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Impact of LE/L cholesterol accumulation on the sequential steps of IAV host cell entry. (A) A431-WT and A431-AnxA6 cells were infected with IAV (PR8M; MOI of 50) at 4°C for 1 h. An acidic bypass was applied to induce fusion at the plasma membrane, and infection through endosomal uptake was blocked by bafilomycin A1 (Baf) treatment. Cells were further incubated for 8 h, stained for NP, and analyzed by FACS for NP-positive cells. Data are expressed as the mean percentages SEM of NP-positive cells from three independent experiments. (B) A431-WT and A431-AnxA6 cells were infected with IAV (PR8M; MOI of 10). M1 protein levels were monitored by Western blotting, and blots were probed for tubulin to verify equal levels of loading. Mean M1 signal intensities SEM of results from three independent experiments were calculated relative to the mean M1 intensity detected in A431-WT cells 30 min p.i. (C) A431-WT and A431-AnxA6 cells were infected with IAV (31; MOI of 20). Cells were fixed and stained 1 h p.i. with the A1 antibody to detect the acid-induced conformation of HA (HAac). The signal intensity per cell was quantified by ImageJ analysis. Mean values SEM were calculated from 55 individual cells per condition from at least two independent experiments. ns, not significant (two-way ANOVA followed by Tukey's multiple-comparison test).
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To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral conte...
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... of the viral genome into the cytosol occurs during passage through the late endosomal compartment. Hence we speculated that LE/L cholesterol imbalance might interfere with viral uncoating and/or membrane fusion. Therefore, a viral envelope/ endosomal membrane fusion assay utilizing IAV particles labeled with the two lipo- philic dyes 3,3=-dioctadecyl-5,5=-di(4-sulfophenyl)oxacarbocyanine (SP-DiOC18) and oc- tadecyl rhodamine b chloride (R18), initially described by Sakai et al. (26), was employed to identify potential fusion defects. Upon fusion, the dyes diffuse out of the viral envelope and into the endosomal membrane, leading to dequenching and a subse- quent increase in SP-DiOC18 fluorescence. This elevated SP-DiOC18 signal was de- creased in infected A549-WT cells transiently overexpressing IFITM3 compared to nontransfected control cells, verifying that the assay was suited for detection of antiviral restriction through host cell factors (Fig. S4A). Bafilomycin A1, which blocks endosomal acidification, an essential prerequisite for successful IAV membrane fusion, almost completely abolished SP-DiOC18 dequenching in both A549-WT and A549- IFITM3KO cells (Fig. 5A). Remarkably, the IFN treatment resulted in a decrease in dequenching in the A549-WT cells, whereas in the A549-IFITM3KO cells the opposite effect, i.e., an increase in dequenching, was found. Importantly, in both cell lines, U18666A treatment significantly caused a pronounced decrease in SP-DiOC18 de- quenching, indicating that IAV membrane fusion within cholesterol-laden endosomes was impaired. Similar results were observed when A431-WT cells and A431-AnxA6 cells were compared for IAV-endosome fusion, and the impaired virus fusion with cholesterol-laden endosomal membranes was still observed in A431-AnxA6 cells that were subjected to small interfering RNA (siRNA) treatment to silence IFITM3 expression (Fig. 5B). Because these results contradicted previously published findings on the impact of LE/L cholesterol on viral envelope/endosomal membrane fusion (6), we applied the respective single-dye dequenching approaches used in the study by Desai et al. Notably, in this assay, neither use of SP-DiOC18 nor use of 1,1=-dioctadecyl- 3,3,3=,3=-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate (DiD) yielded signif- icant differences in the efficiencies of IAV/endosomal membrane fusion between A431-AnxA6 cells and A431-WT cells, although robust bafilomycin A1-mediated inhi- Late Endosomal Cholesterol Protects against IAV Entry bition could be detected (Fig. S4B). This suggests that single-dye fusion assays display reduced ...
