CRISPR Screening Identifies Human Host Pathways That Facilitate Coronavirus Infection

The human coronavirus family consists of 7 known pathogens, for which there are no approved vaccines or specific therapeutic options. Although the seasonal human coronaviruses, OC43, HKU1, 229E, and NL63, only cause mild respiratory infections, three highly pathogenic coronaviruses that emerged in the past two decades revealed the pandemic potential of this family of viruses.

It is known that severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV) can cause acute respiratory distress syndrome and even death. Mortality rates for these viruses ranged from 10 to 40%.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the current COVID-19 pandemic, has a lower death rate but is much more transmissible than SARS-CoV-1 and MERS-CoV. So far, it has been responsible for nearly 33 million cases and 996,000 deaths worldwide.

Given the severity of the impact of these viruses on human health, we must understand the mechanism behind the invasion of host cell machinery by SARS-CoV-2 and other coronaviruses during infection. Identifying the host factors common to several coronaviruses could help develop therapies that can combat current and future pandemics caused by coronaviruses.

CRISPR screens of infected cells reveal different viral entry factors

In a recent prepress article published in prepress bioRxiv, * Researchers from the University of California, San Francisco, Gladstone Institutes, Chan Zuckerberg Biohub and Synthego Corporation, San Francisco, discuss how they identified some host factors common to 3 coronaviruses with the help of the gene editing tool, CRISPR.

The team of researchers performed parallel CRISPR screens of the entire genome of cells infected with SARS-CoV-2 and two seasonal common cold coronaviruses: OC43 and 229E. They were able to identify the different viral entry factors for the 3 viruses: ACE2 for SARS-CoV-2, glycosaminoglycans for OC43 and aminopeptidase N for 229E. Additionally, they also found that phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis are critical host pathways that support infection by these three coronaviruses.

TMEM106B, the lysosomal protein, was unique to SARS-CoV-2 infection. By pharmacological inhibition of cholesterol homeostasis and phosphatidylinositol phosphate biosynthesis, the authors were able to reduce the replication of all three coronaviruses.

Genome-wide CRISPR KO screens in human cells identify important host factors for SARS-CoV-2, HCoV-229E, and HCoV-OC43 infection. (a) Schematic of CRISPR KO screens for the identification of coronavirus host factors. Huh7.5.1-Cas9 (with the bicistronic construct ACE2-IRES-TMPRSS2 for SARS-CoV-2 and without the 229E and OC43 screen) were mutagenized using a whole genome sgRNA library. Mutant cells were infected with each coronavirus separately and virus resistant cells were harvested 10-14 days after infection (dpi). The 347 abundance of each sgRNA in the initial and selected population was determined by high throughput sequencing and a gene enrichment analysis was performed. (bd) Enrichment of genes from CRISPR screens for (b) SARS-CoV-2, (c) 229E and (d) infection by OC43. Enrichment scores were determined by MaGECK analysis and genes were colored according to biological function. SARS-CoV-2 was done once. Screens 229E and OC43 were run twice and the combined MaGECK scores are shown.

Data Suggests Unique Entry Factors, But Common Host Pathways for 3 Coronaviruses

The results of the study highlight that, while all three coronaviruses depend on unique input factors, they have a common set of host pathways that aid in infection. Genes related to cholesterol homeostasis were highlighted on all screens and in the spread of the network.

“Due to their involvement in multiple cellular processes, including vesicular trafficking and autophagy, it remains to be determined whether coronaviruses sequester this pathway during entry or for the generation of double-membrane vesicles necessary for viral transcription / replication complexes.”

Consistent with the findings, 2 SARS-CoV-2 interactomes revealed viral proteins that bind to SCAP, which has been identified as a host factor that is critical for the replication of the SARS-CoV-2 virus. The researchers argue that given SCAP’s role in viral replication, viral proteins likely positively regulate SCAP activity and cholesterol levels. These findings provide key insights into understanding the life cycle of coronaviruses and important directions for the development of host-directed therapies to combat coronavirus infection.

Previous studies have linked cellular cholesterol homeostasis to viral entry and membrane fusion in bunya and hantavirus infections. This suggests a pro-viral role in different virus families. The team’s findings agreed with this hypothesis. They were able to reduce infection with pseudotyped viruses with SARS-CoV-1 and CoV-2 peaks by treatment with 25-hydroxycholesterol, which blocks SREBP processing and inhibits cholesterol synthesis.

The results are also consistent with those of a recent drug reuse analysis that identified more than 100 compounds, including PIKfyve inhibitors, protease inhibitors, and modulators of cholesterol homeostasis, that inhibited SARS-CoV-2 replication. The functional genomic data obtained from this study suggest that the observed effects of these compounds were potentially due to the inhibition of crucial host factors.

“Our results corroborate previously implicated host pathways, uncover new aspects of virus-host interaction, and identify targets for host-directed antiviral therapy.”

According to the team, the study offers critical information about host pathways, usually hijacked by coronavirus. They identified the phosphatidylinositol kinase complex PIK3C3 as a possible therapeutic target for SARS-CoV-2 based on the 229E and OC43 screens, highlighting the value of parallel CRISPR detection to develop new therapies against SARS-CoV-2 and other viruses. Coronaviridae family.

*Important news

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.