FIG 5
Restoration of late endosomal cholesterol export restores IAV titers. (A and B) A431-A6 cells transiently expressing NPC1-YFP (A) or NPC1 P692S-YFP (B) were infected with FPV (MOI of 0.1 for 24 h). Progeny virus titers were determined by standard plaque assay. (C) A549 cells transiently overexpressing GFP or AnxA6-GFP were infected with FPV (MOI of 0.1 for 24 h). Exogenous cholesterol was added to the medium at 2 h p.i. Progeny virus titers were determined by standard plaque assay and expressed as percentages of virus titers released from GFP-expressing control cells. Statistically significant differences of the mean values Ϯ SEM calculated from at least three independent experiments were assessed by two-tailed t test (A, B) or by one-way ANOVA followed by Tukey’s multiple comparison test (C). *, P Յ 0.05; **, P Յ 0.01; ***, P Յ 0.001; n.s., not significant.
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Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca(2+)-regulated membrane-bi...
Contexts in source publication
Context 1
... microscopy (data not shown). At 24 h after transfec- tion, cells were infected with FPV at an MOI of 0.1, and the infec- tious titers were measured with a standard plaque assay technique 24 h postinfection (p.i.). In support of our hypothesis, the over- expression of wild-type NPC1 partially restored progeny virus ti- ters in A431-A6 cells (Fig. 5A). To further underscore this finding, we analyzed viral replication upon overexpression of the loss-of- function NPC1 P692S mutant (having a change of proline to ser- ine at position 692), which cannot bind cholesterol and inhibits cholesterol export from late endosomes (39-41). Indeed, while IAV replication can be rescued by ...
Context 2
... of the loss-of- function NPC1 P692S mutant (having a change of proline to ser- ine at position 692), which cannot bind cholesterol and inhibits cholesterol export from late endosomes (39-41). Indeed, while IAV replication can be rescued by overexpression of wild-type NPC1, the P692S mutant was not able to reestablish viral titers in A431-A6 cells (Fig. 5B). P962S also significantly impaired IAV replication in A431wt cells (not shown), further suggesting that cholesterol pools from late endosomes are required for efficient virus replication and ...
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... cholesterol to replenish plasma membrane cholesterol. Next, cholesterol-treated and nontreated A549 cells were infected with FPV at an MOI of 0.1, and the infectious titers were measured with the standard plaque assay technique. Indeed, cholesterol replen- ishment completely restored the progeny virus titers in A6-GFP- overexpressing A549 cells (Fig. 5C), further supporting a role for AnxA6 to modulate cholesterol-dependent steps during viral replication. In conclusion, restoration of the cellular cholesterol balance via cholesterol replenishment using exogenous choles- terol or the ectopic expression of wild-type NPC1 in AnxA6- overexpressing cells improves the ability of IAV to ...
Citations
... One possibility would be that the exposure of VPs to cyclodextrins could sequester cholesterol from the viral particles, which may, in turn, disrupt lipid rafts and alter the fusogenic properties of the viral envelope. This most probably disorganizes the integrity of HA-VPs, as previously described [28,29]. At the same time, the same compounds only slightly affect the infectivity of the spike-VPs, suggesting that the SARS-CoV-2 envelope is resistant to these compounds. ...
Objective: Many severe acute respiratory infections are caused by viral pathogens, and viruses are responsible for a large number of deaths worldwide. Among the most common respiratory viruses are the influenza A virus (IAV) and, more recently, the SARS-CoV-2 that emerged in 2019 and caused the most significant human pandemic of the beginning of the 21st century. Both IAV and SARS-CoV-2 share clinical features and a common transmission route through the emission of viral particles via aerosols and droplets. These penetrate the host after entry from the nose and mouth or an indirect mode of transmission via contact contamination of different media. These facts prompted us to investigate the possibility of designing a soft cream with a virucidal activity targeted against IAV and SARS-CoV-2. Methods: We first investigated the action of chemical compounds known to have antiviral properties such as cyclodextrin, or algae extracts containing sulfated polysaccharides, on cultured cells infected with lentiviral viral particles pseudotyped (VP) with either proteins HA (hemagglutinin) and NA (neuraminidase) from IAV or the G protein from the vesicular stomatitis virus or spike-bearing particles in order to select molecules with antiviral activities in human embryonic kidney (HEK293T) cells. Results: Our results show that some cyclodextrin-containing creams can significantly reduce the stability of HANA- and spike-bearing particles when they are applied prior to challenge with a viral inoculum on skin. Conclusions: We observed some specificities of these creams towards either IAV or SARS-CoV-2, indicating that the neutralization of viral activity is correlated with the mechanism of receptor interaction and entry of these two pathogens.
... 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). ...
... 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). ...
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.
