key: cord-0909477-ztyin1xk
authors: Nchioua, Rayhane; Schundner, Annika; Kmiec, Dorota; Bozzo, Caterina Prelli; Zech, Fabian; Koepke, Lennart; Frick, Manfred; Sparrer, Konstantin M. J.; Kirchhoff, Frank
title: IFITM dependency of SARS-CoV-2 variants of concern
date: 2021-11-17
journal: bioRxiv
DOI: 10.1101/2021.11.17.468942
sha: 0b3e3cb1c09b47cea470e06487bfc9e09ddd7499
doc_id: 909477
cord_uid: ztyin1xk

We have recently shown that a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020) hijacks interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection. Here, we examined whether SARS-CoV-2 “variants of concern” (VOCs), including the currently dominating delta variant, maintained the dependency on IFITMs for efficient replication. Depletion of IFITM2 reduced viral RNA production from 31- (B.1.1.7) to 755-fold (P.1). In comparison, silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, silencing of IFITM2 generally reduced infectious virus production in Calu-3 cells to near background levels. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for replication of genuine SARS-CoV-2 VOCs, including the Delta variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of 25 coronavirus disease 2019 , has caused a devastating pandemic. The reasons for 26 the efficient spread of SARS-CoV-2 are not fully understood but clearly involve the ability to 27 efficiently infect and propagate in human cells. Viral entry depends on binding of the viral 28 Spike (S) protein to the cellular angiotensin-converting enzyme (ACE) 2 receptor and 29 proteolytic processing of the S precursor into the active S1 and S2 subunits 1 . However, 30 additional host factors may affect the efficiency of SARS-CoV-2 entry and play key roles in 31 viral transmission and pathogenesis. 32

We recently demonstrated that interferon-induced transmembrane proteins (IFITMs 1, 2, and 33

3) are required for efficient SARS-CoV-2 infection 2 . This was unexpected, as IFITMs are a 34 family of IFN stimulated genes (ISGs) that are well-known to protect cells against infection by 35 various viral pathogens, such as influenza A, rhabdo-, and human immunodeficiency viruses 3 . 36 Inhibitory effects were also reported for highly pathogenic coronaviruses, including SARS-37

CoV-2 4,5 . However, most evidence was obtained using cells artificially overexpressing IFITM 38 proteins and pseudo-particles containing the S protein of SARS or MERS coronaviruses. 39

The antiviral mechanism of IFITMs is thought to involve alterations in the rigidity and 40 curvature of the cellular membrane, affecting viruses in a broad, unspecific way. In contrast, 41 the SARS-CoV-2 enhancing effect involves specific interactions between the S protein and the 42 N-terminal region of IFITMs, especially IFITM2 2 , suggesting that the virus hijacks IFITMs. 43

Consequently, knock-down of endogenous IFITM2 expression in human lung cell lines 44 reduced viral entry and infectious virus production by several orders of magnitude. In addition, 45 IFITM-derived peptides or IFITM targeting antibodies protected gut organoids and 46 cardiomyocytes against infection and cytopathic effects of SARS-CoV-2 2 . 47 

Correspondence to Frank Kirchhoff (Frank. Kirchhoff@uni-ulm.de) . 148

The authors declare no competing interests. IFITM2  CTRL  IFITM2  CTRL  IFITM2  CTRL  IFITM2  CTRL  IFITM2  CTRL IFITM1  IFITM2  IFITM3  CTRL IFITM1  IFITM2  IFITM3  CTRL IFITM1  IFITM2  IFITM3  CTRL IFITM1  IFITM2  IFITM3 a-IFITM2 qRT-PCR. N (nucleoprotein) transcript levels were determined in supernatants collected from SARS-CoV-2 infected Calu-3 cells 48 h post-infection as previously described 6 . Total RNA was isolated using the Viral RNA Mini Kit (Qiagen, Cat#52906) according to the manufacturer's instructions. RT-qPCR was performed as previously described 7 using TaqMan

Vero E6 cells (Cercopithecus aethiops derived epithelial kidney, ATCC) and TMPRSS2-expressing Vero E6 cells (kindly provided by the National Institute for Biological Standards and Control (NIBSC), No. 100978) were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco, Cat#41965039) supplemented with 2.5% (upon and after viral infection) or 10% (during all other times) heat-inactivated FBS (Gibco, Cat#10270106), 100 units/ml penicillin, 100 µg/ml streptomycin (ThermoFisher, Cat#15140122), 2 mM L-glutamine (Gibco, Cat#25030081), 1 mM sodium pyruvate

Cat#10131-019 ) (for TMPRSS2-expressing Vero E6 cells). Caco-2 cells (human epithelial colorectal adenocarcinoma

Human induced Alveolar Type 2 cells (iATII) were differentiated from BU3 NKX2-1 GFP ;SFTPC tdTomato induced pluripotent stem cells 1 (iPCSs, kindly provided by Darrell Kotton, Boston University and Boston Medical Center) and maintained as alveolospheres embedded in 3D Matrigel in CK+DCI media, as previously described 2 . For infection studies, iATII cells were cultured as 2D cultures on Matrigel-coated plates in CK+DCI medium + 10 µM Y-27632 (Tocris, Cat#1254) for

Synthetic SARS-CoV-2-RNA (Twist Bioscience, Cat#102024) or RNA isolated from BetaCoV/France/IDF0372/2020 viral stocks quantified via this synthetic RNA (for low Ct samples) were used as a quantitative standard to obtain viral copy numbers. All reactions were run in duplicates

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

One h post-treatment, cells were infected with SARS-CoV-2 VOCs with an MOI of 0.5. 6 h post-infection, cells were washed once with PBS and supplemented with fresh medium. Thereafter, day 0 wash CTRL was harvested

2, 48 h post infection) or iATII (MOI 0.5, 48h post infection) cells or uninfected controls were washed in PBS and subsequently lysed in Western blot lysis buffer (150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 0.1% NP40, 500 μM Na3VO4, 500 μM NaF, pH 7.5) supplemented with protease inhibitor cocktail (Roche, Cat#11697498001)

); 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). Membranes were scanned using an Odyssey infrared imager and band intensities were quantified in Image

000 Caco-2 cells were seeded in 96-well F-bottom plates. One day later, infectious supernatants were serially diluted and added to the cells. Cells were then incubated for 5 days and monitored for CPE. TCID50/mL was calculated according to Reed and Muench

Flow cytometric analysis. 60,000 iATII cells or Calu-3 cells were incubated for 1 h at 4°C with equal protein concentrations of control rabbit IgG (Diagenode, Cat#C15410206) or 1/200 dilution of rabbit anti-ACE2 (Abcam, Cat#ab166755)

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells

Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells

Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM

The Sequence Alignment/Map format and SAMtools

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans

Detection of SARS-CoV-2 in human breastmilk

Supplementary Figure 1. ACE2 expression and impact of IFITM siRNA knock-down on SARS-CoV-2 replication in Calu-3 cells. (A) Flow cytometric analysis of surface ACE2 expression in Calu-3 and iATII cells. (B) Standard curve (left) and raw qRT-PCR CT values (right) obtained using supernatants of Calu-3 cells collected 2 days post-infection. Bars represent mean values (+/-SEM) of four to three independent experiments each measured in technical duplicates