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The role of host factors in the replication of emerging senecavirus A (SVA) which induced porcine idiopathic vesicular disease (PIVD) distributed worldwide remains obscure. Here, interferon-induced transmembrane (IFITM) protein 1 and 2 inhibit SVA replication by positive feedback with RIG-I signaling pathway was reported. The expression levels of IFITM1 and IFITM2 increased significantly in SVA infected 3D4/21 cells. Infection experiments of cells with over and interference expression of IFITM1 and IFITM2 showed that these two proteins inhibit SVA replication by regulating the expression of interferon beta (IFN-β), IFN-stimulated gene 15 (ISG-15), interleukin 6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), IFN regulatory factor-3 (IRF3), and IRF7. Further results showed that antiviral responses of IFITM1 and IFITM2 were achieved by activating retinoic acid-inducible gene I (RIG-I) signaling pathway which in turn enhanced the expression of IFITM1 and IFITM2. It is noteworthy that conserved domains of these two proteins also paly the similar role. These findings provide new data on the role of host factors in infection and replication of SVA and help to develop new agents against the virus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human interferon-induced transmembrane (IFITM) proteins inhibit the fusion of a broad spectrum of enveloped viruses, both when expressed in target cells and when present in infected cells. Upon expression in infected cells, IFITMs incorporate into progeny virions and reduce their infectivity by a poorly understood mechanism. Since only a few envelope glycoproteins (Envs) are present on HIV-1 particles, and Env clustering has been proposed to be essential for optimal infectivity, we asked if IFITM protein incorporation modulates HIV-1 Env clustering. The incorporation of two members of the IFITM family, IFITM1 and IFITM3, into HIV-1 pseudoviruses correlated with a marked reduction of infectivity. Super-resolution imaging of Env distribution on single HIV-1 pseudoviruses did not reveal significant effects of IFITMs on Env clustering. However, IFITM3 reduced the Env processing and incorporation into virions relative to the control and IFITM1-containing viruses. These results show that, in addition to interfering with the Env function, IFITM3 restricts HIV-1 Env cleavage and incorporation into virions. The lack of notable effect of IFITMs on Env clustering supports alternative restriction mechanisms, such as modification of the properties of the viral membrane.

Canine influenza virus (CIV) is an emerging virus that is associated with major hidden hazards to the canine population and public health. Until now, how canine uses its innate immunity to restrict CIV replication is seldomly investigated. Recently, studies on interferon-inducible transmembrane (IFITM) of several major hosts of influenza virus (human, chicken, duck, pig) indicated it can potently restrict the viral replication. Here, the gene locus of five previously annotated canine IFITM (caIFITM) genes was determined on chromosome 18 using multiple bioinformatics strategies, provisionally designated as caIFITM1, caIFITM2a, caIFITM2b, caIFITM3, and caIFITM5. An analysis on protein sequences between caIFITM and its homologs indicated they shared the same conserved amino acids important for the antiviral activity. Expression profile analysis showed that caIFITM was constitutively expressed in tissues and MDCK cell line. After treatment with interferon or infection with influenza virus, the expression level of caIFITM increased with different degrees in vitro . An animal challenge study demonstrated CIV infection resulted in upregulation of caIFITM in beagles. caIFITMs had a similar subcellular localization to their human homologs. caIFITM1 was present at the cell surface and caIFITM3 was present perinuclearly and colocalized with LAMP1-containing compartments. Finally, we generated A549 cell lines stably expressing caIFITM and challenged them with influenza virus. The result demonstrated caIFITM1, caIFITM2a, caIFITM2b, and caIFITM3 had a potent antiviral activity against influenza virus. Our study will help better understand the evolutional pattern of IFITM and its role in the host's defense against virus infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Ou, Cai, Lai, Zhong, Yin and Li.)

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.

The CD225 superfamily regulates vesicular membrane fusion events essential to neurotransmission, immunity, development, and metabolism. Its importance to physiology is reinforced by the identification of polymorphisms associated with disease. This article highlights the shared features that drive the function of CD225 proteins such as interferon-inducible transmembrane proteins 3 (IFITM3) and proline-rich transmembrane protein 2 (PRRT2) and is intended to catalyze efforts towards characterizing the lesser-known family members., Competing Interests: Declaration of Interests No interests are declared., (Published by Elsevier Ltd.)