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... of the viral genome into the cytosol occurs during passage through the late endosomal compartment. Hence we speculated that LE/L cholesterol imbalance might interfere with viral uncoating and/or membrane fusion. Therefore, a viral envelope/ endosomal membrane fusion assay utilizing IAV particles labeled with the two lipo- philic dyes 3,3=-dioctadecyl-5,5=-di(4-sulfophenyl)oxacarbocyanine (SP-DiOC18) and oc- tadecyl rhodamine b chloride (R18), initially described by Sakai et al. (26), was employed to identify potential fusion defects. Upon fusion, the dyes diffuse out of the viral envelope and into the endosomal membrane, leading to dequenching and a subse- quent increase in SP-DiOC18 fluorescence. This elevated SP-DiOC18 signal was de- creased in infected A549-WT cells transiently overexpressing IFITM3 compared to nontransfected control cells, verifying that the assay was suited for detection of antiviral restriction through host cell factors (Fig. S4A). Bafilomycin A1, which blocks endosomal acidification, an essential prerequisite for successful IAV membrane fusion, almost completely abolished SP-DiOC18 dequenching in both A549-WT and A549- IFITM3KO cells (Fig. 5A). Remarkably, the IFN treatment resulted in a decrease in dequenching in the A549-WT cells, whereas in the A549-IFITM3KO cells the opposite effect, i.e., an increase in dequenching, was found. Importantly, in both cell lines, U18666A treatment significantly caused a pronounced decrease in SP-DiOC18 de- quenching, indicating that IAV membrane fusion within cholesterol-laden endosomes was impaired. Similar results were observed when A431-WT cells and A431-AnxA6 cells were compared for IAV-endosome fusion, and the impaired virus fusion with cholesterol-laden endosomal membranes was still observed in A431-AnxA6 cells that were subjected to small interfering RNA (siRNA) treatment to silence IFITM3 expression (Fig. 5B). Because these results contradicted previously published findings on the impact of LE/L cholesterol on viral envelope/endosomal membrane fusion (6), we applied the respective single-dye dequenching approaches used in the study by Desai et al. Notably, in this assay, neither use of SP-DiOC18 nor use of 1,1=-dioctadecyl- 3,3,3=,3=-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate (DiD) yielded signif- icant differences in the efficiencies of IAV/endosomal membrane fusion between A431-AnxA6 cells and A431-WT cells, although robust bafilomycin A1-mediated inhi- Late Endosomal Cholesterol Protects against IAV Entry bition could be detected (Fig. S4B). This suggests that single-dye fusion assays display reduced ...
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... cholesterol accumulation inhibits IAV/endosome membrane fusion. To unravel the details of the actual step that was negatively affected by increased LE/L cholesterol levels, we first performed acid-bypass infection assays. This technique forces the fusion of IAV particles with the host cell plasma membrane (23) and leads to a direct transfer of the viral genome into the cytosol, thus circumventing the endocytic path- way. Strikingly, there was no difference in the numbers of infected A431-WT and A431-AnxA6 cells (Fig. 4A), suggesting that LE/L cholesterol accumulation acted on the endosomal uptake of IAV. We next performed a comprehensive analysis of the sequen- tial events during host-cell entry. In line with our previous work (13), the levels of efficiency of IAV endosomal uptake, as judged by the levels of the virion-associated matrix protein M1 (24), were similar in A431-WT and A431-AnxA6 cells (Fig. 4B). Because the acidic environment encountered in the LE/L induced conformational changes in HA that critically affect the fusogenic capacity, we next used an antibody specific for the fusion-active conformation (25). As expected from the findings indicating that LE/L pH was not affected, no significant differences in the amount of signal were detected in A431-WT and A431-AnxA6 cells (Fig. 4C). In contrast, hardly any signal could be detected in cells treated with bafilomycin A1, an inhibitor of the vacuolar ATPase required for endosomal acidification, which therefore served as a positive ...
