SARS-CoV-2 Envelope-mediated Golgi pH dysregulation interferes with ERAAP retention in cells

Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) trims peptide precursors in the ER for presentation by major histocompatibility (MHC)-I molecules to surveying CD8+ T-cells. This function allows ERAAP to regulate the nature and quality of the peptide repertoire and, accordingly, the resulting immune responses. We recently showed that infection with murine cytomegalovirus leads to a dramatic loss of ERAAP levels in infected cells. In mice, this loss is associated with the activation of QFL T-cells, a subset of T-cells that monitor ERAAP integrity and eliminate cells experiencing ERAAP dysfunction. In this study, we aimed to identify host factors that regulate ERAAP expression level and determine whether these could be manipulated during viral infections. We performed a CRISPR knockout screen and identified ERp44 as a factor promoting ERAAP retention in the ER. ERp44s interaction with ERAAP is dependent on the pH gradient between the ER and Golgi. We hypothesized that viruses that disrupt the pH of the secretory pathway interfere with ERAAP retention. Here, we demonstrate that expression of the Envelope (E) protein from Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) leads to Golgi pH neutralization and consequently decrease of ERAAP intracellular levels. Furthermore, SARS-CoV-2-induced ERAAP loss correlates with its release into the extracellular environment. ERAAPs reliance on ERp44 and a functioning ER/Golgi pH gradient for proper localization and function led us to propose that ERAAP serves as a sensor of disturbances in the secretory pathway during infection and disease.

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Numerous host factors of SARS-CoV-2 have been identified by screening approaches, but delineating their molecular roles during infection and whether they can be targeted for antiviral intervention remains a challenge. Here we use Perturb-seq, a single-cell CRISPR screening approach, to investigate how CRISPR interference of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our data reveal two classes of host factors with pronounced phenotypes: factors required for the response to interferon and factors required for entry or early infection. Among the latter, we have characterized the NF-{kappa}B inhibitor I{kappa}B (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides high-throughput functional validation of host factors of SARS-CoV-2 and describes their roles during viral infection in both infected and bystander cells.

Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection

SARS-CoV-2 can cause a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of the host factors mediating viral infection or restriction is critical to elucidate SARS-CoV-2 host-pathogen interactions and the progression of COVID-19. To this end, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. These screens uncovered proviral and antiviral host factors across highly interconnected host pathways, including components implicated in clathrin transport, inflammatory signaling, cell cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high-molecular weight glycoproteins, as a prominent viral restriction network. We demonstrate that multiple membrane-anchored mucins are critical inhibitors of SARS-CoV-2 entry and are upregulated in response to viral infection. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and suggests interactions between SARS-CoV-2 and airway mucins of COVID-19 patients as a host defense mechanism.

Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre-symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved [~]100 copies/L sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.

Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.