month period and was treated twice with remdesivir and convalescent plasma with resolution of symptoms. The patient's lack of seroconversion and prolonged course illustrate the importance of humoral immunity in resolving SARS-CoV-2 infection. This case highlights challenges in managing immunocompromised hosts, who may act as persistent shedders and sources of transmission.

SARS-CoV-2 receptor networks in diabetic and COVID-19-associated kidney disease.

COVID-19 morbidity and mortality are increased via unknown mechanisms in patients with diabetes and kidney disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Because ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of kidney biopsies from healthy living donors and patients with diabetic kidney disease revealed ACE2 expression primarily in proximal tubular epithelial cells. This cell-specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin-angiotensin-aldosterone system inhibitors in diabetic kidney disease. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing proximal tubular epithelial cells in diabetic kidney disease (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The diabetic kidney disease ACE2-positive proximal tubular epithelial cell module overlapped with expression patterns seen in SARS-CoV-2-infected cells. Similar cellular programs were seen in ACE2-positive proximal tubular epithelial cells obtained from urine samples of 13 hospitalized patients with COVID-19, suggesting a consistent ACE2-coregulated proximal tubular epithelial cell expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19-related kidney damage.