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... cholesterol accumulation inhibits IAV/endosome membrane fusion. To unravel the details of the actual step that was negatively affected by increased LE/L cholesterol levels, we first performed acid-bypass infection assays. This technique forces the fusion of IAV particles with the host cell plasma membrane (23) and leads to a direct transfer of the viral genome into the cytosol, thus circumventing the endocytic path- way. Strikingly, there was no difference in the numbers of infected A431-WT and A431-AnxA6 cells (Fig. 4A), suggesting that LE/L cholesterol accumulation acted on the endosomal uptake of IAV. We next performed a comprehensive analysis of the sequen- tial events during host-cell entry. In line with our previous work (13), the levels of efficiency of IAV endosomal uptake, as judged by the levels of the virion-associated matrix protein M1 (24), were similar in A431-WT and A431-AnxA6 cells (Fig. 4B). Because the acidic environment encountered in the LE/L induced conformational changes in HA that critically affect the fusogenic capacity, we next used an antibody specific for the fusion-active conformation (25). As expected from the findings indicating that LE/L pH was not affected, no significant differences in the amount of signal were detected in A431-WT and A431-AnxA6 cells (Fig. 4C). In contrast, hardly any signal could be detected in cells treated with bafilomycin A1, an inhibitor of the vacuolar ATPase required for endosomal acidification, which therefore served as a positive ...
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... cholesterol accumulation inhibits IAV/endosome membrane fusion. To unravel the details of the actual step that was negatively affected by increased LE/L cholesterol levels, we first performed acid-bypass infection assays. This technique forces the fusion of IAV particles with the host cell plasma membrane (23) and leads to a direct transfer of the viral genome into the cytosol, thus circumventing the endocytic path- way. Strikingly, there was no difference in the numbers of infected A431-WT and A431-AnxA6 cells (Fig. 4A), suggesting that LE/L cholesterol accumulation acted on the endosomal uptake of IAV. We next performed a comprehensive analysis of the sequen- tial events during host-cell entry. In line with our previous work (13), the levels of efficiency of IAV endosomal uptake, as judged by the levels of the virion-associated matrix protein M1 (24), were similar in A431-WT and A431-AnxA6 cells (Fig. 4B). Because the acidic environment encountered in the LE/L induced conformational changes in HA that critically affect the fusogenic capacity, we next used an antibody specific for the fusion-active conformation (25). As expected from the findings indicating that LE/L pH was not affected, no significant differences in the amount of signal were detected in A431-WT and A431-AnxA6 cells (Fig. 4C). In contrast, hardly any signal could be detected in cells treated with bafilomycin A1, an inhibitor of the vacuolar ATPase required for endosomal acidification, which therefore served as a positive ...
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... order to investigate the influence of the pharmacologically increased LE/L cho- lesterol level in more detail, we followed the progress of the fusion over time (Fig. 4C). U18666A-treated cells showed lower values at all times measured during the 6 h after infection. However, the analysis of the dequenching rates revealed that the increases in signal intensity were approximately parallel in both cases. This most likely reflects the well-known fact that the bulk of the fusion occurs within the first hour after infection (25) and that the propagation of the dye in the host membrane after fusion is not disturbed by ...
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... Influenza A viruses (IAVs) are common contagious pathogens involved in global pandemics and seasonal epidemics that pose a substantial global health burden and are transmitted among humans and animals, including poultry, pigs, dogs, and horses 1,2 . Due to individual differences in the immune response (susceptibility, duration, and intensity), IAV infection is more severe in some groups than in others, which may be related to host genetic factors affecting viral replication 3 . ...
