PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

Abstract

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1–4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR–Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal β-barrel domain—but not lipid scramblase activity—was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

A genome-wide arrayed CRISPR screen identifies PLSCR1 as an intrinsic barrier to SARS-CoV-2 entry that recent virus variants have evolved to resist

“…Furthermore, we extend the recent characterization of PLSCR1 as an antiviral against SARS-CoV-2 impacting COVID-19 outcomes (Fig. 1) [18,23,24].…”

“…While the liver is not the primary target organ of SARS-CoV-2 infection, human hepatocellular carcinoma Huh-7.5 cells naturally express SARS-CoV-2 dependency factors, including the receptor angiotensin converting enzyme 2 (ACE2), and proved unexpectedly useful in SARS-CoV-2 research [18,[25][26][27][28][29][30][31][32]. Huh-7.5 cells do not produce IFN during SARS-CoV-2 infection, but they do induce an antiviral state in response to IFN pretreatment (Fig 2A-2C, Supp Tables 1-4), making them a convenient model to control the exposure of the cells to IFN.…”

Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection

“…HeLa cells were grown on coverslips for 24 to 48 hours before fixation in 4% paraformaldehyde (PFA). Anti-GBP1 (1B1; Santa Cruz; 1:500 dilution) and GBP2 (1E5; Origene; 1:200 dilution) antibodies were labeled for 2 hours at room temperature by goat anti-mouse Fab AF647 (Jackson ImmunoResearch, PA) and goat anti-rabbit IgG CF660C (Biotium, CA) at 1:200 dilution as described ( 29 ).…”

“…Sample mounting, image acquisition, and data processing were mostly performed as previously described ( 29 ) except that the imaging speed was 200 Hz with 642-nm laser intensity of ~12.5 kW/cm 2 . Typically, 3000×100~200 frames were recorded.…”

“…We first examined the human GBP1 coat complex surrounding cytosolic bacteria using 4Pi single-molecule switching (4Pi-SMS) nanoscopy and fast, live three-dimensional structured illumination microscopy (3D-SIM) with an OMX-SR Blaze imaging instrument equipped with high-speed galvanometers. 4Pi-SMS is a dual-objective, single-molecule localization, super-resolution microscope that resolves 3D structures to ~20 nm (200 Å) isotropically throughout entire mammalian cells ( 28 , 29 ); it enabled us to detect single GBP molecules on the surface of virulent Stm inside human cells. 3D-SIM imaging with the OMX-SR instrument is capable of ~180 frames per second, ensuring that coat complex assembly could be followed throughout the entire bacterial encapsulation process.…”