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Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane-Associated Protein IFITM3 and Modified Variants.

ABSTRACT: Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.

SUBMITTER: Harmand TJ

PROVIDER: S-EPMC5658968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane-Associated Protein IFITM3 and Modified Variants.

Project description:Interferon-induced transmembrane protein?3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5?kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15?mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.

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Antiviral Protection by IFITM3 In Vivo.

Project description:Purpose of Review:Interferon-induced transmembrane protein 3 (IFITM3) is a cellular restriction factor that blocks fusion between virus and host membranes. Here, we provide an introduction to IFITM3 and the biochemical regulation underlying its antiviral activity. Further, we analyze and summarize the published literature examining phenotypes of IFITM3 knockout mice upon infections with viral pathogens and discuss the controversial association between single nucleotide polymorphisms (SNPs) in the human IFITM3 gene and severe virus infections. Recent Findings:Recent publications show that IFITM3 knockout mice experience more severe pathologies than wild-type mice in diverse virus infections, including infections with influenza A virus, West Nile virus, Chikungunya virus, Venezuelan equine encephalitis virus, respiratory syncytial virus, and cytomegalovirus. Likewise, numerous studies of humans of Chinese ancestry have associated the IFITM3 SNP rs12252-C with severe influenza virus infections, though examinations of other populations, such as Europeans, in which this SNP is rare, have largely failed to identify an association with severe infections. A second SNP, rs34481144-A, found in the human IFITM3 promoter has also recently been reported to be a risk allele for severe influenza virus infections. Summary:There is significant evidence for a protective role of IFITM3 against virus infections in both mice and humans, though additional work is required to identify the range of pathogens restricted by IFITM3 and the mechanisms by which human SNPs affect IFITM3 levels or functionality.

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Total Chemical Synthesis of Modified Histones.

Project description:In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.

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Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity

Project description:The interferon-induced transmembrane proteins (IFITMs) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

| S-SCDT-10_15252-EMBJ_2022112234 | biostudies-other

Hydrophobic variants of ganglio-tripod amphiphiles for membrane protein manipulation.

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| S-EPMC3855929 | biostudies-literature

IFITM3 requires an amphipathic helix for antiviral activity.

Project description:Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion.

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Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media.

Project description:The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C2mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several "gold-standard" ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0-7.5, resulting in ligation yields of up to 80-95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current "gold-standard" NCL methods.

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The N-terminal region of IFITM3 modulates its antiviral activity by regulating IFITM3 cellular localization.

Project description:Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses.

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Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

Project description:The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

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