key: cord-0706243-0419edvn
authors: Shi, Guoli; Kenney, Adam D.; Kudryashova, Elena; Zani, Ashley; Zhang, Lizhi; Lai, Kin Kui; Hall‐Stoodley, Luanne; Robinson, Richard T.; Kudryashov, Dmitri S.; Compton, Alex A.; Yount, Jacob S.
title: Opposing activities of IFITM proteins in SARS‐CoV‐2 infection
date: 2020-12-03
journal: EMBO J
DOI: 10.15252/embj.2020106501
sha: 18d9367122397380500969849393721fd24569c8
doc_id: 706243
cord_uid: 0419edvn

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxΦ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.

Severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 is the causative agent of the respiratory and multi-organ-associated disease known as COVID-19 Zhu et al, 2020) . Understanding virus-host interactions and immune responses will be critical for explaining the high variation in severity of COVID-19 observed in humans (Zhang et al, 2020a) . Type I interferon (IFN) is a critical component of the innate immune system that induces expression of hundreds of genes, many of which encode proteins with antiviral activities that block specific steps in viral replication cycles (Chemudupati et al, 2019; Schoggins et al, 2011) . Among these are genes that encode the IFN-induced transmembrane proteins (IFITMs), including IFITM1, IFITM2, and IFITM3.

IFITMs have been shown to block membrane fusion of diverse enveloped viruses by using an amphipathic helix domain to mechanically alter membrane lipid order and curvature in a manner that disfavors virus fusion (Chesarino et al, 2017; Guo et al, 2020; Li et al, 2013; Lin et al, 2013; Rahman et al, 2020) . While IFITMs have been shown in most instances to inhibit CoV infections (Bertram et Accepted Article Huang et al, 2011; Wrensch et al, 2014; , in some cases they enhance infections (Zhao et al, 2014; Zhao et al, 2018) . For example, IFITM2 and IFITM3 enhance cellular entry of human CoV-OC43 (Zhao et al., 2014) . Similarly, infections by virus-like particles containing SARS-CoV or MERS-CoV glycoprotein (pseudovirus) are increased upon expression of an IFITM3 variant with enhanced plasma membrane localization versus endosomal localization (Zhao et al., 2018) . Given that polymorphisms in the genes encoding IFITM2 and IFITM3 have been proposed to increase their plasma membrane localization (Chesarino et al, 2014a; Everitt et al, 2012; Jia et al, 2014; Wu et al, 2017) , and that an additional polymorphism in the IFITM3 gene promoter results in significant loss of IFITM3 expression (Allen et al, 2017) , determining whether IFITMs affect SARS-CoV-2 cellular infections should be informative for understanding susceptibility and resistance of human cells to infection.

Published experimental results are contradictory with regard to the effect of IFITMs on SARS-CoV-2 cell entry. SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus infection was inhibited by human IFITM2 and IFITM3, but not IFITM1, in an overexpression screen of IFN effector proteins (Zang et al, 2020) . In contrast, IFITM3 overexpression did not inhibit infection of Spike-containing pseudovirus in a separate study . IFITMs have also been reported to inhibit cell-tocell fusion induced by SARS-CoV-2 Spike protein (Buchrieser et al, 2020) , though a conflicting report did not observe inhibition of Spike-mediated cell fusion when IFITM2 or IFITM3 were expressed in target cells (Zang et al., 2020) . Overall, the impact of IFITMs on SARS-CoV-2 infections remains unclear and has yet to be examined with bona fide SARS-CoV-2. Herein, we measure the effects of mouse and human IFITMs on SARS-CoV-2 infections using both pseudovirus and genuine virus systems. We further utilize a series of mutants affecting critical functional amino acid motifs within IFITM3, and identify pro-and anti-viral effects that we delineate in terms of distinct cellular locations and mechanisms of action.

