NEW YORK – By performing genome-wide functional genetic screens, researchers in Belgium have identified host factors that are required for SARS-CoV-2 infection as well as others that are shared by various coronaviruses. These factors could be used to develop drugs to treat SARS-CoV-2 infection or even outbreaks of future zoonotic coronaviruses.
In a study published on Monday in Nature Genetics, researchers at KU Leuven described their use of genetic screens of SARS-CoV-2 and human coronavirus (HCoV) 229E, which can cause mild upper respiratory illnesses. These screens uncovered virus-specific as well as shared host factors, including TMEM41B and PI3K type 3. The researchers also found that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells.
Specifically, TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, which suggested that it plays a role in viral entry. Further, when the researchers performed single-cell RNA-sequencing of airway cells from patients with COVID-19, they found that TMEM106B expression correlated with SARS-CoV-2 infection.
"The lysosomal protein TMEM106B is a crucial host factor supporting SARS-CoV-2 infection. We show that the virus requires this protein for infection of multiple human cell lines and found that TMEM106B likely plays a role in the entry phase of the viral life cycle," first author Jim Baggen, a postdoctoral researcher in Dirk Daelemans' group at KU Leuven, said in an email. "The essential role of TMEM106B in SARS-CoV-2 infection makes this protein an interesting potential target for antiviral drugs. Although small molecule inhibitors of TMEM106B do not yet exist, the development of such inhibitors could potentially lead to drugs serving as treatment for COVID-19."
The researchers began by performing a series of genome-wide CRISPR-based genetic screens to identify host factors required for SARS-CoV-2 and HCoV-229E infection. They identified PI3K type 3 as a common host factor for SARS-CoV-2, HCoV-229E, and another of the so-called common cold HCoVs, OC43. Further, a high-stringency SARS-CoV-2 screen identified one significantly enriched gene, TMEM106B, encoding a poorly characterized protein that is involved in lysosome function and implicated in neurodegenerative disorders. In a validation screen, the knockout of TMEM106B conferred the strongest resistance to SARS-CoV-2, and significant resistance was observed for two individually tested sgRNAs targeting PIK3C3, TMEM41B, EXT1, OSBPL9, or TMEM30A.
Overall, the researchers said, the results demonstrated that SARS-CoV-2 infection requires TMEM106B as well as genes involved in heparan sulfate synthesis (EXT1), autophagy (PIK3C3 and TMEM41B), and lipid transport (OSBPL9 and TMEM30A). PI3K type 3 is a druggable common anti-coronavirus target.
PIK3C3 encodes the catalytic subunit of the PI3K complex, which plays a role in processes such as endocytic trafficking and the initiation and maturation of autophagosomes. Because inhibitors directly targeting this protein are already available, they tested the activity of the structurally distinct inhibitors VPS34-IN1, VPS34-IN2, SAR405, and autophinib against different coronaviruses and found that PI3K type 3 inhibitors showed antiviral activity against SARS-CoV-2 as well as HCoV-229E and HCoV-OC43.
In a follow-up experiment, the researchers further investigated the effect of TMEM106B knockout on SARS-CoV-2 infection in multiple human cell lines. Knocking the gene out in liver cells prevented SARS-CoV-2-induced cytopathic effects and reduced virus replication. Subsequent complementation with sgRNA-resistant TMEM106B cDNA restored the cytopathic effect and infectivity, confirming the specificity of the TMEM106B knockout, they said. This was further confirmed in lung-derived cell lines and primary human bronchial epithelial cells, but the researchers noted that HCoV-229E production was not affected in these cells, confirming the essential nature of TMEM106B specifically for SARS-CoV-2 infection.
The emergence of multiple SARS-CoV-2 variants, such as Variant VOC202012/01 (also known as lineage B.1.1.7 or the UK variant), will require further study to establish whether they still need TMEM106B and other host factors, the researchers noted. With the exception of spike deletion HV69-70, they said, the UK variant contains at least 24 different mutations that are absent in the strains they used in this study.
"Our manuscript shows that TMEM106B supports infection with at least two different SARS-CoV-2 strains, as well as SARS-CoV, the causative agent of SARS outbreaks in 2003," Baggen said. "We are currently investigating whether additional SARS-CoV-2 strains (such as the variants of concern) also depend on TMEM106B for infection. Future research should establish whether TMEM106B is a conserved host factor among viruses belonging to the same species or genus as SARS-CoV-2. If so, inhibitors of TMEM106B might potentially be used to combat future outbreaks of new pathogenic coronaviruses belonging to these groups."
He and his colleagues also added that their scRNA-seq analysis of airway cells from COVID-19 patients showed higher TMEM106B expression levels in airway epithelium of infected individuals compared to noninfected controls, which may suggest that high TMEM106B expression increases the susceptibility to SARS-CoV-2, causing higher infection rates. This would be in line with the researchers' in vitro data showing increased infectivity with TMEM106B overexpression. They also noted that it might be beneficial to study whether TMEM106B could serve as prognostic biomarker for severity of COVID-19.