Littérature scientifique sur le sujet « IFITM-2 »

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 (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3. Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.

The interferon-induced transmembrane proteins (IFITMs), widely acting against invading viruses are ubiquitously expressed on the cellular membranes, were previously known for their prominent role in tumorigenesis. Studies productively showed that the entry restriction on SARS-CoV spike glycoprotein agreeably involved the action of frontier IFITM1, 2 and 3. On the contrary, overexpression of IFITM3 has been reported in Invasive ductal breast carcinoma (IDC) tissue specimens where lentivirus-delivered shRNA resulted in targeted silencing of IFITM3 mRNA expression. Despite acting protective against virus infection, expression of IFITM favors cancer migration as seen in IDC. The existence of such a phenomenon wherein a choice is made by the selection pressure on IFITM allele frequency in human population between opposing roles of the protein, needs to be untangled.

Our first line of defense against viral pathogens is the innate immune system. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defense through the formation of the IFIT `Interactome', a multiprotein complex made up of IFIT1, IFIT2, IFIT3 and several other host factors1. Through IFIT1, this complex has the ability to distinguish self from non-self nucleic acids such as virus-derived RNA bearing 5´-triphosphate or viral mRNA lacking 2´-O methylation on the first two nucleotides1,2. We have limited information on the architecture of this complex, its role in innate immunity, and its activity downstream of RNA binding remain unclear. To better understand the mechanisms of Interactome formation, we are investigating the structure of its core, namely the IFIT1-IFIT2-IFIT3 complex. Since it is challenging to crystallize the complex as a whole, likely due to its size and heterogeneity, we are also targeting the structure of individual components and co-crystals of interacting domains. A crystal structure of human IFIT2 is available, and our lab has solved the structure of N-terminal human IFIT1 and, more recently, N-terminal IFIT3. In this study, we aim to characterize the interaction between IFIT1 and IFIT2, and between IFIT3 and IFIT2, through gel-filtration binding assays, in vitro pull-downs and deletion mutations. Preliminary results on the expression and purification of IFIT2-deletion mutants will be presented, as well as purification of IFIT subcomplexes. Understanding the molecular mechanisms behind IFIT-mediated virus elimination will help us unravel the complexities of these interactions and significantly advance our fundamental knowledge of innate immunity, paving the way for designing novel immunotherapeutics, which could potentially complement anti-cancer strategies that rely on oncolytic RNA viruses.

Primordial germ cells (PGCs) are the precursors of the gametes. In the mouse, PGCs are specified within the proximal epiblast in response to signals from the extraembryonic membranes during early gastrulation. Epiblast cells competent to form PGCs express Ifitm3. A subset of these cells then express Blimp1, a marker of PGC precursors. Once lineage-restricted, PGCs express Stella. Germ cells entering the gonads express VASA protein, which is a component of the germ plasm in animals in which germ cells are specified by the inheritance of maternal determinatives. Almost all of the research on mammalian PGC specification has used the mouse as a model and it is tacitly assumed that findings in the mouse will apply to mammals in general. We are using the tammar wallaby as a marsupial model for PGC specification. Eutherians and marsupials diverged 125–148 million years ago, so comparisons between the two will provide insights into the evolution of the control of mammalian PGC specification. There are IFITM clusters in both the human (chromosome 11) and mouse (chromosome 7). In the mouse, IFITM1, 2 and 3 are expressed in PGCs, whereas IFITM4 and 5 are not (1). Only one IFITM member, IFITM5, is annotated in the opossum Ensemble database. We have cloned one tammar IFITM member and identified at least one other putative member in the tammar trace archive database. We have also cloned tammar BLIMP1 and VASA, both of which show high sequence conservation with other mammals. RT–PCR profiles for both genes during tammar gastrulation are similar to those for the mouse. In contrast, no marsupial STELLA orthologueue has been identified in either the opossum or tammar genomes. These findings suggest that some but not all of the signals and mechanisms involved in eutherian PGC specification are also applicable to marsupials. (1) Lange UC, Saitou M, Western P, Barton SC and Surani MA (2003) BMC Dev. Biol. Epub 2003 Mar 19

Rift Valley Fever virus (RVFV) and Toscana virus (TOSV) are two pathogenic arthropod-borne viruses responsible for zoonotic infections in both humans and animals; as such, they represent a growing threat to public and veterinary health. Interferon-induced transmembrane (IFITM) proteins are broad inhibitors of a large panel of viruses belonging to various families and genera. However, little is known on the interplay between RVFV, TOSV, and the IFITM proteins derived from their naturally infected host species. In this study, we investigated the ability of human, bovine, and camel IFITMs to restrict RVFV and TOSV infection. Our results indicated that TOSV was extremely sensitive to inhibition by all the animal IFITMs tested, while RVFV was inhibited by human IFITM-2 and IFITM-3, but not IFITM-1,and exhibited a more heterogeneous resistance phenotype towards the individual bovine and camel IFITMs tested. Overall, our findings shed some light on the complex and differential interplay between two zoonotic viruses and IFITMs from their naturally infected animal species.

