key: cord-0814259-c5y2lygj authors: Bozzo, Caterina Prelli; Nchioua, Rayhane; Volcic, Meta; Wettstein, Lukas; Weil, Tatjana; Krüger, Jana; Heller, Sandra; Conzelmann, Carina; Müller, Janis; Gross, Rüdiger; Zech, Fabian; Schütz, Desiree; Koepke, Lennart; Stuerzel, Christina M; Schüler, Christiane; Stenzel, Saskia; Braun, Elisabeth; Weiß, Johanna; Sauter, Daniel; Münch, Jan; Stenger, Steffen; Sato, Kei; Kleger, Alexander; Goffinet, Christine; Sparrer, Konstantin M.J.; Kirchhoff, Frank title: IFITM proteins promote SARS-CoV-2 infection of human lung cells date: 2020-08-18 journal: bioRxiv DOI: 10.1101/2020.08.18.255935 sha: b9aaefb5163fc62a9484c61fe373874eff5c8e42 doc_id: 814259 cord_uid: c5y2lygj Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) restrict numerous viral pathogens and are thought to prevent infection by severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudoparticle infection of cells artificially overexpressing IFITMs. Here, we confirmed that overexpression of IFITMs blocks pseudoparticle infections mediated by the Spike proteins of β-coronaviruses including pandemic SARS-CoV-2. In striking contrast, however, endogenous IFITM expression promoted genuine SARS-CoV-2 infection in human lung cells both in the presence and absence of interferon. IFITM2 was most critical for efficient entry of SARS-CoV-2 and enhanced virus production from Calu-3 cells by several orders of magnitude. IFITMs are expressed and further induced by interferons in the lung representing the primary site of SARS-CoV-2 infection as well as in other relevant tissues. Our finding that IFITMs enhance SARS-CoV-2 infection under conditions approximating the in vivo situation shows that they may promote viral invasion during COVID-19. HIGHLIGHTS Overexpression of IFITM1, 2 and 3 restricts SARS-CoV-2 infection Endogenous IFITM1, 2 and 3 boost SARS-CoV-2 infection of human lung cells IFITM2 is critical for efficient entry of SARS-CoV-2 in Calu-3 cells ( Figures S2B, S2C) . Taken together, our results show that all three IFITMs prevent SARS-CoV-2 S/ACE2-mediated attachment and membrane fusion in single round pseudotype infection assays. 151 It has been reported that overexpression of IFITMs inhibits the function of the S proteins of the two 180 To determine whether IFITMs also prevent infection by genuine SARS-CoV-2, we infected 181 HEK293T cells overexpressing ACE2 alone or together with IFITMs with a wildtype isolate of only observed at a dilution of 10 -2 , while silencing of IFITM3 expression reduced the CPE caused by 242 infectious SARS-CoV-2 down to a dilution of 10 -3 . No virus-induced CPE was observed at any 243 dilution upon depletion of IFITM2 or all three IFITM proteins ( Figure S4D ). This result indicates that lack of IFITM2 expression reduced infectious SARS-CoV-2 yield by more than four orders of 245 magnitude. Our finding that IFITM2 had the strongest effect agrees with the results of the qPCR 246 assays. Notably, titration experiments showed that IFITMs do not promote genuine SARS-CoV-2 247 infection in HEK239T cells over a broad range of expression levels ( Figure S4E ). Thus, the opposing The exact reasons for the opposing effects of overexpressed and endogenous IFITMs exposure to 300 single cycle SARS-CoV-2 S pseudotyped virus and wildtype SARS-CoV-2 remain to be determined. 301 Previous studies suggested that the subcellular localization, membrane curvature, endocytic activity The authors declare no competing interests. IFITM-Family Proteins: The Cell's First Line of Antiviral Defense Severe respiratory illness caused by a novel 649 coronavirus Imbalanced host response to SARS-CoV-2 drives 653 development of COVID-19 A sensitive and specific enzyme-based assay 655 detecting HIV-1 virion fusion in primary T lymphocytes High-efficiency transformation of mammalian cells by plasmid 657 DNA Regulation of the trafficking and 659 antiviral activity of IFITM3 by post-translational modifications The broad-spectrum antiviral functions of IFIT and IFITM 661 proteins PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells More than meets the I: The diverse antiviral and 737 cellular functions of interferon-induced transmembrane proteins Opposing activities of IFITM proteins in SARS-CoV-740 2 infection IFITM proteins -Cellular inhibitors of 742 viral entry Intracellular detection of viral nucleic acids Vpu modulates DNA repair to suppress 747 innate sensing and hyper-integration of HIV-1 IFITM proteins inhibit entry driven by the 749 Spike protein: Evidence for Cholesterol-Independent Mechanisms Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3 Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking 756 membrane fusion Antiviral Protection by IFITM3 In Vivo Interferon 760 induction of IFITM proteins promotes infection by human coronavirus OC43 Identification of Residues Controlling Restriction versus Enhancing Activities of 764 IFITM Proteins on Entry of Human Coronaviruses IFITM genes, variants, and their roles 766 in the control and pathogenesis of viral infections LY6E Restricts the Entry of Human Coronaviruses, Including the Currently Pandemic CoV-2 A pneumonia outbreak associated with a new coronavirus of probable bat origin Figure S2 (related to Figure 2). Spectrum and determinants of the Spike-mediated fusion inhibition of IFITMs Schematic depiction of the Split-GFP fusion assay. (B) Proximity ligation assay of ACE2 and SARS-CoV-2 Spike in HeLa cells. Cells were transfected with siRNA (CTRL, IFITM1 and IFITM3) and infected with VSV(luc)ΔG*SARS-CoV-2-S for 2 h at 4°C. Lines represent means of n=(77-84 cells)±SEM. (C) Exemplary images of the PLA The inset depicts a magnification, membrane outline of a cell depicted by a dotted white line. Scale bar, 20 µm. (D) Alignment of the Spike amino acid sequences from SARS-CoV-2 Alanine substitutions are color coded. Blue, Ubiquitination-negative mutant. Red, Palmitoylation negative mutant. Pink, Y20A. orange, NtΔ21AA20. (F) Quantification of the entry of VSV(luc)ΔG*-SARS-CoV-2-S by luciferase activity in HEK293T cells transiently expressing indicated proteins (IFITM mutants) and infected 24 h post-transfection with the VSVpp (MOI 0.025) for 16 h. Bars represent means of n=3±SEM. (G) Quantification of the entry of HIV(Fluc)Δenv*-SARS-CoV-2-S by luciferase activity in HEK293T cells stably expressing indicated proteins (IFITM mutants) and ACE2 Standard curve (left) and raw qRT-PCR CT values (right) corresponding to the SARS-CoV-2 RNA copy numbers per ml shown in Figure panel A. The bar diagram shows mean raw qRT-PCR Figure S4 (related to Figure 2). Impact of endogenous and transient IFIM expression on SARS-CoV-2 replication Exemplary immunoblots of whole cell lysates of Calu-3 cells transiently transfected with siRNA either control (si.NT) or targeting IFITM1, 2 3 (si.IFITM1, 2, 3) as indicated D) CPE (white) after 72h caused by infection of monolayers of Vero cells with serial dilutions of Calu-3 supernatants form Figure 4D. Cells were stained with crystal violet (blue). (E) SARS-CoV-2 RNA production from HEK239T cells transient expressing ACE2 and increasing levels of the indicated IFITM proteins. Quantification of viral N gene RNA by qRT-PCR in the supernatant of HEK293T was performed 48 h post-infection with