IFITM DEPENDENCY of SARS-CoV-2 VARIANTS of CONCERN

ABSTRACT

Background: We recently showed that genuine SARS-CoV-2 hijacks endogenously expressed interferon-induced transmembrane proteins, especially IFITM2, as entry cofactors for efficient infection (Prelli Bozzo, Nchioua et al., Nat. Com., 2021). This came as a surprise, since IFITMs have been reported to inhibit entry of numerous enveloped viruses, including SARS-CoV-2. However, most data were obtained using IFITM overexpression and pseudoparticle infection assays. In our initial study, we used a SARS-CoV-2 strain isolated in the Netherlands in February 2020 (NL-02-2020). Since then several "variants of concern" (VOCs) have emerged that show increased transmission fitness and evasion of vaccine-induced immunity. These VOCs contain various alterations in their Spike (S) proteins that may alter their dependency on entry cofactors. Here, we examined whether SARS-CoV-2 VOCs, including the currently dominating Delta variant, still depend on IFITMs for efficient infection and replication. Methods: To determine the role of IFITMs in infection of SARS-CoV-2 VOCs, we silenced IFITM1, 2, or 3 expression in Calu-3 cells using siRNAs and infected them with NL-02-2020 as well VOCs B.1.1.7, B.1.351, P.1 and B.1.617.2, also referred to as Alpha, Beta, Gamma and Delta variants, respectively. Viral entry and replication were quantified by qRT-PCR as well as TCID50 analysis. In addition, we determined the inhibitory effect of an α-IFITM2 antibody on VOC infection in iPSC-derived human alveolar epithelial type 2 (iAT2) cells. Results: Depletion of IFITM2 reduced viral RNA production from 31-(B.1.1.7) to 754-fold (P.1). In comparison, KD of IFITM1 generally had little effect, while silencing of IFITM3 resulted in 2-to 20-fold reduction of viral RNA yields by the four VOCs. An antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iAT2 cells. Conclusion: Endogenously expressed IFITM proteins (especially IFITM2) are important cofactors for entry and replication of SARS-CoV-2 VOCs, including the Delta variant that currently dominates the COVID-19 pandemic.