Influenza A virus (IAV) has developed multiple tactics to hinder the innate immune response including the epigenetic regulation during IAV infection, but the novel epigenetic factors and their mechanism in innate immunity remain well studied. Here, through a non-biased high-throughput sgRNA screening of 1041 known epigenetic modifiers in a cellular model of IAV-induced interferon-beta (IFN-β) production, we identified nei endonuclease VIII-like 1 (NEIL1) as a critical regulator of IFN-β in response to viral infection. Further studies showed that NEIL1 promoted the replication of the influenza virus by regulating the methylation of cytonuclear IFN-β promoter (mainly CpG-345), inhibiting the expression of IFN-β and IFN-stimulating genes. The structural domains of NEIL1, especially the catalytic domain, were critical for the suppression of IFN-β production, but the enzymatic activity of NEIL1 was dispensable. Furthermore, our results revealed that NEIL1 relied on interactions with the N-and C-terminus of the nucleoprotein (NP) of IAV, and NEIL1 expression facilitated the entry of the NP into the nucleus, which further enhanced the stability of the viral ribonucleoprotein (vRNP) complex and thus contributed to IAV replication and transcription. These findings reveal an enzyme-independent mechanism of host NEIL1 that negatively regulates IFN-β expression, thereby facilitating IAV propagation. Our study provides new insights into the roles of NEIL1, both in directly promoting virus replication and in evading innate immunity in IAV infection. Influenza A viruses (IAVs) are common contagious pathogens involved in global pandemics and seasonal epidemics that pose a substantial global health burden and are transmitted among humans and animals, including poultry, pigs, dogs, and horses 1,2. Due to individual differences in the immune response (susceptibility, duration, and intensity), IAV infection is more severe in some groups than in others, which may be related to host genetic factors affecting viral replication 3. Multiple genome-wide screening methods used in different studies have also identified a large number of host factors that may contribute to IAV infection 4-6. Validation studies have revealed host factors critical for IAV that act at different stages of viral replication, including entry and antiviral responses 4. Currently, pathogens have evolved a variety of epigenetic strategies to enhance their survival and proliferation. These strategies include the direct modification of host proteins and chromatin via viral-specific gene products, which in turn reduces the sensitivity of antigen-specific receptors and signaling pathways, and modulates the expression of both immune system activators and suppressors. On the other hand, the host can also overcome pathogen-induced epigenetic changes and continue to mount an effective anti-pathogen immune response 7,8. The innate immune system is the host's first line of defense against viral infection, and interferons (IFNs) are considered an important component of the innate immune response 9,10. Type I IFNs (IFN-I) include IFN-a and β, as well as the less well-characterized IFNs τ and ω. Accumulating evidence suggests IFN-I are required for an antiviral response acting, in part, to prevent viral replication 11,12. Recent reports suggest that blocking IFN-I signaling in virus-induced chronic infections reduces immunosuppression and accelerates clearance of persistent infections 13,14. Gao et al. 15 compared the induction of IFN-β during infection with the IAV strain H5N1 and found that the expression of IFN-I was significantly inhibited in infections with the highly pathogenic H5N1 strain. Therefore, precise regulation of IFN-I production is critical to maintaining host homeostasis. Transcrip-tional regulation of IFN-I has long been a focus of research due to its unique function in generating an antiviral response 16 ; and IFN-β promoter is one of the most well-characterized and understood promoters of mammalian gene
... The organic layer was dried over Na 2 SO 4 and the solvent was removed under reduced pressure to provide the desired product. (60). To a solution of 44a (75.0 mg, 279 μmol) in dry DMF (3.00 mL/mmol) cooled to 0°C, DIPEA (5.00 equiv, 1.39 mmol) and HATU (2.00 equiv, 557 μmol) were added sequentially. ...
... The IFITM3-VAPA interaction results in increased cholesterol levels in multivesicular bodies and late endosomes. This is believed to block the infection of VSV and IAV by inhibiting viral release from the endosomes [99,100]. However, conflicting data cast a shadow on the role of VAPA in IFITM-mediated viral restriction. ...
