Revealing the key advocate: PLSCR1 identified as a potent anti-SARS-CoV-2 factor in COVID-19 immunity

In a recent study published in NatureThe researchers identified the role against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of phospholipid scramblase 1 (PLSCR1), an interferon-gamma (IFNγ)-induced cell-autonomous restriction factor.

Their identification will help improve the understanding of protective immunity in the human IFN response.

The study used genome-wide clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated protein 9 screens from human lung epithelial cells and hepatocytes ectopically expressing angiotensin-converting enzyme 2 (ACE2) to study the function of PLSCR1 against live SARS-CoV. -2 before and after IFNγ stimulation.

Study: PLSCR1 is a cell-autonomous defense factor against SARS-CoV-2 infection. Image Credit: AndriiVodolazhskyi / Shutterstock.com

Background

In many plants, bacteria, and animals, including humans, cell-autonomous immunity helps fight viral and other infections, e.g. Shigella flexneri and Mycobacterium tuberculosis. IFNγ, a type II cytokine, is a well-recognized agent for mobilizing cell-autonomous immunity in most nucleated cells.

Therefore, studies have found that increased expression of IFN types I and III (IFNα\β and IFNλ) confers protection against coronavirus disease 2019 (COVID-19) in adults and children.

In contrast, genetic lesions in IFN signaling and IFN type I and II autoantibodies are responsible for up to 20% of critical COVID-19 cases. T cells also recognize COVID-19 vaccines and SARS-CoV-2 variants of concern (COVs) by secreting IFNγ.

Until now, the focus of research has been to use neutralizing antibodies (nAbs) as COVID-19 therapies. Recent evidence that IFNγ therapy rescued immunocompromised patients with COVID-19 for whom convalescent plasma or remdesivir treatment failure prompted the research community to investigate whether IFNγ could orchestrate the defense against SARS-CoV-2.

About the study

In the present study, the researchers used human hepatoma Huh7.5 and A549-ACE2 cells (which mimic host cell targets within the respiratory tract) to test the potency of IFNγ in restricting live infection by SARS-CoV- 2, which they confirmed using CRISPR-Cas9 engineering. .

They then used genome-wide loss-of-function (LoF) screens to identify which IFNγ-induced restriction factors conferred this effect against SARS-CoV-2.

Investigators used a GeCKO version 2.0 single-guide ribonucleic acid (sgRNA) library to transduce each cell type and then performed puromycin selection to ensure stable integration. This library had 122,441 sgRNAs spanning 19,050 genes.

They used SARS-CoV-2 expressing mNeonGreen (mNG) to infect sgRNA-integrated cells treated with recombinant human IFNγ. Then, with the help of fluorescence activated cell sorting (FACS), the researchers separated the SARS-CoV-2-infected cells into mNGhigh and mNGlow populations, where the former were permissive while the latter were restrictive. Next, they performed next-generation sequencing of sgRNA frequencies.

In addition, the team identified PLSCR1 which identified which step in the SARS-CoV-2 life cycle and how it blocked coronavirus invasion via either a cathepsin-dependent endosomal fusion pathway or a cathepsin-dependent cell surface fusion pathway. transmembrane serine protease 2 (TMPRSS2).

In addition, the team used a NanoLuc split-reporter-based assay to test the fusion of virus and endosome in the host cell cytosol. Furthermore, they combined nanoscale imaging with protein mutagenesis to identify the membrane determinants needed to distinguish how PLSCR1 prevented SARS-CoV-2 fusion.

Results and conclusion

The current study had several important findings. First, the authors found that after exposure to recombinant human IFNγ type II, Huh7.5 cells restricted SARS-CoV-2 in a signal transducer and activator of transcription-1 (STAT1)-dependent manner.

Second, the IFNγ-induced PLSCR1-restricted SARS-CoV-2 ancestral strain, USA-WA1/2020; however, it was also effective against Delta and Omicron BA.1 VOCs. Its antiviral activity remained robust even against other highly pathogenic. coronavirushaving bats and mice as hosts.

Third, in PLSCR1-knockout cells, SARS-CoV-2 spike(S)-mediated cell-cell fusion was significantly increased, whereas PLSCR1 overexpression reduced this response. These observations confirmed that PLSCR1 directly prevented membrane fusion caused by the S protein of SARS-CoV-2.

Similarly, in A549-ACE2 cells, PLSCR1-enriched foci appeared within 30 min of viral infection and viral particles detected by anti-S antibodies completely overlapped. PLSCR1 acted against SARS-CoV-2 entry and targeted viral entry mediated by S.

Interestingly, it interfered with SARS-CoV-2 uptake into the endocytic and TMPRSS2-dependent fusion pathways prior to the release of viral RNA into the host cell cytosol, i.e., at a late-entry step. Thus, PLSCR1 The mutations could make people highly prone to severe COVID-19, as confirmed by a recent report.

Structural modeling predicted that PLSCR1 had a flexible N-terminal domain and a 12-stranded membrane β-barrel burying the C-terminal hydrophobic helix.

Once attached to the target plasma membrane via palmitoylation, PLSCR1 used its C-terminal β barrel to disrupt virus-host cell fusion, known as fusogenic lockdown, as revealed by molecular dynamics simulation (MDS) analysis. ) of nanoseconds.

PLSCR1 likely occupied plasma membrane microdomains used by coronaviruses to target distinct subpopulations of SARS-CoV-2 vesicles. Given its palmitoyl anchorage, it operated on high and low curvature membranes (endosome and plasma membrane) to inhibit S.

Conclusion

Overall, this study provided a mechanistic framework for PLSCR1 in blocking SARS-CoV-2-S-mediated fusion by virulent coronaviruses.

It highlights the β-barrel of PLSCR1 as an important structural determinant of cell-autonomous immunity to the large family of coronaviruses.