To explore the effects of human IFITM1, IFITM2, and IFITM3 on SARS-CoV-2 infection, we transfected the SARS-CoV-2 receptor, ACE2, into HEK293T cells stably expressing FLAG-tagged IFITM constructs. We then infected the transfected cells with SARS-CoV-2 Spike-pseudotyped HIV-production, indicating inhibition of Spike-mediated infection, with IFITM1 expression exhibiting particularly strong inhibition (Fig 1A) . We also observed that all three of the IFITMs inhibited SARS-CoV Spike-pseudotyped lentivirus infection (HIV-SARS-1) (Fig 1B) , with IFITM3 appearing to exhibit relatively more antiviral activity against HIV-SARS-1 than HIV-SARS-2 (Fig 1A,B) . We confirmed uniform expression of each of the FLAG-tagged IFITM constructs by flow cytometry (Fig 1C) . We further confirmed the ability of these IFITM constructs to limit HIV-SARS-2 infection in transientlytransfected Caco-2 cells, which naturally express ACE2 (Fig 1D) . Furthermore, Caco-2 cells express basal IFITM3 protein at relatively high levels, while IFITM1 and IFITM2 are nearly absent.

Knockdown of IFITM3 by siRNA transfection increased HIV-SARS-2 infection (Fig 1E) . IFN treatment of Caco-2 cells strongly inhibited HIV-SARS-2 infection, which could be partially reversed with siRNA-mediated knockdown of IFITM3 (Fig 1E,F) . These results indicate that basal, IFNinduced, and ectopically expressed IFITM3 inhibit HIV-SARS-2 infection of Caco-2 cells, and that, while multiple IFN stimulated genes are likely involved in repression of infection, IFITM3 is among those critical factors. In sum, our experiments indicate that IFITMs, both when expressed endogenously or ectopically, are restrictive of SARS-CoV-2 Spike-mediated virus infection

We next sought to validate results from pseudotyped virus infections using genuine SARS-CoV-2 at Biosafety Level 3. We utilized HEK293T cell lines stably expressing IFITMs (Fig 2A) that were transiently transfected with ACE2-GFP and measured percent infection via staining for viral N protein and flow cytometry. IFITM proteins are unique from many classical interferon effectors in that they affect virus entry processes rather than intracellular virus replication (Bailey et al, 2014; Feeley et al, 2011) . Analysis of virus protein production early in infection as done by flow cytometry thus provides a measure of whether virus was able to fuse with cellular membranes and continue the replicative process. Consistent with past reports suggesting low endogenous ACE2 expression in HEK293T cells, we found that infection was limited to cells that were ACE2-GFP positive (Fig 2B) . We thus employed a gating strategy that focused on ACE2-GFP positive cells for measuring percent infection.

Similar to results with Spike-pseudotyped lentivirus, we found that human IFITM1, IFITM2, and IFITM3 were each capable of inhibiting SARS-CoV-2 infections (Fig 2C,D) . Additionally, we examined a cell line expressing a palmitoylation-deficient IFITM3 triple cysteine mutant (C71A, C72A, C105A; termed Palm) that has been shown to lose activity against influenza virus and other viruses (Benfield et al, 2020; Hach et al, 2013; John et al, 2013; McMichael et al, 2017; Percher et al, 

This article is protected by copyright. All rights reserved 2016; Yount et al, 2012; Yount et al, 2010) . Remarkably, IFITM3-Palm maintained restriction of SARS-CoV-2 (Fig 2D) . Given the highly unusual result with the IFITM3-Palm cell line, we sought to authenticate the cell line by performing an influenza virus infection alongside one of our SARS-CoV-2 infections. Indeed, we confirmed the well-established results that IFITM3 strongly restricts influenza virus infection in a palmitoylation-dependent manner (Fig 2E) (Chesarino et al., 2017; Chesarino et al, 2014b; Hach et al., 2013; McMichael et al., 2017; Melvin et al, 2015; Percher et al., 2016; Yount et al., 2012; Yount et al., 2010) . These results suggest that IFITMs provide palmitoylationindependent restriction of SARS-CoV-2 that is distinct from other viruses.