... Annexin involvement in microbe-host interactions also includes an Anx-mediated reshaping of the organelle or cellular membrane the pathogen relies upon for infection. This is exemplified in the AnxA1governed regulation of endocytic transport (see above) that is favorable for endosomal trafficking of influenza A virus 122 and the AnxA6controlled endolysosomal cholesterol content (see above), which impairs the fusion of enveloped viruses at this entry site 123 . Exploring the role of annexins at such levels of increasing complexity might also uncover new targets for the development of entirely new types of drugs and treatments. ...
Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca ²⁺ levels. Through this they act as Ca ²⁺ -regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca ²⁺ -regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.
... Anxa6 is a ubiquitously expressed annexin present in the plasma membrane, mitochondria, endocytic and exocytic vesicles, as well as lipid droplets. This protein interacts with both proteins and lipids in the regulation of cholesterol transport, signal complex disassembly, cytoskeleton rearrangements, the stress response, cell growth and motility, differentiation, lipid and glucose homeostasis, endocytosis, exocytosis and viral infections [69][70][71][72]. Interestingly, Anxa6 is upregulated during B and T cell differentiation, along with IL-2 [73]. ...
A generally accepted hypothesis for the initial activation of an immune or autoimmune response argues that alarmins are released from injured, dying and/or activated immune cells, and these products complex with receptors that activate signal transduction pathways and recruit immune cells to the site of injury where the recruited cells are stimulated to initiate immune and/or cellular repair responses. While there are multiple diverse families of alarmins such as interleukins (IL), heat-shock proteins (HSP), Toll-like receptors (TLR), plus individual molecular entities such as Galectin-3, Calreticulin, Thymosin, alpha-Defensin-1, RAGE, and Interferon-1, one phylogenetically conserved family are the Annexin proteins known to promote an extensive range of biomolecular and cellular products that can directly and indirectly regulate inflammation and immune activities. For the present report, we examined the temporal expression profiles of the 12 mammalian annexin genes (Anxa1-11 and Anxa13), applying our temporal genome-wide transcriptome analyses of ex vivo salivary and lacrimal glands from our C57BL/6.NOD-Aec1Aec2 mouse model of Sjögren’s Syndrome (SS), a human autoimmune disease characterized primarily by severe dry mouth and dry eye symptoms. Results indicate that annexin genes Anax1-7 and -11 exhibited upregulated expressions and the initial timing for these upregulations occurred as early as 8 weeks of age and prior to any covert signs of a SS-like disease. While the profiles of the two glands were similar, they were not identical, suggesting the possibility that the SS-like disease may not be uniform in the two glands. Nevertheless, this early pre-clinical and concomitant upregulated expression of this specific set of alarmins within the immune-targeted organs represents a potential target for identifying the pre-clinical stage in human SS as well, a fact that would clearly impact future interventions and therapeutic strategies.
... Therefore, this assay can uncover the regulation of MCS proteins during a given biological process, helping to formulate functional hypotheses of multi-faceted organelle regulation. Additional validation using microscopy or proximity ligation assays, as shown in this study, may be 24,48,72,96, 120 hpi, respectively, corresponding to 3 independent experiments; ***p ≤ 0.0001 by one-way ANOVA to Mock). B Images of fibroblasts expressing mito-RFP-ER (Tether, yellow) and labeled for ER (cyan), mitochondria (red), and IE1 (magenta). ...
Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by the ancient beta-herpesvirus human cytomegalovirus (HCMV). Integration with super-resolution microscopy and comparisons to herpes simplex virus (HSV-1), Influenza A, and beta-coronavirus HCoV-OC43 infections reveals time-sensitive contact regulation that allows switching anti- to pro-viral organelle functions. We uncover a stabilized mitochondria-ER encapsulation structure (MENC). As HCMV infection progresses, MENCs become the predominant mitochondria-ER contact phenotype and sequentially recruit the tethering partners VAP-B and PTPIP51, supporting virus production. However, premature ER-mitochondria tethering activates STING and interferon response, priming cells against infection. At peroxisomes, ACBD5-mediated ER contacts balance peroxisome proliferation versus membrane expansion, with ACBD5 impacting the titers of each virus tested.
... For example, IAV-induced autophagy is dependent on M2 ion channel activity, and SERCA is proportional to autophagic flux, therefore, IAV M2 induces autophagic flux arrest by modulating host factor SECRA activity (Peng et al., 2021). The host factor AnX6 disrupts virus release by interacting with the M2 CT region (Musiol et al., 2013). The host factor YWHAG does not affect the binding, entry, replication, and transcription of IAV in the early stages of infection. ...
