SARS-CoV-2 variants of concern remain dependent on IFITM2 for efficient replication in human lung cells
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Abstract
It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several “Variants of Concern” (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant.
IMPORTANCE
Recent results showed that an early SARS-CoV-2 isolate requires endogenously expressed IFITM proteins for efficient infection. However, whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that out-competed the original strains and currently dominate the pandemic remained to be determined. Here, we demonstrate that depletion of endogenous IFITM2 expression almost entirely prevents the production of infectious Alpha, Beta, Gamma and Delta VOC SARS-CoV-2 virions in a human lung cell line. In comparison, silencing of IFITM1 had little impact, while knock-down of IFITM3 had intermediate effects on viral replication. Finally, an antibody targeting the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs including the currently dominant Delta variant are dependent on IFITM2 for efficient replication suggesting that IFITM proteins play a key role in viral transmission and pathogenicity.
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SciScore for 10.1101/2021.11.17.468942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization First, 1 μl random hexamers (50 ng/μl), 1 μl dNTPs mix (10 mM each), and 11 μl template RNA (diluted 1:10 in DNase/RNase free water) were mixed, incubated at 65°C for 5 min and placed on ice for 1 min. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Inhibition by IFITM2 antibody and Remdesivir: 30,000 iATII cells were seeded as single cells in 96-well plates coated for 1 h at 37 °C with 0.16 mg/ml Matrigel (Corning, Cat#356238) diluted in DMEM/F12 (Thermo Fisher, Cat#11330032), 24 h later cells were treated with increasing concentrations (20, … SciScore for 10.1101/2021.11.17.468942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization First, 1 μl random hexamers (50 ng/μl), 1 μl dNTPs mix (10 mM each), and 11 μl template RNA (diluted 1:10 in DNase/RNase free water) were mixed, incubated at 65°C for 5 min and placed on ice for 1 min. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Inhibition by IFITM2 antibody and Remdesivir: 30,000 iATII cells were seeded as single cells in 96-well plates coated for 1 h at 37 °C with 0.16 mg/ml Matrigel (Corning, Cat#356238) diluted in DMEM/F12 (Thermo Fisher, Cat#11330032), 24 h later cells were treated with increasing concentrations (20, 40, 80 μg/ml) of α-IFITM2 (Cell Signaling, Cat#13530 S) or Remdesivir (Selleck Chemicals Cat#S8932) (10 μM). IFITM2suggested: (Cell Signaling Technology Cat# 13530, RRID:AB_2798248)α-IFITM2suggested: NoneProteins were stained using primary antibodies against IFITM1 (α-IFITM1, Cell Signaling Cat#13126S, 1:1,000), IFITM2 (α-IFITM2 Cell Signaling Cat#13530 S, 1:1,000), IFITM3 (α-IFITM3 Cell Signaling Cat#59212S, 1:1,000), ACE2 (Rabbit polyclonal anti-ACE2 Abcam, Cat#ab166755, 1:1,000); rat anti-GAPDH (Biolegend Cat#607902, 1:1,000) and SARS CoV-2 N (anti-SARS-CoV-2 N Sino Biologicals Cat#40588-V08B, 1:1,000) and Infrared Dye labeled secondary antibodies (LI-COR IRDye). IFITM1suggested: Noneα-IFITM1suggested: NoneIFITM3suggested: Noneα-IFITM3suggested: NoneACE2suggested: Noneanti-GAPDHsuggested: (BioLegend Cat# 607902, RRID:AB_2734503)anti-SARS-CoV-2suggested: NoneExperimental Models: Cell Lines Sentences Resources Caco-2 cells (human epithelial colorectal adenocarcinoma, kindly provided by Prof. Holger Barth (Ulm University)) were grown in the same media as Vero E6 cells but with supplementation of 10% heat-inactivated FBS. Caco-2suggested: NoneSARS-CoV-2 strains were propagated on Vero E6 (NL-02-2020, Delta), VeroE6 overexpressing TMPRSS2 (Alpha), CaCo-2 (Beta) or Calu-3 (Gamma) cells. Vero E6suggested: None6 h after the second transfection, Calu-3 cells were infected with the various SARS-CoV-2 variants at an MOI of 0.05. Calu-3suggested: BCRJ Cat# 0264, RRID:CVCL_0609)Software and Algorithms Sentences Resources The obtained sequenced reads were demultiplexed and mapped against the SARS-CoV-2 reference genome (NC_045512.2) with BWA-MEM3. BWA-MEM3suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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