We additionally sought to confirm the effects of IFITMs on SARS-CoV-2 by transfecting IFITM constructs into stable HEK293T-ACE2-GFP cells. To measure SARS-CoV-2 infection, we employed a flow cytometry gating strategy in which cells that were expressing the highest levels of ACE2-GFP as well as IFITMs were analyzed for percent infection (Fig 3A,B) . We again found that human IFITMs inhibit SARS-CoV-2 infection, though effects of transiently transfected IFITM2 did not reach statistical significance (Fig 3C,D) . We also confirmed via transient transfection that IFITM3-Palm is active in restricting the virus (Fig 3C,D) . We further found that, consistent with past results examining endogenous ACE2 (Huang et al., 2011) , IFITM expression did not affect levels of exogenously expressed ACE2-GFP (Fig 3E,F) . We additionally examined mouse Ifitm1-3 through transient transfection of HEK293T-ACE2-GFP cells and found that restriction of SARS-CoV-2 infection is a conserved activity of mouse and human IFITMs, and that restriction by mouse IFITM3 occurred independently of palmitoylation like its human counterpart (Fig 4A-C) . As an additional investigation of restriction of SARS-CoV-2 by endogenous IFITMs, WT, IFITM3 KO, and IFITM-locus deficient (IFITMdel) mouse embryonic fibroblasts (MEFs) were transduced with adenovirus expressing both ACE2 and GFP, followed by infection with SARS-CoV-2 (Fig 5A-D) . IFITMdel MEFs were significantly more susceptible to SARS-CoV-2 compared to WT or IFITM3 KO cells (Fig 5D) .

Additionally, both IFITM3 KO and IFITM-locus deletion prevented type I IFN from inhibiting SARS-CoV-2 infections to the level observed in WT cells (Fig 5C,D) .

We additionally examined mouse and human IFITM3 mutants that impact protein localization. Y20 is a critical amino acid within a Yxx endocytosis motif (20-YEML-23 in human IFITM3), and has been shown in several studies to be required for active endocytosis of human and mouse IFITM3 (Chesarino et al., 2014a; Jia et al, 2012; Jia et al., 2014; McMichael et al, 2018) . Mutation of Y20 or Accepted Article modifying this residue via phosphorylation by Src kinases results in accumulation of IFITM3 at the plasma membrane (Fig 3G, 4D ) (Chesarino et al., 2014a; Compton et al, 2016; Jia et al., 2012; Jia et al., 2014; McMichael et al., 2018) . Human IFITM3-Y20A caused an increase in SARS-CoV-2 infection as compared to vector control cells, suggesting that IFITM3 at the plasma membrane promotes, rather than inhibits, infection (Fig 3C,D) . In contrast, mouse IFITM3-Y20A did not increase infection compared to vector control cells (Fig 4B,C) , revealing a species-specific distinction between mouse and human IFITM3 in their ability to promote SARS-CoV-2 infection. To confirm that enhancement of infection was due to disruption of the 20-YEML-23 endocytosis motif, we tested an additional construct, human IFITM3-L23Q, which similarly accumulates at the plasma membrane due to disruption of the critical  residue of the endocytosis signal (Fig 3G) (Chesarino et al., 2014a) .

IFITM3-L23Q expression also increased SARS-CoV-2 infection, confirming the unusual ability of IFITM3 to enhance infection of this virus (Fig 3C,D) . Overall, these results demonstrate that while mouse and human IFITMs generally restrict infection, human IFITM3 can promote infection when its localization is shifted toward the plasma membrane.

We next examined whether IFITM3 affects SARS-CoV-2 Spike-mediated membrane fusion in a cell-to-cell fusion assay. U2OS cells co-transfected with Spike and mCherry expression plasmids were mixed with Calu-3 target cells that were co-transfected with plasmids encoding EGFP and IFITMs. After 24 h co-culture, mCherry and EGFP double positive multi-nucleated cell syncytia could be observed dependent upon the presence of Spike protein, demonstrating Spike-mediated fusion between the two cell types (Fig 6A) . Upon transfection of IFITM3 into Calu-3 target cells, double positive syncytia remained readily observable upon visual inspection, though quantification of nuclei number within these double positive syncytia suggested that IFITM3 may have a modest ability to limit S-mediated cell-to-cell fusion (Fig 6A,B) . In contrast, transfection of IFITM3-Y20A or IFITM3-L23Q resulted in an increase in the size of double positive syncytia as compared to vector control or WT IFITM3 transfections, which was confirmed by quantification of nuclei per syncytia (Fig 6A,B) .