Influenza A virus (IAV) poses a serious threat to human life and property. The IAV matrix protein 2 (M2) is significant in viral budding. Increasing studies have proven the important roles of host factors in IAV replication. In this study, immunoprecipitation combined with mass spectrometry revealed that the host protein tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG), which belongs to the 14-3-3 protein scaffold family, interacts with M2. Their interactions were further confirmed by co-immunoprecipitation (Co-IP), immunofluorescence, and confocal microscopy of virus-infected HeLa cells. Moreover, we constructed YWHAG-KO and YWHAG-overexpressing cells and found that YWHAG knockout significantly increased viral production, whereas its overexpression reduced the titer of virus progeny. Therefore, YWHAG is a negative regulatory factor during IAV infection. Further, YWHAG knockout or overexpression had no effect on the binding, entry, or viral RNA replication in the early stages of the virus life cycle. On the contrary, it impaired the release of virions at the plasma membrane as determined using transmission electron microscopy and suppressed the M2-mediated budding of the influenza virus. Importantly, the H158F mutation of YWHAG was found to affect interaction with M2 and its budding. Collectively, our work demonstrates that YWHAG is a novel cellular regulator that targets and mediates the interaction and release of M2.
... Given that cholesterol is involved in viral membrane fusion, viral genome release, and viral budding, this may explain the efficacy of cholesterol-lowering drugs, gemfibrozil, and lovastatin in reducing the stability and infectivity of progeny virus (Bajimaya et al., 2017). It was also demonstrated that overexpression of Annexin A6 as well as the addition of U18666A, a hydrophobic polyamine, was able to reduce cholesterol levels in the plasma membrane and inhibit viral replication (Musiol et al., 2013). ...
Annually, the influenza virus causes 500,000 deaths worldwide. Influenza-associated mortality and morbidity is especially high among the elderly, children, and patients with chronic diseases. While there are antivirals available against influenza, such as neuraminidase inhibitors and adamantanes, there is growing resistance against these drugs. Thus, there is a need for novel antivirals for resistant influenza strains. Host-directed therapies are a potential strategy for influenza as host processes are conserved and are less prone mutations as compared to virus-directed therapies. A literature search was performed for papers that performed viral–host interaction screens and the Reactome pathway database was used for the bioinformatics analysis. A total of 15 studies were curated and 1717 common interactors were uncovered among all these studies. KEGG analysis, Enrichr analysis, STRING interaction analysis was performed on these interactors. Therefore, we have identified novel host pathways that can be targeted for host-directed therapy against influenza in our review.
... The requirement of cholesterol for IAV internalization is not well understood, probably due to the confusion caused by three difficulties. First, quantification of internalized IAV was quite challenging (15): most methods measured the extent of IAV cell entry at much later stages and thus failed to accurately estimate the amounts of internalized IAV (16)(17)(18)(19). Second, experimental results were often obtained without rigorously differentiating CME and CIE. ...
Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that the sphingomyelin (SM)-sequestered cholesterol, but not the accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol-independent. Whereas, the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein which activates actin nucleation, is recruited to IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry.
Importance:
IAV infects the cells by harnessing cellular endocytic machineries. Better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies, and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol-independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results would provide new insights into IAV infection and pathway/cargo-specific involvement of cholesterol pool(s).
... Annexin A6 (AnxA6), can interact with the cytoplasmic tail of M2 at the plasma membrane and inhibit virus budding [58]. AnxA6 can also cause sequestration of cholesterol in late endosomes, impairing IAV endosomal fusion and release of vRNP [59], and reduces the cholesterol availability for viral envelopes, thus impairing viral progeny [60]. M2 can also bind to Na + /K + -ATPase β1 subunit (ATP1B1), an interaction which is essential for viral replication [61]. ...
The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed “Viroporins”. They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.
... Lipid rafts play a significant role in the influenza virus life cycleserving as a site for the gathering of viral proteins. Lack of interaction with raft microdomains impairs influenza virus infectivity (Zhang et al., 2000) and budding (Musiol et al., 2013). It was shown that an AnxA6 overexpression leads to reduced reproduction of influenza A virus in cell culture accompanied by a significant decrease in cholesterol levels both in the cell plasma membrane and the membrane of progeny virions (Ma et al., 2012). ...
Using cell cultures of human origin for the propagation of influenza virus is an attractive way to preserve its glycosylation profile and antigenic properties, which is essential in influenza surveillance and vaccine production. However, only few cell lines are highly permissive to influenza virus, and none of them are of human origin. The barrier might be associated with host restriction factors inhibiting influenza growth, such as AnxA6 protein counteracting the process of influenza virion packaging. In the presented work we explore the CRISPR-Cas9 mediated knockout of ANXA6 gene as a way to overcome the host restriction barrier and increase the susceptibility of human cell line to influenza infection. By CRISPR-Cas9 genome editing we modified HEK293FT cells and obtained several clones defective in the ANXA6 gene. The replication of the influenza A virus in original HEK293FT cells and the HEK293FT-ANXA6-/- mutant cells was compared in growth curve experiments. By combination of methods including TCID assay and flow cytometry we showed that accumulation of influenza A virus in the mutant HEK293FT-ANXA6-/- cells significantly exceeded the virus titer in the original HEK293FT cells.
