Thu. Jan 20th, 2022

The causative agent responsible for the unprecedented 2019 coronavirus disease (COVID-19) pandemic is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The mechanism of action of SARS-CoV-2 involves the binding of the viral spike (S) protein to the cellular angiotensin converting enzyme (ACE) 2 receptor leading to proteolytic treatment of the S precursor to the active S1 and S2 subunits. However, there are other factors responsible for SARS-CoV-2 entry, prevalence, and pathogenesis.

In a previous study, researchers have shown that a variant of SARS-CoV-2 isolated in the Netherlands on 12 February 2020 seizes interferon-induced transmembrane (IFITM) proteins, especially IFITM2, as entry co-factors during infection.

The World Health Organization (WHO) has classified four SARS-CoV-2 variants as variants of concern (VOCs): B.1.1.7, B.1.351, P.1 and B.1.617.2. These are also known as alpha, beta, gamma and delta variants, respectively.

In a study recently published on bioRxiv* preprint server, researchers studied IFITM2 dependence on SARS-CoV-2 VOCs, including the delta variant for effective infection.

Study: FITM dependence on SARS-CoV-2 variants of concern.  Image credit: Pong Ch
Study: FITM dependence on SARS-CoV-2 variants of concern. Image credit: Pong Ch

Interferon-induced transmembrane proteins for efficient SARS-CoV-2 infection

IFITMs are a family of IFN-stimulated genes (ISGs) that are known to protect cells against infection with many viruses, such as human immunodeficiency viruses, influenza A, rhabdo, and highly pathogenic coronaviruses, including SARS-CoV-2.

In this study, the researchers used the human epithelial lung cancer cell line Calu-3 because of its ability to siRNA knockdown (KD) of IFITM expression and iPSC-derived alveolar epithelial type II (iATII) cells as a model for the primary target cells of SARS-CoV 2 infection in the distal lung.

The Western blot analysis could not detect the ACE2 expression of iATII cells; however, it was easily detectable using fluorescence-activated cell sorting.

Effects of IFITM proteins on replication of SARS-CoV-2 variants.  (A) Mutations in the Spike proteins of SARS-CoV-2 VOCs (Beta, blue; Alpha, green; Gamma, yellow; Delta, red) compared to the SARS-CoV-2 (NL-02-2020) strain.  (B) Immunoblot of Calu-3 and iATII cells left uninfected (c) or infected with the indicated SARS-CoV-2 variants.  Whole cell lysates were stained with the indicated antibodies.  A nonspecific signal was observed in the Calu-3 control pathway stained with the CoV-2 N antibody.  (C) Viral N-RNA levels in the supernatant of Calu-3 cells, collected 48 hours after infection with SARS-CoV-2 (MOI 0.05).  Cells were transfected with control (CTRL) or IFITM-targeted siRNAs as indicated.  Bars represent the average of 3 to 4 independent experiments (± SEM).  (D) Effect of IFITM2 siRNA knock-down on infectious SARS-CoV-2 yields.  Supernatants derived from Calu-3 cells treated with control (CTRL) of IFITM2 siRNA two days after infection with SARS-CoV-2 NL-02-2020 or VOCs were serially diluted and added to Caco-2 cells seeded in 96- well plates.  Five days later, cells were examined for CPE, fixed, and stained with crystal violet.  Productively infected wells appear transparent as cells are eliminated and released by the virus.  (E) Infectious SARS-CoV-2 particles in the supernatant of Calu-3 cells treated with control or IFITM2-targeted siRNAs.  Bars represent the average of an experiment performed with eight technical replicates (± SD) shown in panel D. (F) Quantification of viral N RNA levels in the supernatant of iATII cells treated with α-IFITM2 antibody (20, 40 or 80 μg / ml) or Remdesivir (10 μM) 1 hour before infection (SARS-CoV-2, MOI 0.5), collected 48 hours after infection.  Bars represent the average of three independent experiments.  (G) Mean percentage of reduction in vRNA levels in the supernatants of (E) compared to the untreated control.
Effects of IFITM proteins on replication of SARS-CoV-2 variants. (A) Mutations in the Spike proteins of SARS-CoV-2 VOCs (Beta, blue; Alpha, green; Gamma, yellow; Delta, red) compared to the SARS-CoV-2 (NL-02-2020) strain. (B) Immunoblot of Calu-3 and iATII cells left uninfected (c) or infected with the indicated SARS-CoV-2 variants. Whole cell lysates were stained with the indicated antibodies. A nonspecific signal was observed in the Calu-3 control pathway stained with the CoV-2 N antibody. (C) Viral N-RNA levels in the supernatant of Calu-3 cells, collected 48 hours after infection with SARS-CoV-2 (MOI 0.05). Cells were transfected with control (CTRL) or IFITM-targeted siRNAs as indicated. Bars represent the average of 3 to 4 independent experiments (± SEM). (D) Effect of IFITM2 siRNA knock-down on infectious SARS-CoV-2 yields. Supernatants derived from Calu-3 cells treated with control (CTRL) of IFITM2 siRNA two days after infection with SARS-CoV-2 NL-02-2020 or VOCs were serially diluted and added to Caco-2 cells seeded in 96- well plates. Five days later, cells were examined for CPE, fixed, and stained with crystal violet. Productively infected wells appear transparent as cells are eliminated and released by the virus. (E) Infectious SARS-CoV-2 particles in the supernatant of Calu-3 cells treated with control or IFITM2-targeted siRNAs. Bars represent the average of an experiment performed with eight technical replicates (± SD) shown in panel D. (F) Quantification of viral N RNA levels in the supernatant of iATII cells treated with α-IFITM2 antibody (20, 40 or 80 μg / ml) or Remdesivir (10 μM) 1 hour before infection (SARS-CoV-2, MOI 0.5), collected 48 hours after infection. Bars represent the mean of three independent experiments. (G) Mean percentage of reduction in vRNA levels in the supernatants of (E) compared to the untreated control.