These results provide confirmation of infection experiments indicating that IFITM3 enriched at the plasma membrane due to endocytosis-impairing mutations enhances SARS-CoV-2 Spike-mediated membrane fusion.

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Our results suggest that IFITM3 is able to inhibit SARS-CoV-2 infection overall, but that shifting human IFITM3 localization away from the endosomal system and toward the plasma membrane results in enhancement of infection (Fig 2, 3) . Given that SARS-CoV-2 has dual cellular entry pathways involving Spike proteolytic activation either at the plasma membrane or within endosomes (Hoffmann et al, 2020; Shang et al, 2020) , these findings may indicate that IFITM3 has distinct effects on the virus dependent upon the location at which virus fuses with cell membranes.

Plasma membrane-associated processing of Spike involves TMPRSS2 protease, and it has been reported that TMPRSS2 overexpression increases the proportion of virus able to fuse at the cell surface (Hoffmann et al., 2020) . Further, it is reported that high TMPRSS2 levels allow human coronaviruses to avoid restriction by IFITMs (Bertram et al., 2013; . In line with these data, we found that TMPRSS2 overexpression decreased the ability of IFITM3 to inhibit SARS-CoV-2 infection (Fig 7A-C) . Interestingly, IFITM3 slightly enhanced infection in the presence of TMPRSS2 overexpression in 2 out of 3 experiments (Fig 7B) . Inhibition of infection by IFITM3-Palm and IFITM1 was also counteracted by TMPRSS2, though enhancement of infection was not observed for these constructs (Fig 7A-C) .

The IFITM3 amphipathic helix domain located at amino acid positions 59-68 is required for inhibition of influenza virus infection (Chesarino et al., 2017) . This helix was recently shown to be able to alter mechanical properties of lipid membranes, such as curvature and lipid order, thus inhibiting viral membrane fusion Rahman et al., 2020) . In the context of SARS-CoV-2, we found that expression of an amphipathic helix-deleted mutant of IFITM3 (59-68) resulted in a modest enhancement rather than inhibition of infection (Fig 7A-C) . Likewise, decreasing amphipathicity of the helix by mutation of its three hydrophilic residues (S61A, N64A, T65A), decreased restriction of infection (Fig 7A-C) , further demonstrating that the amphipathicity of IFITM3, and likely its associated membrane-altering properties, underlie restriction of SARS-CoV-2. Notably, both of the amphipathic helix mutants were increased in their ability to enhance SARS-CoV-2 infection when co-expressed with TMPRSS2 (Fig 7A-C) , providing the first clear evidence that IFITM3 enhancement of CoV infection occurs via a mechanism distinct from its amphipathicity-based membrane alterations. These results support a model wherein IFITM3 exerts opposing activities on SARS-CoV-2, including amphipathicity-dependent restriction of virus at endosomes and amphipathicity-independent enhancement of infection at the plasma membrane.

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In the present study we have identified IFITM1, IFITM2, and IFITM3 as restrictors of SARS-CoV-2 infection of cells. Our result showing that IFITM3 inhibits SARS-CoV-2 infection independently of Spalmitoylation is surprising since intact palmitoylation sites are necessary for inhibition of a number of viruses, including influenza virus, dengue virus, CoV-NL63, and CoV-229E (Benfield et al., 2020; Hach et al., 2013; John et al., 2013; McMichael et al., 2017; Percher et al., 2016; Yount et al., 2012; Yount et al., 2010; Zhao et al., 2018) . This conclusion is supported by experiments with three distinct expression systems (stable transduction of human IFITM3-Palm in the pLenti vector and transient transfections of human and mouse IFITM3-Palm in pCMV) (Fig 2,3,4) . Previous work suggests that S-palmitoylation increases the rate at which IFITM3 traffics to influenza viruscontaining endosomes during infection (Spence et al, 2019) . Our results suggest possible differences between influenza virus and SARS-CoV-2 either in their endocytic entry pathways or fusion kinetics, and IFITM constructs may prove useful in dissecting such differences.