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., (© 2020 The Authors.)

Type I interferon (IFN) induced by virus infections during pregnancy can cause placental damage, but the mechanisms and identities of IFN-stimulated genes that are involved in this damage remain under investigation. The IFN-induced transmembrane proteins (IFITMs) inhibit virus infections by preventing virus membrane fusion with cells and by inhibiting fusion of infected cells (syncytialization). Fusion of placental trophoblasts via expression of endogenous retroviral fusogens known as syncytins forms the syncytiotrophoblast, a multinucleated cell structure essential for fetal development. We found here that IFN blocks fusion of BeWo human placental trophoblasts. Stably expressed IFITM1, -2, and -3 also blocked fusion of these trophoblasts while making them more resistant to virus infections. Conversely, stable IFITM knockdowns in BeWo trophoblasts increased their spontaneous fusion and allowed fusion in the presence of IFN while also making the cells more susceptible to virus infection. We additionally found that exogenous expression of IFITMs in HEK293T cells blocked fusion with cells expressing syncytin-1 or syncytin-2, confirming the ability of IFITMs to block individual syncytin-mediated fusion. Overall, our data indicate that IFITMs inhibit trophoblast fusion and suggest that there may be a critical balance between these antifusogenic effects and the beneficial antiviral effects of IFITMs in virus infections during pregnancy., (© 2019 Zani et al.)

The interferon-induced transmembrane proteins 1-3 (IFITM1-3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1-3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein-protein interactions. Coexpression of IFITM1-3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

Interferon inducible transmembrane proteins (IFITMs) are one of several IFN-stimulated genes (ISGs) that restrict entry of enveloped viruses, including flaviviruses, filoviruses and retroviruses. It has been recently reported that in U87 glioblastoma cells IFITM proteins inhibit HIV-1 entry in a co-receptor-dependent manner, that is, IFITM1 is more inhibitory on CCR5 tropic HIV-1 whereas IFITM2/3 confers a greater suppression of CXCR4 counterparts. However, how entry of HIV-1 with distinct co-receptor usage is modulated by different IFITM orthologs in physiologically relevant CD4⁺ T cells and monocytes/macrophages has not been investigated in detail. Here, we report that overexpression of IFITM1, 2 and 3 in human CD4⁺ HuT78 cells, SupT1 cells, monocytic THP-1 cells and U87 cells expressing CD4 and co-receptor CCR5 or CXCR4, suppressed entry of CXCR4 tropic viruses NL4.3 and HXB2, CCR5 tropic viruses AD8 and JRFL, dual tropic 89.6 virus, as well as a panel of 32 transmitted founder (T/F) viruses, with a consistent order of potency, that is, IFITM3 > IFITM2 > IFITM1. Consistent with previous reports, we found that some CCR5-using HIV-1 isolates, such as AD8 and JRFL, were relatively resistant to inhibition by IFITM2 and IFITM3, although the effect can be cell-type dependent. However, in no case have we observed that IFITM1 had a stronger inhibition on entry of any HIV-1 strains tested, including those of CCR5-using T/Fs. We knocked down the endogenous IFITMs in peripheral blood mononuclear cells (PBMCs) and purified CD4⁺ T cells and observed that, while this treatment did greatly enhance the multiple-round of HIV-1 replication but had modest effect to rescue the single-round HIV-1 infection, reinforcing our previous conclusion that the predominant effect of IFITMs on HIV-1 infection is in viral producer cells, rather than in target cells to block viral entry. Overall, our results argue against the idea that IFITM proteins distinguish co-receptors CCR5 and CXCR4 to inhibit entry but emphasize that the predominant role of IFITMs on HIV-1 is in producer cells that intrinsically impair the viral infectivity.

Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes. IMPORTANCE The differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs., (Copyright © 2018 American Society for Microbiology.)

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.

