SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-191. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response2,3. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression4-7, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.

The coding capacity of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs 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; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.

Identification of T-Cell Epitopes Using a Combined In-Silico and Experimental Approach in a Mouse Model for SARS-CoV-2.

Following viral infection, T-cells are crucial for an effective immune response to intracellular pathogens, including respiratory viruses. During the COVID-19 pandemic, diverse assays were required in pre-clinical trials to evaluate the immune response following vaccination against SARS-CoV-2 and assess the response following exposure to the virus. To assess the nature and potency of the cellular response to infection or vaccination, a reliable and specific activity assay was needed. A cellular activity assay based on the presentation of short peptides (epitopes) allows the identification of T cell epitopes displayed on different alleles of the MHC, shedding light on the strength of the immune response towards antigens and aiding in antigen design for vaccination. In this report, we describe two approaches for scanning T cell epitopes on the surface glycoprotein of the SARS-CoV-2 (spike), which is utilized for attachment and entry and serves as an antigen in many vaccine candidates. We demonstrate that epitope scanning is feasible using peptide libraries or computational scanning combined with a cellular activity assay. Our scans identified four CD8 T cell epitopes, including one novel undescribed epitope. These epitopes enabled us to establish a reliable T-cell response assay, which was examined and used in various experimental mouse models for SARS-CoV-2 infection and vaccination. These approaches could potentially aid in future antigen design for vaccination and establish cellular activity assays against uncharacterized antigens of emerging pathogens.

Glucosylceramide synthase inhibitors prevent replication of SARS-CoV-2 and influenza virus.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies.

Preliminary nonclinical safety and immunogenicity of an rVSV-ΔG-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs.

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.

rVSV-ΔG-SARS-CoV-2-S vaccine: repeated intramuscular (IM) toxicity, local tolerance, immunogenicity and biodistribution study in NZW rabbits.

BriLife®, a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (106, 107 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials.

Monkeypox DNA levels correlate with virus infectivity in clinical samples, Israel, 2022.

The current monkeypox virus global spread and lack of data regarding clinical specimens' infectivity call for examining virus infectivity, and whether this correlates with results from PCR, the available diagnostic tool. We show strong correlation between viral DNA amount in clinical specimens and virus infectivity toward BSC-1 cell line. Moreover, we define a PCR threshold value (Cq ≥ 35, ≤ 4,300 DNA copies/mL), corresponding to negative viral cultures, which may assist risk-assessment and decision-making regarding protective-measures and guidelines for patients with monkeypox.

Sensitive Immunodetection of Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern 501Y.V2 and 501Y.V1.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may influence the effectiveness of existing laboratory diagnostics. In the current study we determined whether the British (20I/501Y.V1) and South African (20H/501Y.V2) SARS-CoV-2 variants of concern are detected with an in-house S1-based antigen detection assay, analyzing spiked pools of quantitative reverse-transcription polymerase chain reaction-negative nasopharyngeal swab specimens. The assay, combining 4 monoclonal antibodies, allowed sensitive detection of both the wild type and the variants of concern, despite accumulation of several mutations in the variants' S1 region-results suggesting that this combination, targeting distinct epitopes, enables both specificity and the universality.

Nuclear to cytoplasmic shuttling of ERK promotes differentiation of muscle stem/progenitor cells.

The transition between the proliferation and differentiation of progenitor cells is a key step in organogenesis, and alterations in this process can lead to developmental disorders. The extracellular signal-regulated kinase 1/2 (ERK) signaling pathway is one of the most intensively studied signaling mechanisms that regulates both proliferation and differentiation. How a single molecule (e.g. ERK) can regulate two opposing cellular outcomes is still a mystery. Using both chick and mouse models, we shed light on the mechanism responsible for the switch from proliferation to differentiation of head muscle progenitors and implicate ERK subcellular localization. Manipulation of the fibroblast growth factor (FGF)-ERK signaling pathway in chick embryos in vitro and in vivo demonstrated that blockage of this pathway accelerated myogenic differentiation, whereas its activation diminished it. We next examined whether the spatial subcellular localization of ERK could act as a switch between proliferation (nuclear ERK) and differentiation (cytoplasmic ERK) of muscle progenitors. A myristoylated peptide that blocks importin 7-mediated ERK nuclear translocation induced robust myogenic differentiation of muscle progenitor/stem cells in both head and trunk. In the mouse, analysis of Sprouty mutant embryos revealed that increased ERK signaling suppressed both head and trunk myogenesis. Our findings, corroborated by mathematical modeling, suggest that ERK shuttling between the nucleus and the cytoplasm provides a switch-like transition between proliferation and differentiation of muscle progenitors.

Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection.

The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses.

Induction of Superior Systemic and Mucosal Protective Immunity to SARS-CoV-2 by Nasal Administration of a VSV-ΔG-Spike Vaccine.

The emergence of rapidly spreading variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a major challenge to vaccines' protective efficacy. Intramuscular (IM) vaccine administration induces short-lived immunity but does not prevent infection and transmission. New vaccination strategies are needed to extend the longevity of vaccine protection, induce mucosal and systemic immunity and prevent viral transmission. The intranasal (IN) administration of the VSV-ΔG-spike vaccine candidate directly to mucosal surfaces yielded superior mucosal and systemic immunity at lower vaccine doses. Compared to IM vaccination in the K18-hACE2 model, IN vaccination preferentially induced mucosal IgA and T-cells, reduced the viral load at the site of infection, and ameliorated disease-associated brain gene expression. IN vaccination was protective even one year after administration. As most of the world population has been vaccinated by IM injection, we demonstrate the potential of a heterologous IM + IN vaccination regimen to induce mucosal immunity while maintaining systemic immunity. Furthermore, the IM + IN regimen prevented virus transmission in a golden Syrian hamster co-caging model. Taken together, we show that IN vaccination with VSV-ΔG-spike, either as a homologous IN + IN regimen or as a boost following IM vaccination, has a favorable potential over IM vaccination in inducing efficient mucosal immunity, long-term protection and preventing virus transmission.

Discriminative Identification of SARS-CoV-2 Variants Based on Mass-Spectrometry Analysis.

The spread of SARS-CoV-2 variants of concern (VOCs) is of great importance since genetic changes may increase transmissibility, disease severity and reduce vaccine effectiveness. Moreover, these changes may lead to failure of diagnostic measures. Therefore, variant-specific diagnostic methods are essential. To date, genetic sequencing is the gold-standard method to discriminate between variants. However, it is time-consuming (taking several days) and expensive. Therefore, the development of rapid diagnostic methods for SARS-CoV-2 in accordance with its genetic modification is of great importance. In this study we introduce a Mass Spectrometry (MS)-based methodology for the diagnosis of SARS-CoV-2 in propagated in cell-culture. This methodology enables the universal identification of SARS-CoV-2, as well as variant-specific discrimination. The universal identification of SARS-CoV-2 is based on conserved markers shared by all variants, while the identification of specific variants relies on variant-specific markers. Determining a specific set of peptides for a given variant consists of a multistep procedure, starting with an in-silico search for variant-specific tryptic peptides, followed by a tryptic digest of a cell-cultured SARS-CoV-2 variant, and identification of these markers by HR-LC-MS/MS analysis. As a proof of concept, this approach was demonstrated for four representative VOCs compared to the wild-type Wuhan reference strain. For each variant, at least two unique markers, derived mainly from the spike (S) and nucleocapsid (N) viral proteins, were identified. This methodology is specific, rapid, easy to perform and inexpensive. Therefore, it can be applied as a diagnostic tool for pathogenic variants.

Generation of recombinant mAbs to vaccinia virus displaying high affinity and potent neutralization.

Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.

VSV-ΔG-Spike Candidate Vaccine Induces Protective Immunity and Protects K18-hACE2 Mice against SARS-CoV-2 Variants.