Abstract Background IFITM proteins (IFITM-1, -2, and -3) mediate cellular resistance to influenza, dengue, and other viruses. IFITM expression on human platelets has not been previously recognized. Our laboratory recently demonstrated that IFITMs are robustly expressed by human platelets and megakaryocytes after stimulation by pathogens and inflammatory mediators and restrict viral infection. IFITMs, which are interferon inducible, also mediate clathrin localization and associated protein endocytosis. Nevertheless, whether IFITMs regulate protein endocytosis by platelets and megakaryocytes remains unknown. Aims We investigated IFITM expression on murine megakaryocytes and platelets and determined whether IFITMs regulate fibrinogen endocytosis under basal and inflammatory conditions. Methods We examined the expression of IFITMs and clathrin in bone-marrow derived murine megakaryocytes and platelets under basal conditions and following interferon-beta (IFN-β) stimulation. To determine whether upregulation of IFITM causes increased fibrinogen endocytosis, megakaryocytes were stimulated ex vivo with IFN-β and treated with labeled fibrinogen. Endocytosis of labeled fibrinogen was then measured by immunocytochemistry and flow cytometry. To determine whether this response also occurred in vivo, C57Bl/6 mice were injected intraperitoneally (IP) with 50,000 units of IFN-β over four days. On the fourth day, 100 μg of labeled fibrinogen was injected into the tail vein and the amount of endocytosed, labeled fibrinogen in platelets was determined the next day via flow cytometry. Parallel experiments were performed in age and gender matched IFITM-/- mice. Results Bone-marrow derived murine megakaryocytes and platelets basally express IFITMs. Upon IFN-β stimulation, IFITM and clathrin expression significantly increased (p<0.05). Fibrinogen endocytosis by murine megakaryocytes occurred under resting conditions and appeared to be punctate and granular in nature. Upon IFN-b stimulation, fibrinogen endocytosis in megakaryocytes significantly increased compared to unstimulated conditions (p<0.004). The increase in endocytosis appeared independent of changes in αIIbβ3 expression as IFN-β stimulation did not change αIIbβ3 surface protein. Fibrinogen endocytosis after IFN-β stimulation did not increase in megakaryocytes from IFITM-/- mice, suggesting that IFITMs regulate fibrinogen uptake under these conditions. We next determined if fibrinogen endocytosis occurred in platelets isolated from IFITM-/- mice. Platelet counts and activation indices (assessed by JonA staining) were similar in C57Bl/6 mice (WT) and IFITM-/- mice. Nevertheless, the injection of IFN-β IP results in significant increases in fibrinogen endocytosis by platelets in vivo in WT but not IFITM-/- mice (p<0.02). Summary/Conclusions These findings suggest IFITMs, in addition to their anti-viral roles, mediate fibrinogen endocytosis. Further, in settings where inflammatory stimuli such as interferons are increased, enhanced IFITM expression may promote upregulation of fibrinogen endocytosis by platelets and megakaryocytes. Disclosures No relevant conflicts of interest to declare.

Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses bothin vitroandin vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includesIFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. ChickenIFITM2and -3are constitutively expressed in all tissues examined, whereasIFITM1is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

AbstractRibosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.

2014 - 2015<br>Bcl-2-associated athanogene 3 (BAG3) belongs to the family of co-chaperone proteins that interact with the heat shock protein 70 (Hsp70) and is involved in a number of cellular processes including proliferation and apoptosis. BAG3 contains the BAG domain which interacts with the ATPase domain of Hsp70. BAG3 is also characterized by the presence of a WW domain, two conserved Ile-Pro-Val (IPV) motifs and a proline-rich (PXXP) repeat that mediate the binding to partners different from Hsp70. These diverse and multiple interactions underlie the ability of BAG3 to modulate major biological processes such as development, cytoskeleton organization and autophagy. In our laboratory, BAG3 has been recently found in a soluble or membrane-associated form and it has been detected in the serum obtained from patients with pancreatic cancer or heart failure. Moreover, we found that BAG3 is able to bind the cell surface of macrophages and activate the production of inflammatory associated components, such as Nitric Oxide (NO) and Interleukin (IL) -6. To identify novel interacting partners of BAG3 an affinity chromatography on nickel-charged resin was performed, in J774A.1 cells, using recombinant BAG3 (rBAG3) followed by mass spectrometry analysis of the rBAG3-containing complexes. Among these, Interferon- Inducible TransMembrane (IFITM) -2 and Neuropilin (NRP) -1 were the only transmembrane proteins and therefore represented good candidates as receptors for BAG3. Our results show that NRP-1 and IFITM-2 are both essential for the binding of rBAG3 to the cell surface of macrophages and its activation for IL-6 release. We then investigated if BAG3 binding activates some of the signaling pathways known to be involved in macrophage activation. In particular we focused on the phosphatidylinositol 3-kinase (PI3K) and on the p38 pathway that are both involved in Cox-2, iNOS and IL-6 induction in macrophages. We demonstrated that BAG3 signaling is mediated by the receptor complex we identified, since IFITM-2 and/or NRP-1 silencing abrogates BAG3- induced phosphorylation of AKT and p38. We than focus our study on human monocytes, rBAG3 binds the cell surface and induces the release of many pro-inflammatory cytokines and chemokines. Furthermore, we have shown that rBAG3 can bind T lymphocytes cells surface after lipopolysaccharide (LPS) stimulus. All together these findings suggest a role for extracellular BAG3 in immune response. [edited by Author]<br>XIV n.s.