Interferons (IFNs) are antiviral cytokines that defend against viral infections by inducing the expression of interferon-stimulated genes (ISGs). Interferon-inducible transmembrane proteins (IFITMs) 1, 2, and 3 are crucial ISG products and members of the CD225 protein family. Compelling evidence shows that IFITMs restrict the infection of many unrelated viruses by inhibiting the virus–cell membrane fusion at the virus entry step via the modulation of lipid composition and membrane properties. Meanwhile, viruses can evade IFITMs’ restrictions by either directly interacting with IFITMs via viral glycoproteins or by altering the native entry pathway. At the same time, cumulative evidence suggests context-dependent and multifaceted roles of IFITMs in modulating virus infections and cell signaling. Here, we review the diverse antiviral mechanisms of IFITMs, the viral antagonizing strategies, and the regulation of IFITM activity in host cells. The mechanisms behind the antiviral activity of IFITMs could aid the development of broad-spectrum antivirals and enhance preparedness for future pandemics.
... IFITM-3 belongs to the IFITM family (Yánez et al. 2020). FITM3 encodes interferon-induced transmembrane protein 3, which plays a crucial role in conferring immunity to various viral infections and degrading viral components via lysosomes (Bedford et al. 2019;Kühnl et al. 2018). Simultaneously, the IFITM protein is associated with other atopic dermatitis (F. ...
Sjogren’s syndrome (SS) is an autoimmune disorder characterized by dry mouth and dry eyes. Its pathogenic mechanism is currently unclear. This study aims to integrate weighted gene co-expression network analysis (WGCNA) and machine learning to identify key genes associated with SS. We downloaded 3 publicly available datasets from the GEO database comprising the gene expression data of 231 SS and 78 control cases, including GSE84844, GSE48378 and GSE51092, and carried out WGCNA to elucidate differences in the abundant genes. Candidate biomarkers for SS were then identified using a LASSO regression model. Totally 6 machine-learning models were subsequently utilized for validating the biological significance of major genes according to their expression. Finally, immune cell infiltration of the SS tissue was assessed using the CIBERSORT algorithm. A weighted gene co-expression network was built to divide genes into 10 modules. Among them, blue and red modules were most closely associated with SS, and showed significant enrichment in type I interferon signaling, cellular response to type I interferon and response to virus, etc. Combined machine learning identified 5 hub genes, including OAS1, EIF2AK2, IFITM3, TOP2A and STAT1. Immune cell infiltration analysis showed that SS was associated with CD8⁺ T cell, CD4⁺ T cell, gamma delta T cell, NK cell and dendritic cell activation. WGCNA was combined with machine learning to uncover genes that may be involved in SS pathogenesis, which can be utilized for developing SS biomarkers and appropriate therapeutic targets.
... This includes the main cholesterol transporter in LE/Lys, Niemann-Pick type C1 (NPC1), which serves as the entry factor for several filoviruses with Ebola virus using NPC1 in a cholesterolindependent manner (Carette et al, 2011;Cote et al, 2011). In addition, cholesterol accumulation in LE/Lys, using the pharmacological NPC1 inhibitor U18666A, compromised fusion of the influenza lipid envelope with late endosomal membranes (Musiol et al, 2013;Kuhnl et al, 2018;Schloer et al, 2019). Alike, U18666A and other drugs that cause LE/Lys cholesterol accumulation elicit antiviral activity (Sturley et al, 2020) and specifically, U18666A reduced SARS-CoV-2 infection in Vero-E6 and Calu-3a cells (Schloer et al, 2020a). ...
... Blocked endolysosomal cholesterol efflux upon NPC1 inhibition causes cholesterol depletion in other cellular sites (Cubells et al, 2007;Musiol et al, 2013), such as the plasma membrane. Indeed, U18666A treatment or Rab7 inhibition, which also triggers LE/Lyscholesterol accumulation (Meneses-Salas et al, 2020), reduced the number of released influenza virus progeny and lowered cholesterol in the viral envelope, both critical for the success of influenza infection (Musiol et al, 2013;Kuhnl et al, 2018). Yet, coronaviruses assemble at membranes of the ER Golgi intermediate compartment (ERGIC), followed by budding into the lumen and release via exocytosis of cargo vesicles (Stertz et al, 2007). ...