Mechanistically, we found that the IFITM3 amphipathic helix is necessary for its SARS-CoV-2 restrictive capacity (Fig 7) . We previously showed that this helix was also required for restriction of influenza virus and proposed that the helix affected virus fusion by wedging into the inner leaflet of the membrane bilayer, thus generating local membrane curvature that is disfavorable to virus-to-cell fusion (Chesarino et al., 2017) . More recent research has shown that the amphipathic helix is indeed able to mechanically induce membrane curvature and increase lipid order Rahman et al., 2020) . Overall, IFITMs likely restrict SARS-CoV-2 infection by mechanically disfavoring viral membrane fusion reactions occurring at endosomes.

Remarkably, we observed that IFITM3-Y20A and -L23Q constructs enhance SARS-CoV-2 infection (Fig 3) . Since these constructs are well characterized to accumulate at the plasma membrane of cells due to loss of interaction with the AP-2 endocytic adaptor protein complex (Chesarino et al., 2014a; Jia et al., 2014; McMichael et al., 2018) , these data suggest that IFITM3 localization to the plasma membrane enhances infection. Given that WT IFITM3 traffics to the plasma membrane prior to its endocytosis, and since phosphorylation of IFITM3 Y20 by Src kinases also blocks endocytosis (Chesarino et al., 2014a; Jia et al., 2012) , there are likely opposing restrictive and enhancing activities occurring in cells expressing WT IFITM3. It will be interesting to determine whether these balances are shifted in different cell types with distinct endocytic or Accepted Article phosphorylation capacities. Dueling enhancing and inhibiting activities of IFITM3 in SARS-CoV-2 infection are also consistent with the partial restriction of SARS-CoV-2 infection observed in cells stably expressing IFITM3 as compared to the near complete inhibition of influenza virus in the same cells (Fig 2D, E) .

Enhancement of SARS-CoV-2 infection at the plasma membrane does not require the IFITM3 amphipathic helix, as demonstrated by our observation that TMPRSS2 overexpression converts amphipathic helix mutants into strong enhancers of infection (Fig 7) . We did not observe an enhancing effect of human IFITM1, which also has a conserved and functional amphipathic helix domain (Chesarino et al., 2017) , further supporting that enhancement of infection at the plasma membrane is not occurring via amphipathic helix-mediated mechanical membrane alterations (Fig 6) .

These data are in alignment with past reports that IFITM3, but not IFITM1, enhances CoV-OC43

infections (Zhao et al., 2014) . Removal of the IFITM1 extended C-terminal tail converted IFITM1 into an enhancer of specific CoV infections (Zhao et al., 2014; Zhao et al., 2018) . Likewise, and similar to our results with SARS-CoV-2, mutation of human IFITM3 Y20 was also reported to enhance SARS-CoV and MERS-CoV pseudotype virus infections, but not CoV-229E or CoV-NL63 pseudotype infections (Zhao et al., 2018) . Since enhancement of infection does not correlate with specific receptor usage of the different coronaviruses, it is unlikely that IFITMs directly affect the virus receptors. In that regard, we did not observe an effect of IFITM proteins on levels of exogenously expressed ACE2-GFP (Fig 3E,F) . It may be possible that the short IFITM3 C-terminal tail, predicted to be exposed at the cell surface, directly interacts with specific features of CoV surface glycoproteins or with entry factors. It is also interesting to note that mouse IFITM3 has an extended C-terminal tail compared to human IFITM3, consistent with results that mouse IFITM3-Y20A did not enhance infection (Fig 4B,C) . Overall, our results indicate that IFITMs are not likely exerting a broad mechanism of amphipathicity-based membrane alteration to promote infection, but rather are more specifically coopted by specific CoVs, such as SARS-CoV-2, under certain conditions. An early study on a limited cohort suggests that the human IFITM3 rs12552-C SNP, which has been proposed to result in IFITM3 plasma membrane localization (Chesarino et al., 2014a; Everitt et al., 2012; Jia et al., 2012) , is a risk factor for severe COVID-19 (Zhang et al, 2020b) . Larger studies are warranted to examine whether this and other polymorphisms in IFITM3 positively or negatively influence COVID-19 severity.