Interferon-induced transmembrane protein (IFITM) 1, 2 and 3 genes encode a family of interferon (IFN)-induced transmembrane proteins that block entry of a broad spectrum of pathogens. However, the transcriptional regulation of these genes, especially whether there exist any enhancers and their roles during the IFN induction process remain elusive. Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. These findings expand our understanding of the mechanisms underlying the transcriptional regulation of IFITM1, 2 and 3 expression and its ability to mediate IFN signaling., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Interferon-induced transmembrane proteins (IFITMs) restrict the entry of diverse enveloped viruses through incompletely understood mechanisms. While IFITMs are reported to inhibit HIV-1, their in vivo relevance is unclear. We show that IFITM sensitivity of HIV-1 strains is determined by the co-receptor usage of the viral envelope glycoproteins as well as IFITM subcellular localization within the target cell. Importantly, we find that transmitted founder HIV-1, which establishes de novo infections, is uniquely resistant to the antiviral activity of IFITMs. However, viral sensitivity to IFITMs, particularly IFITM2 and IFITM3, increases over the first 6 months of infection, primarily as a result of neutralizing antibody escape mutations. Additionally, the ability to evade IFITM restriction contributes to the different interferon sensitivities of transmitted founder and chronic viruses. Together, these data indicate that IFITMs constitute an important barrier to HIV-1 transmission and that escape from adaptive immune responses exposes the virus to antiviral restriction., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interferon inducible transmembrane proteins (IFITMs) are broad-spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo-, flavi-, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid-induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH-induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV., (© 2016 The Authors. Traffic published by John Wiley & Sons Ltd.)

Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses., (Copyright © 2016. Published by Elsevier Inc.)

Ebolaviruses are highly pathogenic in humans and nonhuman primates and pose a severe threat to public health. The interferon-induced transmembrane (IFITM) proteins can restrict entry of ebolaviruses, influenza A viruses, and other enveloped viruses. However, the breadth and mechanism of the antiviral activity of IFITM proteins are incompletely understood. Here, we employed ebolavirus glycoprotein-pseudotyped vectors and ebolavirus-like particles to address this question. We show that IFITM proteins inhibit the cellular entry of diverse ebolaviruses and demonstrate that type I interferon induces IFITM protein expression in macrophages, major viral targets. Moreover, we show that IFITM proteins block entry of influenza A viruses and ebolaviruses by different mechanisms and provide evidence that antibodies and IFITM proteins can synergistically inhibit cellular entry of ebolaviruses. These results provide insights into the role of IFITM proteins in infection by ebolaviruses and suggest a mechanism by which antibodies, though poorly neutralizing in vitro, might contribute to viral control in vivo., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Background: Higher tissue transcript levels of immune-related markers-including the recently discovered viral restriction factor interferon-induced transmembrane protein (IFITM), which inhibits viral entry and replication-have been reported in the prefrontal cortex in schizophrenia. Interestingly, mouse models of neuroinflammation have higher IFITM levels and deficits in γ-aminobutyric acid (GABA)-related markers that are similar to findings in schizophrenia, suggesting that a shared pathogenetic process might underlie diverse cortical pathology in the disorder. However, the cell types that overexpress IFITM messenger RNA (mRNA) in schizophrenia are unknown, and it is unclear whether higher IFITM mRNA levels are associated with lower GABA-related marker levels in the same schizophrenia subjects., Methods: We used quantitative polymerase chain reaction and in situ hybridization with film and grain counting analyses to quantify IFITM mRNA levels in prefrontal cortex area 9 of 57 schizophrenia and 57 healthy comparison subjects and in antipsychotic-exposed monkeys., Results: Quantitative polymerase chain reaction and in situ hybridization film analysis revealed markedly elevated IFITM mRNA levels (+114% and +117%, respectively) in prefrontal gray matter in schizophrenia. Interestingly, emulsion-dipped, Nissl-stained sections from schizophrenia and comparison subjects revealed IFITM mRNA expression in pia mater and blood vessels. The IFITM grain density over blood vessels was 71% higher in schizophrenia. The IFITM mRNA levels were negatively correlated with GABA-related mRNAs in the same schizophrenia subjects., Conclusions: The finding that schizophrenia subjects with higher IFITM mRNA levels in cortical blood vessels have greater disturbances in cortical GABA neurons suggests that these cell-type distinct pathological disturbances might be influenced by a shared upstream insult that involves immune activation., (© 2013 Society of Biological Psychiatry Published by Society of Biological Psychiatry All rights reserved.)