... Several other factors of the endocytic machinery in LE/Lys linked to cholesterol and NPC1 function could also emerge as druggable antiviral targets. This includes endosomal sorting complex required for transport complexes (Du et al, 2012(Du et al, , 2013, late endosomal annexins (Musiol et al, 2013;Kuhnl et al, 2018;Enrich et al, 2021), and Rab-GTPases (Zang et al, 2020) and their regulators and effectors (Meneses-Salas et al, 2020), but also proteins that link cholesterol and calcium balance in LE/Lys (Lloyd-Evans et al, 2008;Enrich et al, 2021). ...
The rapid development of vaccines to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has been critical to reduce the severity of COVID-19. However, the continuous emergence of new SARS-CoV-2 subtypes highlights the need to develop additional approaches that oppose viral infections. Targeting host factors that support virus entry, replication, and propagation provide opportunities to lower SARS-CoV-2 infection rates and improve COVID-19 outcome. This includes cellular cholesterol, which is critical for viral spike proteins to capture the host machinery for SARS-CoV-2 cell entry. Once endocytosed, exit of SARS-CoV-2 from the late endosomal/lysosomal compartment occurs in a cholesterol-sensitive manner. In addition, effective release of new viral particles also requires cholesterol. Hence, cholesterol-lowering statins, proprotein convertase subtilisin/kexin type 9 antibodies, and ezetimibe have revealed potential to protect against COVID-19. In addition, pharmacological inhibition of cholesterol exiting late endosomes/lysosomes identified drug candidates, including antifungals, to block SARS-CoV-2 infection. This review describes the multiple roles of cholesterol at the cell surface and endolysosomes for SARS-CoV-2 entry and the potential of drugs targeting cholesterol homeostasis to reduce SARS-CoV-2 infectivity and COVID-19 disease severity.
... IFITMs are active against viruses that enter cells through both pH-independent and pH-dependent mechanisms, albeit to a varying degree of efficiency [3,6,13,14]. Accumulating evidence suggests that these proteins block virus entry by making the host cell membranes more rigid and imposing unfavorable membrane curvature that traps viral fusion at a hemifusion stage [15][16][17][18][19][20][21][22]. However, intriguingly, IFITMs have little or no effect on the Murine Leukemia Virus and arenaviruses such as the Lassa virus and lymphocytic choriomeningitis virus [23][24][25]. ...
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.
... For example, proteomic analysis of the S-palmitoylated IFITM3 interactome identified many membrane-associated protein interaction partners, including the p97/VCP ATPase, which contributes to IFITM3 lysosomal turnover and antiviral activity [24,34,35]. Furthermore, yeast two-hybrid screening revealed IFITM3's interaction with vesicle-membrane-protein-associated protein A (VAPA), which has been implicated in cholesterol accumulation in endolysosomal compartments and IFITM3's antiviral activity [36,37]. However, other studies indicate that high cholesterol accumulation in these compartments via U18666A pretreatment or down-regulation of Niemann-Pick C1 (NPC1) does not inhibit IAV hemagglutinin-mediated viral membrane fusion [38]. ...
... For example, proteomic analysis of the S-palmitoylated IFITM3 interactome identified many membrane-associated protein interaction partners, including the p97/VCP ATPase, which contributes to IFITM3 lysosomal turnover and antiviral activity [24,34,35]. Furthermore, yeast two-hybrid screening revealed IFITM3's interaction with vesicle-membrane-proteinassociated protein A (VAPA), which has been implicated in cholesterol accumulation in endolysosomal compartments and IFITM3's antiviral activity [36,37]. However, other studies indicate that high cholesterol accumulation in these compartments via U18666A pretreatment or down-regulation of Niemann-Pick C1 (NPC1) does not inhibit IAV hemagglutininmediated viral membrane fusion [38]. ...
