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

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.

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2 (preprint)

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted an unbiased CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is an essential node in the cellular response to SARS-CoV-2 infection. BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells. BRD2 also controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates ACE2 levels. It is possible that the previously reported interaction between the viral E protein and BRD2 evolved to manipulate the transcriptional host response during SARS-CoV-2 infection. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

SARS-CoV-2 infection of circulating immune cells is not responsible for virus dissemination in severe COVID-19 patients (preprint)

In late 2019 a novel coronavirus (SARS-CoV-2) emerged, and has since caused a global pandemic. Understanding the pathogenesis of COVID-19 disease is necessary to inform development of therapeutics, and management of infected patients. Using scRNAseq of blood drawn from SARS-CoV-2 patients, we asked whether SARS-CoV-2 may exploit immune cells as a 'Trojan Horse' to disseminate and access multiple organ systems. Our data suggests that circulating cells are not actively infected with SARS-CoV-2, and do not appear to be a source of viral dissemination.

Sterically-Confined Rearrangements of SARS-CoV-2 Spike Protein Control Cell Invasion (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and transmission involves a series of processes that may be targeted by vaccines and therapeutics. During transmission, host cell invasion is controlled by a large-scale conformational change of the Spike protein. This conformational rearrangement leads to membrane fusion, which creates transmembrane pores through which the viral genome is passed to the host. During Spike-protein-mediated fusion, the fusion peptides must be released from the core of the protein and associate with the host membrane. Interestingly, the Spike protein possesses many post-translational modifications, in the form of branched glycans that flank the surface of the assembly. Despite the large number of glycosylation sites, until now, the specific role of glycans during cell invasion has been unclear. Here, we propose that glycosylation is needed to provide sufficient time for the fusion peptides to reach the host membrane, otherwise the viral particle would fail to enter the host. To understand this process, an all-atom model with simplified energetics was used to perform thousands of simulations in which the protein transitions between the prefusion and postfusion conformations. These simulations indicate that the steric composition of the glycans induces a pause during the Spike protein conformational change. We additionally show that this glycan-induced delay provides a critical opportunity for the fusion peptides to capture the host cell. This previously-unrecognized role of glycans reveals how the glycosylation state can regulate infectivity of this pervasive pathogen.