IFITM Proteins Research Articles

Role of Viral Envelope Proteins in Determining Susceptibility of Viruses to IFITM Proteins.

Interferon-induced transmembrane proteins (IFITMs) are a family of proteins which inhibit infections of various enveloped viruses. While their general mechanism of inhibition seems to be non-specific, involving the tightening of membrane structures to prevent fusion between the viral envelope and cell membrane, numerous studies have underscored the importance of viral envelope proteins in determining the susceptibility of viruses to IFITMs. Mutations in envelope proteins may lead to viral escape from direct interaction with IFITM proteins or result in indirect resistance by modifying the viral entry pathway, allowing the virus to modulate its exposure to IFITMs. In a broader context, the nature of viral envelope proteins and their interaction with IFITMs can play a crucial role in the context of adaptive immunity, leading to viral envelope proteins that are more susceptible to antibody neutralization. The precise mechanisms underlying these observations remain unclear, and further studies in this field could contribute to a better understanding of how IFITMs control viral infections.

IFITM2 Presents Antiviral Response through Enhancing Type I IFN Signaling Pathway.

Interferon (IFN) helps cells fight viral infections by further inducing the expression of many downstream IFN-stimulated genes (ISGs). Human interferon-inducible transmembrane proteins (IFITM) are one of these ISGs. The antiviral function of human IFITM1, IFITM2, and IFITM3 are well known. In this study, we report that IFITM can significantly inhibit EMCV infectivity in HEK293 cells. Overexpression of IFITM proteins could promote IFN-β production. Meanwhile, IFITMs facilitated type I IFN signaling pathway adaptor MDA5 expression. We detected the binding of IFITM2 to MDA5 in a co-immunoprecipitation assay. It was also found that the ability of IFITM2 to activate IFN-β was significantly inhibited after interfering with MDA5 expression, suggesting that MDA5 may play an important role in the activation of the IFN-β signaling pathway by IFITM2. Moreover, the N-terminal domain plays an active role in the antiviral activity and the activation of IFN-β by IFITM2. These findings suggest that IFITM2 plays a vital role in antiviral signaling transduction. In addition, a positive feed-forward loop between IFITM2 and type I IFN establishes a key role for IFITM2 in enforcing innate immune responses.

Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity.

Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity.

IFITM proteins assist cellular uptake of diverse linked chemotypes.

The search for cell-permeable drugs has conventionally focused on low-molecular weight (MW), nonpolar, rigid chemical structures. However, emerging therapeutic strategies break traditional drug design rules by employing flexibly linked chemical entities composed of more than one ligand. Using complementary genome-scale chemical-genetic approaches we identified an endogenous chemical uptake pathway involving interferon-induced transmembrane proteins (IFITMs) that modulates the cell permeability of a prototypical biopic inhibitor of MTOR (RapaLink-1, MW: 1784 g/mol). We devised additional linked inhibitors targeting BCR-ABL1 (DasatiLink-1, MW: 1518 g/mol) and EIF4A1 (BisRoc-1, MW: 1466 g/mol), uptake of which was facilitated by IFITMs. We also found that IFITMs moderately assisted some proteolysis-targeting chimeras and examined the physicochemical requirements for involvement of this uptake pathway.

IFITM3 promotes NiV envelope protein-mediated entry into MDCK cells and interacts with the fusion subunit of the F protein.

IFITM proteins are a host restriction factor with broad-spectrum antiviral activity, but the role in the paramyxovirus entry remains unclear. Nipah virus (NiV) is a zoonotic virus of the paramyxoviridae with extremely high lethality. Here, we assessed the role of IFITM3 on NiV G and F glycoprotein-mediated virus entry. Using NiV pseudovirus bearing NiV G and F proteins to infect IFITM3-induced MDCK cells, we found that overexpression of IFITM3 promotes NiV G and F proteins-mediated virus entry. Mechanistically, the subcellular distribution showed that F protein completely co-localized with IFITM3, but G protein does not. Immunoprecipitation further indicated that IFITM3 strongly captures F protein rather than G protein. F protein truncation found that the F1 subunit completely co-localized and captures with IFITM3, but not the F2 subunit. Furthermore, IFITM3 strongly binds to F1 truncations containing fusion peptide (FP), and F1 strongly captures IFITM3 truncation with the intramembrane domain (IMD). Together, the results suggest that IFITM3 can promote NiV G and F proteins-mediated virus entry into MDCK cells, and IFITM3 directly interacts with the F1 subunit of NiV F protein dependent on the former's IMD and the latter's FP, which may occur after incorporation of fusion peptides into the cell membrane following virus fusion activation.