This article is protected by copyright. All rights reserved Biosafety All work with live SARS-CoV-2 was performed at Biosafety Level 3 (BSL3) according to standard operating procedures approved by the Ohio State University BSL3 Operations Group and Institutional Biosafety Committee. Samples were removed from the BSL3 facility for flow cytometry analysis only after decontamination with 4% paraformaldehyde for a minimum of 1 h according to an in-house validated and approved method of sample decontamination.

All cells were cultured at 37°C with 5% CO 2 in a humidified incubator and were grown in Dulbecco's HEK293T cells stably expressing human IFITM1, IFITM2, IFITM3, IFITM3-PalmΔ, or empty pLentipuro vector that were used in genuine virus infections were generated and validated as described previously (McMichael et al., 2018) , with the exception that the IFITM3-PalmΔ cell line required three sequential transductions to achieve expression comparable to WT IFITM3 in the majority of the cells.

HEK293T cells stably expressing GFP-tagged human ACE2 (Origene) were generated using the same methodology. HEK293T cell lines stably transduced with FLAG-tagged human IFITM1, IFITM2, IFITM3, or pQCXIP vector control for HIV-SARS-1 and HIV-SARS-2 infection experiments were described previously (Shi et al, 2018) . Type -I interferon (human recombinant interferon beta, NR-3085, BEI Resources) was added to Caco-2 cells at 48h post siRNA transfection at a concentration of 250 IU/mL. Mouse IFN2 (eBiosciences) was added to MEFs at a concentration of 0.1 µg/mL. Cells were incubated with interferon for a 24h period prior to being removed and replaced with virus inoculum.

This article is protected by copyright. All rights reserved SARS-CoV-2 USA-WA1/2020 stock from BEI Resources was diluted 1:10,000 in DMEM and added to confluent Vero cells for 1 h at 37°C. Following the 1 h infection period, virus-containing media was replaced with DMEM containing 4% FBS and incubated at 37°C for 72 h at which point significant cytopathic effect was observed. Cell supernatant containing infectious virus was centrifuged at 1,000

x g for 10 min to remove cell debris, and was aliquoted, flash frozen in liquid nitrogen, and stored at - (Chesarino et al., 2017; Chesarino et al., 2014a; Hach et al., 2013; Percher et al., 2016; Yount et al., 2010) . TMPRSS2 in pCAGGS was a kind gift from Dr. Stefan Pöhlmann (German Primate Center). ACE2-GFP in pLenti-puro was purchased from Origene. Adenovirus expressing ACE2 and GFP was acquired from the Iowa University Viral Vector Core Facility. All transient transfections for SARS-CoV-2 infection experiments were performed using LipoJet transfection reagent (Signagen Laboratories). siRNAs targeting IFITM1 (s16192), IFITM2 (s20771), IFITM3 (s195035) and a non-targeting siRNA control pool (4390844) were purchased from Ambion. siRNA-mediated knockdown was performed by transfecting Caco-2 cells with 20 nM amounts of siRNA and Lipofectamine RNAiMAX (Thermo Fisher). Knockdown efficiency was assessed by Western blot analysis at 72h post transfection.

This article is protected by copyright. All rights reserved Virus-like particles pseudotyped with SARS-CoV-2 or SARS-CoV-1 Spike protein were produced by transfecting HEK293T cells with pNL4-3.Luc.R-E-(obtained from NIH AIDS Reagent Resource; 3418) and pcDNA-SARS-CoV2-C9 or pcDNA-SARS-CoV1-C9 (gifts from Thomas Gallagher) using

TransIT-293 reagent. Cells were incubated with virus-containing medium for 72h, after which cells were lysed with Passive Lysis Buffer (Promega). Luciferase assays were performed using Luciferase Assay System (Promega). Luciferase values were measured with a Tecan Infinite M1000 plate reader.

