CRISPR screens for host factors critical for infection by SARS-CoV-2 variants of concern identify GATA6 as a central modulator of ACE2.

The global spread of SARS-CoV-2 led to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SASR-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

Induced pulmonary comorbidities render CD-1 mice sensitive to SARS-CoV-2

Severe manifestations of COVID-19 are mostly restricted to people with comorbidities. Here we report that induced mild pulmonary morbidities render SARS-CoV-2-refractive CD-1 mice to be susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low-doses of the acute-lung-injury stimulants bleomycin or ricin caused a severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates of >50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart and serum of low-dose-ricin pretreated, as compared to non-pretreated mice. Notably, the deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against SARS-CoV-2 RBD. Thus, viral cell entry in the sensitized mice seems to involve viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. In summary, we present a novel mice-based animal model for the study of comorbidity-dependent severe COVID-19.

A single dose of recombinant VSV-{triangleup}G-spike vaccine provides protection against SARS-CoV-2 challenge

The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-{Delta}G-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-{Delta}G-spike indicated expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in vivo model for COVID-19 was implemented. We show that vaccination of hamsters with recombinant VSV-{Delta}G-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-{Delta}G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

Evaluating the efficacy of RT-qPCR SARS-CoV-2 direct approaches in comparison to RNA extraction

SARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies support the elimination of the extraction step. Herein, we assessed the RNA extraction necessity, by comparing RT-qPCR efficacy in several direct approaches vs. the gold standard RNA extraction, in detection of SARS-CoV-2 from laboratory samples as well as clinical Oro-nasopharyngeal SARS-CoV-2 swabs. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified up to 70% of positive clinical specimens.

The coding capacity of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic 1,2. In order to understand SARS-CoV-2 pathogenicity and antigenic potential, and to develop diagnostic and therapeutic tools, it is essential to portray the full repertoire of its expressed proteins. The SARS-CoV-2 coding capacity map is currently based on computational predictions and relies on homology to other coronaviruses. Since coronaviruses differ in their protein array, especially in the variety of accessory proteins, it is crucial to characterize the specific collection of SARS-CoV-2 proteins in an unbiased and open-ended manner. Utilizing a suite of ribosome profiling techniques 3-8, we present a high-resolution map of the SARS-CoV-2 coding regions, allowing us to accurately quantify the expression of canonical viral open reading frames (ORF)s and to identify 23 novel unannotated viral translated ORFs. These ORFs include upstream ORFs (uORFs) that are likely playing a regulatory role, several in-frame internal ORFs lying within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; rather, virus translation dominates host translation due to high levels of viral transcripts. Overall, our work reveals the full coding capacity of SARS-CoV-2 genome, providing a rich resource, which will form the basis of future functional studies and diagnostic efforts.

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.