IFITM protein regulation and functions: Far beyond the fight against viruses.

Interferons (IFNs) are important cytokines that regulate immune responses through the activation of hundreds of genes, including interferon-induced transmembrane proteins (IFITMs). This evolutionarily conserved protein family includes five functionally active homologs in humans. Despite the high sequence homology, IFITMs vary in expression, subcellular localization and function. The initially described adhesive and antiproliferative or pro-oncogenic functions of IFITM proteins were diluted by the discovery of their antiviral properties. The large set of viruses that is inhibited by these proteins is constantly expanding, as are the possible mechanisms of action. In addition to their beneficial antiviral effects, IFITM proteins are often upregulated in a broad spectrum of cancers. IFITM proteins have been linked to most hallmarks of cancer, including tumor cell proliferation, therapeutic resistance, angiogenesis, invasion, and metastasis. Recent studies have described the involvement of IFITM proteins in antitumor immunity. This review summarizes various levels of IFITM protein regulation and the physiological and pathological functions of these proteins, with an emphasis on tumorigenesis and antitumor immunity.

ABHD16A Negatively Regulates the Palmitoylation and Antiviral Function of IFITM Proteins.

Interferon-inducible transmembrane (IFITM) proteins are small homologous proteins that are encoded by the interferon-stimulated genes (ISGs), which can be strongly induced by interferon (IFN) and provide resistance to invasion by a variety of viral pathogens. However, the exact molecular mechanisms underlying this function have remained elusive. The antiviral activity of IFITMs from different species depends on S-palmitoylation at conserved cysteine residues. However, specific enzymes involved in the dynamic palmitoylation cycle of IFITMs, especially depalmitoylase, have not yet been reported. Here, we demonstrate that α/-hydrolase domain-containing 16A (ABHD16A) is a depalmitoylase and a negative regulator of IFITM protein that can catalyze the depalmitoyl reaction of S-palmitoylated IFITM proteins, thereby decreasing their antiviral activities on RNA viruses. Using the acyl-PEGyl exchange gel shift (APEGS) assay, we identified ABHD16A proteins from humans, pigs, and mice that can directly participate in the palmitoylation/depalmitoylation cycles of IFITMs in the constructed abhd16a-/- cells and ABHD16A-overexpressing cells. Furthermore, we showed that ABHD16A functions as a regulator of subcellular localization of IFITM proteins and is related to the immune system. It is tempting to suggest that pharmacological intervention in IFITMs and ABHD16A can be achieved either through controlling their expression or regulating their activity, thereby providing a broad-spectrum therapeutic strategy for animal viral diseases. IMPORTANCE IFITM protein is the cells first line of antiviral defense that blocks early stages of viral replication; the underlying mechanism might be associated with the proper distribution in cells. The palmitoylation/depalmitoylation cycle can dynamically regulate protein localization, stability, and function. This work is the first one that found the critical enzyme that participates in the palmitoylation/depalmitoylation cycle of IFITM, and this type of palmitoyl loss may be an essential regulation mode for balancing the antiviral functions of the IFN pathway. These findings imply that the pharmacological intervention in IFITM and ABHD16A, either through controlling their expression or regulating their activities, could provide a broad-spectrum therapeutic strategy for animal viral diseases and complications linked to interferon elevation.

S-Palmitoylation and Sterol Interactions Mediate Antiviral Specificity of IFITMs.

Interferon-induced transmembrane proteins (IFITM1, 2, and 3) are important antiviral proteins that are active against many viruses, including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV), and severe acute respiratory syndrome coronavirus (SARS-CoV). IFITM proteins exhibit specificity in activity, but their distinct mechanisms of action and regulation are unclear. Since S-palmitoylation and cholesterol homeostasis are crucial for viral infections, we investigated IFITM interactions with cholesterol by photoaffinity cross-linking in mammalian cells along with molecular dynamic simulations and nuclear magnetic resonance analysis in vitro. These studies suggest that cholesterol can directly interact with S-palmitoylated IFITMs in cells and alter the conformation of IFITMs in membrane bilayers. Notably, we discovered that the S-palmitoylation levels regulate differential IFITM protein interactions with cholesterol in mammalian cells and specificity of antiviral activity toward IAV, SARS-CoV-2, and EBOV. Our studies suggest that modulation of IFITM S-palmitoylation levels and cholesterol interaction influence host susceptibility to different viruses.

SARS-CoV-2 Variants of Concern Hijack IFITM2 for Efficient Replication in Human Lung Cells.