Since the emergence of the original SARS-CoV-2, several variants were described, raising questions as to the ability of recently developed vaccine platforms to induce immunity and provide protection against these variants. Here, we utilized the K18-hACE2 mouse model to show that VSV-ΔG-spike vaccination provides protection against several SARS-CoV-2 variants: alpha, beta, gamma, and delta. We show an overall robust immune response, regardless of variant identity, leading to reduction in viral load in target organs, prevention of morbidity and mortality, as well as prevention of severe brain immune response, which follows infection with various variants. Additionally, we provide a comprehensive comparison of the brain transcriptomic profile in response to infection with different variants of SARS-CoV-2 and show how vaccination prevents these disease manifestations. Taken together, these results highlight the robust VSV-ΔG-spike protective response against diverse SARS-CoV-2 variants, as well as its promising potential against newly arising variants.

Insights from the Infection Cycle of VSV-ΔG-Spike Virus.

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3-4 h post infection (PI), and accumulates as the infection proceeds. By 10-24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.

Imatinib inhibits SARS-CoV-2 infection by an off-target-mechanism.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell-cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection.

HCV Infection Increases the Expression of ACE2 Receptor, Leading to Enhanced Entry of Both HCV and SARS-CoV-2 into Hepatocytes and a Coinfection State.

Recent studies suggest the enhancement of liver injury in COVID-19 patients infected with Hepatitis C virus (HCV). Hepatocytes express low levels of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor, raising the possibility of HCV-SARS-CoV-2 coinfection in the liver. This work aimed to explore whether HCV and SARS-CoV-2 coinfect hepatocytes and the interplay between these viruses. We demonstrate that SARS-CoV-2 coinfects HCV-infected Huh7.5 (Huh7.5HCV) cells. Both viruses replicated efficiently in the coinfected cells, with HCV replication enhanced in coinfected compared to HCV-mono-infected cells. Strikingly, Huh7.5HCV cells were eight fold more susceptible to SARS-CoV-2 pseudoviruses than naive Huh7.5 cells, suggesting enhanced SARS-CoV-2 entry into HCV-preinfected hepatocytes. In addition, we observed increased binding of spike receptor-binding domain (RBD) protein to Huh7.5HCV cells, as well as enhanced cell-to-cell fusion of Huh7.5HCV cells with spike-expressing Huh7.5 cells. We explored the mechanism of enhanced SARS-CoV-2 entry and identified an increased ACE2 mRNA and protein levels in Huh7.5HCV cells, primary hepatocytes, and in data from infected liver biopsies obtained from database. Importantly, higher expression of ACE2 increased HCV infection by enhancing its binding to the host cell, underscoring its role in the HCV life cycle as well. Transcriptome analysis revealed that shared host signaling pathways were induced in HCV-SARS-CoV-2 coinfection. This study revealed complex interactions between HCV and SARS-CoV-2 infections in hepatocytes, which may lead to the increased liver damage recently reported in HCV-positive COVID-19 patients. IMPORTANCE Here, we provide the first experimental evidence for the coexistence of SARS-CoV-2 infection with HCV, and the interplay between them. The study revealed a complex relationship of enhancement between the two viruses, where HCV infection increased the expression of the SARS-CoV-2 entry receptor ACE2, thus facilitating SARS-CoV-2 entry, and potentially, also HCV entry. Thereafter, SARS-CoV-2 infection enhanced HCV replication in hepatocytes. This study may explain the aggravation of liver damage that was recently reported in COVID-19 patients with HCV coinfection and suggests preinfection with HCV as a risk factor for severe COVID-19. Moreover, it highlights the possible importance of HCV treatment for coinfected patients. In a broader view, these findings emphasize the importance of identifying coinfecting pathogens that increase the risk of SARS-CoV-2 infection and that may accelerate COVID-19-related co-morbidities.

Induction of Innate Immune Response by TLR3 Agonist Protects Mice against SARS-CoV-2 Infection.

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.

Genome-wide CRISPR screens identify GATA6 as a proviral host factor for SARS-CoV-2 via modulation of ACE2.

The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show 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 shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent 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.