Genome-scale identification of SARS-CoV-2 and pan-coronavirus host factor networks

Abstract

SUMMARYThe COVID-19 pandemic has claimed the lives of more than one million people worldwide. The causative agent, SARS-CoV-2, is a member of the Coronaviridae family, which are viruses that cause respiratory infections of varying severity. The cellular host factors and pathways co-opted by SARS-CoV-2 and other coronaviruses in the execution of their life cycles remain ill-defined. To develop an extensive compendium of host factors required for infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E), we performed parallel genome-scale CRISPR knockout screens. These screens uncovered multiple host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis, as well as an unexpected requirement for several poorly characterized proteins. We identified an absolute requirement for the VTT-domain containing protein TMEM41B for infection by SARS-CoV-2 and all other coronaviruses. This human Coronaviridae host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus spillover events.HIGHLIGHTSGenome-wide CRISPR screens for SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E coronavirus host factors.Parallel genome-wide CRISPR screening uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles.Coronaviruses co-opt multiple biological pathways, including glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis and anchoring, among others.TMEM41B - a poorly understood factor with roles in autophagy and lipid mobilization - is a critical pan-coronavirus host factor.

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

“…This finding is in contrast to a number of recent studies that performed genome-scale CRISPR-Cas9 screens in VeroE6, Huh-7, Huh-7.5.1, Huh-7.5, and ACE2-overexpressing A549 cells, which failed to identify significant enrichment of the same PPI network ( Baggen et al., 2020 ; Daniloski et al., 2020 ; Heaton et al., 2020 ; Schneider et al, 2020 ; Wang et al., 2020 ; Wei et al., 2020 ; Zhu et al., 2020 ). We speculate that this difference might be due in part to well-established advantages of focused screens, including (1) improved signal-to-noise ratios in the absence of sgRNAs targeting genes that are likely to dominate the screen (e.g., the SARS-CoV-2 cellular receptor, ACE2), (2) increased coverage by including more sgRNAs per gene (ten versus four), (3) feasibility of exploring multiple screening conditions (e.g., different viruses and temperatures), and (4) higher screen representation (i.e., >1,500-fold versus < 750-fold), as well as the lower propensity of Huh-7.5 cells to undergo syncytia formation in relation to ACE2-overexpressing A549 cells, which potentially confounds pooled screening results ( DeWeirdt et al., 2020 ; Doench, 2018 ; Parnas et al., 2015 ; Schneider et al, 2020 ). Thus, deeply probing individual interactome proteins by using a focused sgRNA library coupled with functional readouts across multiple conditions allowed us to identify many host factors required for SARS-CoV-2 infection that were missed in genome-scale CRISPR screens.…”

“…Importantly, these regulators of lipid and cholesterol homeostasis are all constitutively expressed in Huh-7.5 cells ( Figure S1 ), as well as in cells and tissues known to be infected by SARS-CoV-2 ( Figure S4 ). These results are also consistent with our accompanying work ( Schneider et al, 2020 ) demonstrating that regulators of cholesterol homeostasis, including SCAP, SREBF2, membrane-bound transcription factor site 1 protease (MBTPS1), MBTPS2, and secretion associated ras related GTPase 1A (SAR1A) play critical roles during coronavirus infection and further suggest that targeting SCAP might be a potential pan-coronavirus therapeutic strategy.

Figure 5 Pathway focused views of high confidence CRISPR hits (A) Heatmap of Z scores for the union of positively enriched coronavirus host factors across experimental replicates in all infection conditions.

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“…The urgency of the ongoing SARS-CoV-2 pandemic has advanced coronavirus research at unprecedented speed. Several groups performed genome-wide CRISPR-Cas9 screens to identify host factors required by SARS-CoV-2 ( Baggen et al., 2020 ; Daniloski et al., 2020 ; Heaton et al., 2020 ; Schneider et al, 2020 ; Wang et al., 2020 ; Wei et al., 2020 ; Zhu et al., 2020 ) with the hope that these screens would not only uncover the biology underlying this virus, but also identify druggable host factors that could be targeted to prevent virus spread or lessen disease. Other groups conducted drug repurposing screens using compound libraries enriched with FDA-approved or clinical-phase drug candidates to identify off-the-shelf interventions ( Dittmar et al., 2020 ; Zhou et al., 2020 ).…”

BRD2 inhibition blocks SARS-CoV-2 infection by reducing transcription of the host cell receptor ACE2

“…Previous CRISPR screens were performed in cell-based models of SARS-CoV-2 infection that overexpressed an ACE2 transgene 9 , 10 , represented cell types not primarily targeted by SARS-CoV-2 11 , or were non-human cells 12 . While these studies elucidated major features of SARS-CoV-2 biology, we reasoned that the cell lines used would not have enabled the discovery of regulators of ACE2 expression in relevant human cell types.…”

Autophagosome maturation stymied by SARS-CoV-2

“…Coronaviruses are known to interact with the autophagy pathway ( Carmona-Gutierrez et al., 2020 ; Delorme-Axford and Klionsky, 2020 ; Miller et al., 2020 ). Although core autophagy genes don’t seem to be required for coronavirus infection ( Zhao et al., 2007 ; Schneider et al., 2021 ; Hoffmann et al., 2020 ), the nonlipidated form of LC3 (LC3-I) appears to be important for viral replication in the case of mouse hepatitis virus infection ( Reggiori et al., 2010 ). LC3 is one of the proteins marking the membranes of autophagosomes, which viral replication complexes strongly resemble; both are derived from the ER, and both are double-membrane vesicles.…”