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Interaction of IFITM3 with HA 2 subunit mediated by its transmembrane domain. A The truncated variants of IFITM3 protein are depicted. B The expression of IFITM3-truncated mutants was detected by Western blotting. C-D The interaction of IFITM3-truncated variants with H5 2 or H7 2 subunit. HEK293T cells (1 Â 10 6 cell/ well, 6-well plate) were co-transfected with H5 2 or H7 2 -fusion expression plasmids and each IFITM3 truncate respectively (2 μg/well each plasmid, 6-well plate) for 48 h before Co-IP with anti-mCherry beads. The lysates (C) and immunoprecipitates (D) were measured with the indicated antibodies by Western blotting. E Colocalization of IFITM3-truncated mutants (IMD-CIL and CIL-TMD) and H5 2 or H7 2 subunit in HeLa cells. (F-G) The interaction of IFITM3-truncated mutants with the truncated variants of the H5 2 (F) or H7 2 (G) subunit was verified by Co-IP with anti-EGFP magnetic beads. H Co-localization of IFITM3-truncated mutants and the truncated H5 2 or H7 2 subunit variants in HeLa cells.
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Interferon-inducible transmembrane protein 3 (IFITM3) inhibits influenza virus infection by blocking viral membrane fusion, but the exact mechanism remains elusive. Here, we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin (HA). The restriction of IFITM3 on HA-mediated viral entry was co...
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... feature of IFITM3 topology contains five domains: a cytosolic N terminus (NTD), an intramembrane domain (IMD), a cytosolic conserved intracellular loop (CIL) domain, a transmembrane domain (TMD) and extracellular C terminus (CTD) (Fig. 5A) (Y anez et al., 2020). The IMD and CIL domains are highly conserved to form the CD225 domain ( John et al., 2013). Each domain and the correct subcellular localization of IFITM3 are key factors of its antiviral activity ( Rahman et al., 2020). So, it is still a question about which domain is involved in its interaction with the HA 2 subunit. To answer this ...
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... subcellular localization of IFITM3 are key factors of its antiviral activity ( Rahman et al., 2020). So, it is still a question about which domain is involved in its interaction with the HA 2 subunit. To answer this question, a series of EGFP-tagged IFITM3-truncated mutants were constructed to encode various fragments with C-terminally EGFP (Fig. 5A). Also, the EGFP-fused NTD and CIL-TMD fragments were highly expressed, while IMD-CIL-EGFP expression was slightly lower (Fig. 5B). After that, mCherry-tagged HA 2 subunit with NTD, IMD-CIL, and CIL-TMD fragments was expressed in HEK293T cells, and the expression of the interest proteins was detected with anti-mCherry or EGFP antibodies ...
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... about which domain is involved in its interaction with the HA 2 subunit. To answer this question, a series of EGFP-tagged IFITM3-truncated mutants were constructed to encode various fragments with C-terminally EGFP (Fig. 5A). Also, the EGFP-fused NTD and CIL-TMD fragments were highly expressed, while IMD-CIL-EGFP expression was slightly lower (Fig. 5B). After that, mCherry-tagged HA 2 subunit with NTD, IMD-CIL, and CIL-TMD fragments was expressed in HEK293T cells, and the expression of the interest proteins was detected with anti-mCherry or EGFP antibodies by Western blotting (Fig. 5C and D). Immunoprecipitation analyses demonstrated that mCherry-tagged H5 2 and H7 2 have efficiently ...
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... EGFP-fused NTD and CIL-TMD fragments were highly expressed, while IMD-CIL-EGFP expression was slightly lower (Fig. 5B). After that, mCherry-tagged HA 2 subunit with NTD, IMD-CIL, and CIL-TMD fragments was expressed in HEK293T cells, and the expression of the interest proteins was detected with anti-mCherry or EGFP antibodies by Western blotting (Fig. 5C and D). Immunoprecipitation analyses demonstrated that mCherry-tagged H5 2 and H7 2 have efficiently co-precipitated with CIL-TMD fragment of IFITM3 but did not interact with the NTD domain (Fig. 5D). Surprisingly, a weaker interaction between IMD-CIL in IFITM3 with H5 2 was observed, but not with the H7 2 subunit (Fig. 5D). Localization ...
