Autophagy‐linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS‐CoV‐2 entry into human cells

Better understanding on interactions between SARS‐CoV‐2 and host cells should help to identify host factors that may be targetable to combat infection and COVID‐19 pathology. To this end, we have conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen in human lung cancer cells infected with SARS‐CoV‐2‐pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS‐CoV‐2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS‐CoV‐2 viruses confirm that loss‐of‐function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single‐cell RNA‐Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS‐CoV‐2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions. Synopsis Identifying targetable host factors to fight COVID‐19 requires full understanding of virus‐host cell interactions. Here, a genome‐wide loss‐of‐function screen identifies new regulators of the intracellular fate of endocytosed SARS‐CoV‐2 virions. A genome‐wide CRISPR/Cas9‐based screen in human lung cancer cells identifies lysosomal membrane proteins PLAC8 and SPNS1 as required for viral entry. PLAC8 loss reduces SARS‐CoV‐2 infection efficiency, while overexpression boosts it in several lung cancer lines Single‐cell RNA‐Seq analyses reveal a strong enrichment of PLAC8 expression in lung and colon SARS‐CoV‐2 target cells PLAC8 colocalizes with ACE2, but its loss‐of‐function does not affect binding or endocytosis in experiments with recombinant SPIKE receptor binding domain PLAC8 loss‐of‐function causes an expansion of the autophagolysosomal compartment Graphical Abstract Genome‐wide CRISPR screens identify lysosomal efflux transporter SPNS1 and transmembrane protein PLAC8 as regulators of the intracellular fate of endocytosed SARS‐CoV‐2 virions.