Cells were infected with SARS-CoV-2 for 24 hours, after which cells were fixed with 4% paraformaldehyde (Thermo Scientific) for 1 h at room temperature, permeabilized with PBS containing 0.1% TritonX-100, and blocked with PBS/2% FBS. Cells were then stained with mouseproduced anti-SARS-CoV-2 N (Sino Biological, 40143-MM08) and a cocktail of rabbit-produced anti-IFITM2/3 (ProteinTech, 11714-1-AP) and rabbit produced anti-IFITM1 (Cell Signaling Technologies, 13126S). Primary antibody labeling was followed by labeling with goat-anti-mouse-AlexaFluor-555 (Life Technologies, A-21424) and donkey-anti-rabbit-AlexaFluor-647 (Life Technologies, A-31573) secondary antibodies. Flow cytometry data was collected using a FACSCanto II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software. Infected cell gates were set using non-infected control samples.

Using Lipofectamine 3000 (Life Technologies), U2OS cells were co-transfected with pmCherry and either empty pCAGGS vector or SARS-CoV-2 Spike Glycoprotein (BEI Resources, deposited by Dr.

Florian Krammer, Icahn School of Medicine at Mount Sinai); Calu-3 cells were co-transfected with pEGFP and either empty pCMV vector, IFITM3, IFITM3-Y20A, or IFITM3-L23Q. Transfected Calu-3 and U2OS cells were mixed and co-incubated for 24 hours. Following incubation, the co-cultures were contra-stained with Hoechst 33342 and imaged using a Nikon Eclipse Ti-E inverted microscope (Nikon Instruments). Nikon NIS Elements AR software was used to quantify fusion indices calculated as number of nuclei per double-positive (expressing both EGFP and mCherry) cell fusion cluster.

This article is protected by copyright. All rights reserved To examine IFITM3 localization, HEK293T cells grown on coverslips were transiently transfected with empty vector, IFITM3, IFITM3-Y20A, IFITM3-L23Q, mIFITM3, or mIFITM3-Y20A. 24 h post transfection, cells were fixed with 4% paraformaldehyde in PBS, were permeabilized with 0.1% Triton-X100 in PBS, and were incubated with anti-IFITM3 (ProteinTech), followed by washing and incubation with donkey-anti-rabbit Alexa488 secondary antibody (Life Technologies, A-11029).

Coverslips were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher).

Fluorescence images were captured using an Olympus FV 1000 Spectral Confocal system.

This study includes no data deposited in external repositories. 

This article is protected by copyright. All rights reserved C) Stable HEK293T lines as in (A) were infected with SARS-CoV-2 (MOI 1) for 24 h and analyzed for percent infection by flow cytometry. Representative example plots are shown for each cell line.

each performed in triplicate, from cells infected as in (C). Bars represent averages with individual data points shown as circles. Error bars represent SD. # p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.

E) The indicated cell lines were infected with influenza A virus (IAV) for 24h and percent infection was determined by flow cytometry. Graph depicts percent infection of triplicate samples. Bars represent averages with individual data points shown as circles. Error bars represent SD. # p<0.05., NS, not significant, by ANOVA followed by Tukey's multiple comparisons test. 

This article is protected by copyright. All rights reserved HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector

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Interferon induction of IFITM proteins promotes infection by human coronavirus OC43

Identification of Residues Controlling Restriction versus Enhancing Activities of IFITM Proteins on Entry of Human Coronaviruses

Bat SARS-Like WIV1 coronavirus uses the ACE2 of multiple animal species as receptor and evades IFITM3 restriction via TMPRSS2 activation of membrane fusion

A Novel Coronavirus from Patients with Pneumonia in China

We would like to thank Alan Rein for facilitating the production of lentiviral pseudotypes containing SARS-CoV-1 and SARS-CoV-2 Spike proteins. We thank Dr. Eugene Oltz for critical reading of the 

The authors declare that they have no conflict of interest.

GS, ADK, EK, AZ, and LZ performed experiments, analyzed data, and generated figures. KKL provided assistance and expertise in generating pseudotyped lentiviruses. LHS and RTR provided

This article is protected by copyright. All rights reserved critical assistance and expertise in generating and titering SARS-CoV-2 stocks in BSL3 containment, and in developing methodology for analysis of infected cells. Experiments were supervised by DSK, AAC, and JSY. GS, ADK, AAC, and JSY wrote an initial manuscript draft that was critically edited and approved by all authors.