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... was expressed in HEK293T cells, and the expression of the interest proteins was detected with anti-mCherry or EGFP antibodies by Western blotting (Fig. 5C and D). Immunoprecipitation analyses demonstrated that mCherry-tagged H5 2 and H7 2 have efficiently co-precipitated with CIL-TMD fragment of IFITM3 but did not interact with the NTD domain (Fig. 5D). Surprisingly, a weaker interaction between IMD-CIL in IFITM3 with H5 2 was observed, but not with the H7 2 subunit (Fig. 5D). Localization analyses showed that the H5 2 subunit showed co-localization with the IFITM3 CIL-TMD fragment but not the IMD-CIL fragment, while the H7 2 subunit had partial co-localization (Fig. 5E). These ...
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... by Western blotting (Fig. 5C and D). Immunoprecipitation analyses demonstrated that mCherry-tagged H5 2 and H7 2 have efficiently co-precipitated with CIL-TMD fragment of IFITM3 but did not interact with the NTD domain (Fig. 5D). Surprisingly, a weaker interaction between IMD-CIL in IFITM3 with H5 2 was observed, but not with the H7 2 subunit (Fig. 5D). Localization analyses showed that the H5 2 subunit showed co-localization with the IFITM3 CIL-TMD fragment but not the IMD-CIL fragment, while the H7 2 subunit had partial co-localization (Fig. 5E). These results seemed to imply that the transmembrane structure of IFITM3 plays an important role in its interaction with ...
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... with the NTD domain (Fig. 5D). Surprisingly, a weaker interaction between IMD-CIL in IFITM3 with H5 2 was observed, but not with the H7 2 subunit (Fig. 5D). Localization analyses showed that the H5 2 subunit showed co-localization with the IFITM3 CIL-TMD fragment but not the IMD-CIL fragment, while the H7 2 subunit had partial co-localization (Fig. 5E). These results seemed to imply that the transmembrane structure of IFITM3 plays an important role in its interaction with ...
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... ΔFP_H5 2 , ΔTM_H5 2 , ΔFP_H7 2 , or ΔTM_H7 2 was co-precipitated with IMD-CIL or CIL-TMD fragment of IFITM3. Co-immunoprecipitation experiments showed that the FP-deficient H5 2 subunit was coprecipitated with IMD-CIL (CD225 domain) and CIL-TMD domain (Fig. 5F). Similarly, the TM-deficient H5 2 subunit was also captured by IMD-CIL (CD225 domain) or CIL-TMD domain, respectively, but the affinity between them remained weak ( Fig. 5F) and consistent with that shown in Fig. 5D. Localization analyses showed that TM-deficient or FPdeficient H5 2 subunit showed partial co-localization with the ...
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... or CIL-TMD fragment of IFITM3. Co-immunoprecipitation experiments showed that the FP-deficient H5 2 subunit was coprecipitated with IMD-CIL (CD225 domain) and CIL-TMD domain (Fig. 5F). Similarly, the TM-deficient H5 2 subunit was also captured by IMD-CIL (CD225 domain) or CIL-TMD domain, respectively, but the affinity between them remained weak ( Fig. 5F) and consistent with that shown in Fig. 5D. Localization analyses showed that TM-deficient or FPdeficient H5 2 subunit showed partial co-localization with the IFITM3 IMD-CIL fragment and FP-deficient H5 2 subunit co-localization with IFITM3 CIL-TMD fragment, while TM deletion mutation resulted in the disappearance of this ...
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... experiments showed that the FP-deficient H5 2 subunit was coprecipitated with IMD-CIL (CD225 domain) and CIL-TMD domain (Fig. 5F). Similarly, the TM-deficient H5 2 subunit was also captured by IMD-CIL (CD225 domain) or CIL-TMD domain, respectively, but the affinity between them remained weak ( Fig. 5F) and consistent with that shown in Fig. 5D. Localization analyses showed that TM-deficient or FPdeficient H5 2 subunit showed partial co-localization with the IFITM3 IMD-CIL fragment and FP-deficient H5 2 subunit co-localization with IFITM3 CIL-TMD fragment, while TM deletion mutation resulted in the disappearance of this co-localization (Fig. 5H). Meanwhile, the FPdeficient H7 ...