... This step is followed by an extension of the fusion diaphragm, and finally the membranes separate to form a fusion pore through which the virus' genetic material escapes into the host cytosol [43]. Initially, it was suggested that IFITM3 blocks virus infection by disrupting cholesterol homeostasis in cells, leading to the accumulation of cholesterol in late endosomes and multivesicular bodies [36,37]. However, this is highly contentious, since other studies showed that cholesterol accumulation does not inhibit virus infection [38]. ...
Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important host antiviral defense factors. They are active against viruses like the influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In this review, we focus on IFITM3 S-palmitoylation, a reversible lipid modification, and describe its role in modulating IFITM3 antiviral activity. Our laboratory discovered S-palmitoylation of IFITMs using chemical proteomics and demonstrated the importance of highly conserved fatty acid-modified Cys residues in IFITM3 antiviral activity. Further studies showed that site-specific S-palmitoylation at Cys72 is important for IFITM3 trafficking to restricted viruses (IAV and EBOV) and membrane–sterol interactions. Thus, site-specific lipid modification of IFITM3 directly regulates its antiviral activity, cellular trafficking, and membrane-lipid interactions.
... Unlike influenza viruses, the entry of RSV into the cytoplasm is pH insensitive [36]; therefore, this mechanism cannot explain the observed anti-RSV activity of azelastine-HCl. The interference of CADs with the lysosomal function also leads to the accumulation of cholesterol inside the late endosomal-lysosomal cell compartments [37], which is a documented host-cell protective mechanism inhibiting influenza A virus escape [38]. ...
The Coronavirus Disease 2019 (COVID-19) pandemic and the subsequent increase in respiratory viral infections highlight the need for broad-spectrum antivirals to enable a quick and efficient reaction to current and emerging viral outbreaks. We previously demonstrated that the antihistamine azelastine hydrochloride (azelastine-HCl) exhibited in vitro antiviral activity against SARS-CoV-2. Furthermore, in a phase 2 clinical study, a commercial azelastine-containing nasal spray significantly reduced the viral load in SARS-CoV-2-infected individuals. Here, we evaluate the efficacy of azelastine-HCl against additional human coronaviruses, including the SARS-CoV-2 omicron variant and a seasonal human coronavirus, 229E, through in vitro infection assays, with azelastine showing a comparable potency against both. Furthermore, we determined that azelastine-HCl also inhibits the replication of Respiratory syncytial virus A (RSV A) in both prophylactic and therapeutic settings. In a human 3D nasal tissue model (MucilAirTM-Pool, Epithelix), azelastine-HCl protected tissue integrity and function from the effects of infection with influenza A H1N1 and resulted in a reduced viral load soon after infection. Our results suggest that azelastine-HCl has a broad antiviral effect and can be considered a safe option against the most common respiratory viruses to prevent or treat such infections locally in the form of a nasal spray that is commonly available globally.
... IFITMs are active against viruses that enter cells through both pH-independent and pH-dependent mechanisms, albeit to a varying degree of efficiency [3,6,16,17]. Accumulating evidence suggests that these proteins block virus entry by making the host cell membranes more rigid and imposing unfavorable membrane curvature that traps viral fusion at a hemifusion stage [18][19][20][21][22][23][24][25]. However, intriguingly, IFITMs have little or no effect on the Murine Leukemia Virus and arenaviruses, such as the Lassa virus and lymphocytic choriomeningitis virus [26][27][28]. ...
The 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 Env glycoproteins 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 verified by Western blotting 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 markedly reduced the Env processing and incorporation into virions relative to control and IFITM1-containing viruses. These results suggest that IFITM1 and IFITM3 restrict progeny HIV-1 infectivity via distinct mechanisms. We propose that IFITM1 interferes with viral fusion by altering the properties of the viral membrane or Env functionality by means other than disrupting Env clusters, while IFITM3 also targets Env processing and incorporation into progeny virions.
... 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]. In addition to blocking virus entry into cells, IFITMs have been shown to interfere with the spread of newly produced virus particles by incorporating into virions [54][55][56][57]. ...
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.