ABSTRACTIt 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 lung cells, cardiomyocytes, and gut organoids. 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 five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (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 dominant Omicron variant.IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, 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 Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.

A novel domain within the CIL regulates egress of IFITM3 from the Golgi and reveals a regulatory role of IFITM3 on the secretory pathway.

The InterFeron-Induced TransMembrane proteins (IFITMs) are members of the dispanin/CD225 family that act as broad viral inhibitors by preventing viral-to-cellular membrane fusion. In this study, we uncover egress from the Golgi as an important step in the biology of IFITM3 by identifying the domain that regulates this process and that similarly controls the egress of the dispanins IFITM1 and PRRT2, protein linked to paroxysmal kinesigenic dyskinesia. In the case of IFITM3, high levels of expression of wild-type, or mutations in the Golgi egress domain, lead to accumulation of IFITM3 in the Golgi and drive generalized glycoprotein trafficking defects. These defects can be relieved upon incubation with Amphotericin B, compound known to relieve IFITM-driven membrane fusion defects, as well as by v-SNARE overexpression, suggesting that IFITM3 interferes with membrane fusion processes important for Golgi functionalities. The comparison of glycoprotein trafficking in WT versus IFITMs-KO cells indicates that the modulation of the secretory pathway is a novel feature of IFITM proteins. Overall, our study defines a novel domain that regulates the egress of several dispanin/CD225 members from the Golgi and identifies a novel modulatory function for IFITM3.

The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

IFITM proteins inhibit migration and invasion of human trophoblasts.

IFITM proteins inhibit migration and invasion of human trophoblasts.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Functional Heterogeneity of Mammalian IFITM Proteins against HIV-1.

Interferon-induced transmembrane proteins (IFITMs) are a family of interferon-inducible proteins that inhibit a broad range of viruses by interfering with viral-to-cellular membrane fusion. The antiviral activity of IFITMs is highly regulated by several posttranslational modifications and by a number of protein domains that modulate steady-state protein levels, trafficking, and antiviral effectiveness. Taking advantage of the natural diversity existing among IFITMs of different animal species, we have compared 21 IFITMs for their ability to inhibit HIV-1 at two steps, during virus entry into cells (target cell protection) and during the production of novel virion particles (negative imprinting of virion particles' infectivity). We found a high functional heterogeneity among IFITM homologs with respect to both antiviral modalities, with IFITM members that exhibit enhanced viral inhibition, while others have no ability to block HIV-1. These differences could not be ascribed to known regulatory domains and could only be partially explained through differential protein stability, implying the existence of additional mechanisms. Through the use of chimeras between active and inactive IFITMs, we demonstrate that the cross talk between distinct domains of IFITMs is an important contributor of their antiviral potency. Finally, we identified murine IFITMs as natural variants competent for target cell protection, but not for negative imprinting of virion particles' infectivity, suggesting that the two properties may, at least in principle, be uncoupled. Overall, our results shed new light on the complex relationship between IFITMs and viral infection and point to the cross talk between IFITM domains as a novel layer of regulation of their activity. IMPORTANCE IFITMs are broad viral inhibitors capable of interfering with both early and late phases of the replicative cycle of many different viruses. By comparing 21 IFITM proteins issued from different animal species for their ability to inhibit HIV-1, we have identified several that exhibit either enhanced or impaired antiviral behavior. This functional diversity is not driven by differences in known domains and can only be partly explained through differential protein stability. Chimeras between active and inactive IFITMs point to the cross talk between individual IFITM domains as important for optimal antiviral activity. Finally, we show that murine IFITMs are not capable of decreasing the infectivity of newly produced HIV-1 virion particles, although they retain target cell protection abilities, suggesting that these properties may be, in principle, disconnected. Overall, our results shed new light on the complex layers of regulation of IFITM proteins and enrich our current understanding of these broad antiviral factors.

Ferret Interferon (IFN)-Inducible Transmembrane Proteins Are Upregulated by both IFN-α and Influenza Virus Infection.

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.

The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets.

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.

Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

Opposing activities of IFITM proteins in SARS-CoV-2 infection.

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 YxxФ 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.

Syncytia formation by SARS-CoV-2-infected cells.

Severe cases of COVID‐19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS‐CoV‐2‐infected cells express the Spike protein (S) at their surface and fuse with ACE2‐positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon‐induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S‐mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell‐free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS‐CoV‐2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.

IFITM3 Reduces Retroviral Envelope Abundance and Function and Is Counteracted by glycoGag.

Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3.IMPORTANCE The viral envelope glycoprotein, known as "Env" in Retroviridae, is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.