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... Fig. 5F) and consistent with that shown in Fig. 5D. Localization analyses showed that TM-deficient or FPdeficient H5 2 subunit showed partial co-localization with the IFITM3 IMD-CIL fragment and FP-deficient H5 2 subunit co-localization with IFITM3 CIL-TMD fragment, while TM deletion mutation resulted in the disappearance of this co-localization (Fig. 5H). Meanwhile, the FPdeficient H7 2 subunit was co-precipitated with the CIL-TMD domain Fig. 2. Subcellular localization analysis of HA 2 subunit in late endosomes. A Schematic diagram of HA. HA precursor is divided into two subunits by proteolytic cleavage. HA 2 contains fusion peptide (FP), stem region (stalk), transmembrane domain ...
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... not with the IMD-CIL domain, and TM deletion mutant (ΔTM_H7 2 ) did not bind with the CIL-TMD domain in IFITM3 (Fig. 5G). In terms of subcellular localization, TM-deficient or FP-deficient H7 2 subunit partly co-localized with IFITM3 IMD-CIL or CIL-TMD domain (Fig. 5H), providing favorable conditions for the interaction between them. To sum up, the transmembrane domain of IFITM3 was required for IFITM3-HA 2 subunit interaction. According to the above ...
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... not with the IMD-CIL domain, and TM deletion mutant (ΔTM_H7 2 ) did not bind with the CIL-TMD domain in IFITM3 (Fig. 5G). In terms of subcellular localization, TM-deficient or FP-deficient H7 2 subunit partly co-localized with IFITM3 IMD-CIL or CIL-TMD domain (Fig. 5H), providing favorable conditions for the interaction between them. To sum up, the transmembrane domain of IFITM3 was required for IFITM3-HA 2 subunit interaction. According to the above data, the transmembrane domain of IFITM3 and HA protein is supported to mediate their ...
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The viral hemagglutinins of conventional influenza A viruses (IAVs) bind to sialylated glycans on host cell surfaces for attachment and subsequent infection. In contrast, hemagglutinins of bat-derived IAVs target major histocompatibility complex class II (MHC-II) for cell entry. MHC-II proteins from various vertebrate species can facilitate infecti...
Citations
... To investigate whether IFITM3 may directly interact with virus envelope proteins as previously reported, [31][32][33] we constructed fusion expression plasmids of Gn/Gc (M), individual Gn or Gc with C-terminally EGFP tag, and IFITM3 fused with mCherry in its C-terminal. Laser confocal microscope analysis showed that IFITM3 colocalized with M precursor protein ( Figure 5A) and individual Gn ( Figure 5B), but no obvious colocalization was observed with individual Gc ( Figure 5C). ...
Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick‐borne hemorrhagic fever disease with high fatality rate of 10%–20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad‐spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti‐SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti‐SFTSV function by blocking Gn/Gc‐mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3‐Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3‐Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs‐mediated anti‐SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.
... Nonetheless, distinct patterns of sensitivity to IFITM3 have been described, depending on the viral serotype [12]. Focusing on the inhibitory effect of IFITM3 on viral entry in target cells, a recent study showed that IFITM3 interacted with the membrane fusion subunit of hemagglutinin (HA2) via their transmembrane domain [58]. This interaction occurred in late endosomes and lysosomes and could be a possible mechanism of antiviral action for IFITM3 ( Figure 2B). ...
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.
... The N914Q mutant was constructed using the Q5® Site-Directed Mutagenesis Kit (New England Biolab, USA). The WT and mutant pseudoviruses were prepared as previously described 62 . Briefly, HEK293T/17 cells were cotransfected with pLentiCMV-Luc2, pCMV-dR8.2-dvpr ...
The severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne human-infecting bunyavirus, which utilizes two envelope glycoproteins, Gn and Gc, to enter host cells. However, the structure and organization of these glycoproteins on virion surface are not yet known. Here we describe the structure of SFTSV determined by single particle reconstruction, which allows mechanistic insights into bunyavirus assembly at near-atomic resolution. The SFTSV Gn and Gc proteins exist as heterodimers and further assemble into pentameric and hexameric peplomers, shielding the Gc fusion loops by both intra- and inter-heterodimer interactions. Individual peplomers are associated mainly through the ectodomains, in which the highly conserved glycans on N914 of Gc play a crucial role. This elaborate assembly stabilizes Gc in the metastable prefusion conformation and creates some cryptic epitopes that are only accessible in the intermediate states during virus entry. These findings provide an important basis for developing vaccines and therapeutic drugs.
