Tuesday, February 9, 2021

Removing the host factors.

Highlights

  • •
    Genome-wide CRISPR screens for SARS-CoV-2, HCoV-229E, and HCoV-OC43 host factors
  • •
    Screens correctly identified divergent entry factors for the three coronaviruses
  • •
    Cholesterol and phosphatidylinositol pathways are shared host dependency factors
  • •
    Pharmacological inhibition of host factors reduces coronavirus replication

Summary

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted 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 identified 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 kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.

Graphical Abstract

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Keywords

Introduction

The Coronaviridae family includes seven known human pathogens for which there are no approved vaccines and only limited therapeutic options. The seasonally circulating human coronaviruses (HCoV) OC43, HKU1, 229E, and NL63 cause mild, common cold-like respiratory infections in humans (
). However, three highly pathogenic coronaviruses emerged in the last two decades, highlighting the pandemic potential of this viral family (
; 
; 
). Infection with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV) can lead to acute respiratory distress syndrome and death, with fatality rates between 10%–40% (
). SARS-CoV-2, though less deadly, is far more transmissible than SARS-CoV-1 and MERS-CoV and has been responsible for over 50 million cases and 1.2 million deaths globally as of November 2020 (
; 
). Because of the severity of their impact on global health, it is critical to understand how SARS-CoV-2 and other coronaviruses hijack the host cell machinery during infection and apply this knowledge to develop new therapeutic strategies.
Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a genome length of approximately 30 kb. Upon receptor binding and membrane fusion, the viral RNA is released into the cytoplasm, where it is translated to produce viral proteins. Subsequently, the viral replication/transcription complexes form on double-membrane vesicles and generate genome copies. These are then packaged into new virions via a budding process, through which they acquire the viral envelope, and the resulting virions are released from infected cells (
). During these steps, specific cellular proteins are hijacked and play crucial roles in the viral life cycle. For example, the angiotensin-converting enzyme 2 (ACE2) is exploited as the viral entry receptor for NL63, SARS-CoV-1, and SARS-CoV-2 (
; 
; 
). Additionally, cellular proteases, such as TMPRSS2, cathepsin L, and furin, are important for the cleavage of the viral spike (S) protein of several coronaviruses thereby mediating efficient membrane fusion with host cells (
; 
; 
; 
; 
). Systematic studies have illuminated virus-host interactions during the later steps of the viral life cycle. For example, proteomics approaches revealed comprehensive interactomes between individual coronavirus proteins and cellular proteins (
; 
; 
). Additionally, biotin labeling identified candidate host factors based on their proximity to coronavirus replicase complexes (
). While these studies uncovered physical relationships between viral and cellular proteins, they do not provide immediate information about the importance of these host components for viral replication.
An orthogonal strategy is to screen for mutations that render host cells resistant to viral infection using CRISPR-based mutagenesis. These screens identify host factors that are functionally required for viral infection and could be targets for host-directed therapies (
). In this study, we have performed a genome-wide CRISPR knockout (KO) screen using SARS-CoV-2 (USA/WA-1 isolate) in human cells. Importantly, we expanded our functional genomics approach to distantly related Coronaviridae members in order to probe for commonalities and differences across the family. This strategy can reveal potential pan-coronavirus host factors and thus illuminate targets for antiviral therapy to combat the current and potential future outbreaks. We conducted comparative CRISPR screens for SARS-CoV-2 and two seasonally circulating common cold coronaviruses, OC43 and 229E. Our results corroborate previously implicated host pathways, uncover new aspects of virus-host interaction, and identify targets for host-directed antiviral treatment.

Results

 CRISPR KO Screens Identify Common and Virus-Specific Candidate Host Factors for Coronavirus Infection

Phenotypic selection of virus-resistant cells in a pooled CRISPR KO screen is based on survival and growth differences of mutant cells upon virus infection. We chose Huh7.5.1 hepatoma cells as they were uniquely susceptible to all tested coronaviruses. We readily observed drastic cytopathic effect during OC43 and 229E infection (Figure S1A). Huh7.5.1 also supported SARS-CoV-2 replication but exhibited limited virus-induced cell death (Figures S1B and S1C). To improve the selection conditions for the SARS-CoV-2 CRISPR screen, we overexpressed ACE2 and/or TMPRSS2, which are present at low levels in wild-type (WT) Huh7.5.1 cells (Figure S1D). This led to increased viral uptake of a SARS-CoV-2 S-pseudotyped lentivirus, confirming the important function of ACE2 and TMPRSS2 for SARS-CoV-2 entry (Figure S1E). We ultimately used Huh7.5.1 cells harboring a bicistronic ACE2-IRES-TMPRSS2 construct for the SARS-CoV-2 screen as these cells sustained efficient infection that led to widespread cell death while still allowing the survival of a small number of cells (Figures S1C and S1F). The generated CRISPR KO libraries in Huh7.5.1 and Huh7.5.1-ACE2-IRES-TMPRSS2 cells had virtually complete single-guide RNA (sgRNA) representation prior to the start of the virus challenge but, as expected, were depleted of cells containing sgRNAs against commonly essential fitness genes within 7 days post-library transduction (Figures S1G and S1H) (
).
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Figure S1Optimization of Phenotypic Selection of Coronavirus-Infected Huh7.5.1 Cells and Quality Control Metrics for CRISPR Screens, Related to 
The three CRISPR screens—for resistance to SARS-CoV-2, 229E, and OC43—identified a compendium of critical host factors across the human genome (Figure 1A; Table S1). The overall performance of the screens was robust as indicated by the enrichment of multiple individual sgRNAs against the top 10 scoring genes from each screen (Figure S1I). Importantly, the known viral entry receptors ranked among the top hits: ACE2 for SARS-CoV-2 and aminopeptidase N (ANPEP) for 229E (Figures 1B and 1C) (
; 
). OC43, unlike the other coronaviruses, does not have a known proteinaceous receptor but primarily depends on sialic acid or glycosaminoglycans for cell entry (
; 
); consistent with this fact, multiple heparan sulfate biosynthetic genes (B3GALT6, B3GAT3, B4GALT7, EXT1, EXT2, EXTL3, FAM20B, NDST1, SLC35B2, UGDH, XYLT2) were identified in our OC43 screen (Figures 1D and S2A). Several of these genes were also markedly enriched in the SARS-CoV-2 screen (Figures 1B and S2A), which is consistent with a recent report that SARS-CoV-2 requires both ACE2 and cellular heparan sulfate for efficient infection (
). Overall, the identification of the expected entry factors validates the phenotypic selection of our host factor screens.
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Figure 1Genome-wide Loss-of-Function Screens in Human Cells Identify Host Factors Important for Infection by SARS-CoV-2, 229E, and OC43
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