The results of the study

The results showed that the SARS-CoV-2 Delta variant of concern compared to NL-02-2020 in Calu-3 cells generated 3 times higher levels of viral RNA.

The results of the study showed that there was a reduction in viral RNA production from 31- (Alpha) to 754-fold (Gamma) by depletion of endogenous IFITM2 expression in Calu-3 cells.

Furthermore, it was noted that the KD of IFITM1 had a small effect; however, attenuation of IFITM3 resulted in 2- to 31-fold depletion of viral RNA production.

Interestingly, the production of infectious SARS-CoV-2 particles in Calu-3 cells was reduced to almost background levels by attenuating IFITM2. Furthermore, the replication of SARS-CoV-2 VOC in iATII was reduced by the antibody targeting the N-terminus of IFITM2 in a dose-dependent manner.

IFITMs (especially IFITM2) are also critical cofactors for efficient replication of current SARS-CoV-2 VOCs including the dominant Delta variant. “

Conclusions

The results of the study suggest that endogenous IFITMs (especially IFITM2) are important cofactors for the efficient replication of SARS-CoV-2 VOCs, including the dominant Delta variant. It was noted that the SARS-CoV-2 Delta variant replicated at a 30-fold higher rate compared to the early NL-02-2020 isolate in iATII cells. Thus, the dependence on IFITM2 maintained by VOCs can be further explored as a measure of therapeutic or preventive approaches.

Effective inhibition of SARS-CoV-2 Delta VOC by an α-IFITM2 antibody illustrates that IFITM2 may play a crucial role in SARS-CoV-2 transmission as well as pathogenesis.

“Our observation that IFITM2 dependence is maintained by VOCs also further underscores that this cellular ‘antiviral’ factor represents an interesting target for therapeutic or preventive approaches.”

*Important message

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

Journal reference:

  • IFITM dependence on SARS-CoV-2 variants is a cause for concern. Rayhane Nchioua, Annika Schundner, Dorota Kmiec, Caterina Prelli Bozzo, Fabian Zech, Lennart Koepke, Manfred Frick, Konstantin MJ Sparrer, Frank Kirchhoff, bioRxiv, 2021. doi: https://doi.org/10.1101/2021.11.17.468942, https://www.biorxiv.org/content/10.1101/2021.11.17.468942v1

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