... The mechanism of a broad-spectrum restriction of enveloped virus entry by IFITMs is not fully understood. It is believed that different IFITMs inhibit virus fusion through a common mechanism that does not generally involve specific interactions with viral proteins or their cellular receptors, although a few instances of such interactions have been reported [44][45][46]. Current models for IFITM-mediated restriction include (1) rendering the cell membranes more rigid and thereby trapping viral fusion at a hemifusion stage [47][48][49][50][51][52] and (2) accelerating the degradation of incoming viruses by transporting them to lysosomes [53]. ...
Interferon-induced transmembrane proteins (IFITMs) block the fusion of diverse enveloped viruses, likely through increasing the cell membrane’s rigidity. Previous studies have reported that the antiviral activity of the IFITM family member, IFITM3, is antagonized by cell pretreatment with rapamycin derivatives and cyclosporines A and H (CsA and CsH) that promote the degradation of IFITM3. Here, we show that CsA and CsH potently enhance virus fusion with IFITM1- and IFITM3-expressing cells by inducing their rapid relocalization from the plasma membrane and endosomes, respectively, towards the Golgi. This relocalization is not associated with a significant degradation of IFITMs. Although prolonged exposure to CsA induces IFITM3 degradation in cells expressing low endogenous levels of this protein, its levels remain largely unchanged in interferon-treated cells or cells ectopically expressing IFITM3. Importantly, the CsA-mediated redistribution of IFITMs to the Golgi occurs on a much shorter time scale than degradation and thus likely represents the primary mechanism of enhancement of virus entry. We further show that rapamycin also induces IFITM relocalization toward the Golgi, albeit less efficiently than cyclosporines. Our findings highlight the importance of regulation of IFITM trafficking for its antiviral activity and reveal a novel mechanism of the cyclosporine-mediated modulation of cell susceptibility to enveloped virus infection.
... IFITMs have also been found to interact with HIV-1 envelope glycoproteins (Env) and to disrupt their proteolytic processing (62). Similarly IFITM3 has been found to interact directly with IAV hemagglutinin (HA) (63), while interactions between IFITM proteins and SARS-CoV-2 spike have also been described and may influence infection outcome (64). ...
... One study noted that IFITM3 expression displaced IAV HA from viral particles, which in turn sensitized the virus to neutralizing antibodies (68). At present, it is unclear whether this mechanism involves direct binding between IFITMs and HA (63) or if it will be applicable to other viruses. ...
Interferon induced transmembrane proteins (IFITMs) are broad spectrum antiviral factors that inhibit the entry of a wide range of clinically important pathogens including influenza A virus, HIV-1 and Dengue virus. IFITMs are thought to act primarily by antagonising virus-cell membrane fusion in this regard. However, recent work on these proteins has uncovered novel post-entry viral restriction mechanisms. IFITMs are also increasingly thought to have a role regulating immune responses, including innate antiviral and inflammatory responses as well as adaptive T cell and B cell responses. Further, IFITMs may have pathological activities in cancer wherein IFITM expression can be a marker of therapeutically resistant and aggressive disease courses. In this review, we summarise the respective literatures concerning these apparently diverse functions with a view to identifying common themes and potentially yielding a more unified understanding of IFITM biology.
Influenza virus has been one of the most prevalent and researched viruses globally. Consequently, there is ample information available about influenza virus lifecycle and pathogenesis. However, there is plenty yet to be known about the determinants of influenza virus pathogenesis and disease severity. Influenza virus exploits host factors to promote each step of its lifecycle. In turn, the host deploys antiviral or restriction factors that inhibit or restrict the influenza virus lifecycle at each of those steps. Two broad categories of host restriction factors can exist in virus-infected cells: (1) encoded by the interferon-stimulated genes (ISGs) and (2) encoded by the constitutively expressed genes that are not stimulated by interferons (non-ISGs). There are hundreds of ISGs known, and many, e.g., Mx, IFITMs, and TRIMs, have been characterized to restrict influenza virus infection at different stages of its lifecycle by (1) blocking viral entry or progeny release, (2) sequestering or degrading viral components and interfering with viral synthesis and assembly, or (3) bolstering host innate defenses. Also, many non-ISGs, e.g., cyclophilins, ncRNAs, and HDACs, have been identified and characterized to restrict influenza virus infection at different lifecycle stages by similar mechanisms. This review provides an overview of those ISGs and non-ISGs and how the influenza virus escapes the restriction imposed by them and aims to improve our understanding of the host restriction mechanisms of the influenza virus.
