ABSTRACT
A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.
Subject(s)
Antigens, CD/genetics , Host-Pathogen Interactions/genetics , Interferon Regulatory Factors/genetics , Interferon Type I/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Animals , Antigens, CD/chemistry , Antigens, CD/immunology , Binding Sites , Cell Line, Tumor , Chlorocebus aethiops , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/virology , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Gene Expression Regulation , Golgi Apparatus/genetics , Golgi Apparatus/immunology , Golgi Apparatus/virology , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Interferon Regulatory Factors/classification , Interferon Regulatory Factors/immunology , Interferon Type I/immunology , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/immunology , Signal Transduction , Vero Cells , Viral Proteins/chemistry , Viral Proteins/immunology , Virus Internalization , Virus Release/genetics , Virus Release/immunology , Virus Replication/genetics , Virus Replication/immunologyABSTRACT
As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.
Subject(s)
HIV Infections , HIV-1 , Bayes Theorem , HIV Infections/metabolism , HIV-1/genetics , Humans , Proteome/metabolism , ProteomicsABSTRACT
SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.
Subject(s)
Amiloride/pharmacology , COVID-19 Drug Treatment , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effectsABSTRACT
The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , HIV Infections/virology , Human Immunodeficiency Virus Proteins/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Proteome/metabolism , Sorting Nexins/metabolism , Vesicular Transport Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , HIV Infections/genetics , HIV Infections/metabolism , HIV-1/physiology , HeLa Cells , Human Immunodeficiency Virus Proteins/genetics , Humans , Microscopy, Electron , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Transport , Protein Tyrosine Phosphatases, Non-Receptor/genetics , Proteome/analysis , Sorting Nexins/chemistry , Sorting Nexins/genetics , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics , Viroporin Proteins/geneticsABSTRACT
Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.
Subject(s)
Bacteriophage M13/genetics , Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Neoplasms/therapy , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Bacteriophage M13/immunology , Capsid Proteins/genetics , Capsid Proteins/immunology , Cell Line, Tumor , Dependovirus/immunology , Endosomes/immunology , Endosomes/virology , Genetic Vectors/administration & dosage , Genetic Vectors/immunology , Humans , Lysosomes/immunology , Lysosomes/virology , Mice , Neoplasms/genetics , Oligopeptides/genetics , Oligopeptides/immunology , Proof of Concept Study , Rats , Transduction, Genetic/methods , Virus Internalization , Xenograft Model Antitumor AssaysABSTRACT
The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses, and its activity is counteracted by the glycosylated Gag (glycoGag) protein of murine leukemia virus (MLV), the S2 protein of equine infectious anemia virus (EIAV), and the Nef protein of human immunodeficiency virus type 1 (HIV-1). Determining the regions within SERINC5 that provide restrictive activity or Nef sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and which is reminiscent of an acidic-cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef, while it has no effect on the intrinsic activity of the protein as an inhibitor of infectivity. These effects correlated with enhanced removal of the ΔEDTEE mutant relative to that of wild-type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis indicated that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.IMPORTANCE Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically, HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen "arms race" is a compensatory change in the host antiviral protein as it evolves to escape the effects of viral antagonists. This is often reflected in a genetic signature, positive selection, which is conspicuously missing in SERINC5 Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.
Subject(s)
Membrane Proteins/chemistry , nef Gene Products, Human Immunodeficiency Virus/metabolism , Alleles , Amino Acid Motifs , Cytoplasm/metabolism , Gene Deletion , Glycosylation , HEK293 Cells , HIV-1 , HeLa Cells , Humans , Infectious Anemia Virus, Equine/metabolism , Jurkat Cells , Moloney murine leukemia virus/metabolism , Mutation , Protein Domains , nef Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
HIV-1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune-evasion, triggering ubiquitin-mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP-1 and AP-2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α-chain. CD8-VpuCD was rapidly endocytosed in a clathrin- and AP-2-dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the ß-TrCP binding site and a leucine-based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV-dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP-2 and showed that this is enhanced by serine-phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (µ) subunits of AP-2 and AP-1; this interaction was dependent on serine-phosphorylation of Vpu and on basic residues in the µ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Endocytosis , Human Immunodeficiency Virus Proteins/metabolism , Phosphoserine/metabolism , Viral Regulatory and Accessory Proteins/metabolism , HeLa Cells , Humans , PhosphorylationABSTRACT
Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate many cell processes by injecting effector proteins from the bacteria into the host cell via a Type III secretion system. In this paper we report that the effector protein EspG disrupts recycling endosome function. In particular, we found that following transferrin binding and endocytosis EspG reduces recycling of the transferrin receptor (TfR), the prototypical recycling protein, from an intracellular location to the cell surface, resulting in an accumulation of TfR within the cell. The surface levels of three receptors [TfR, epidermal growth factor receptor (EGFR) and ß1 integrin] were tested and found to be reduced dependent on EspG translocation. Furthermore, disruption of recycling endosome function and the reduced surface presentation of receptors was dependent on the previously reported RabGAP activity and ARF binding ability of EspG. This paper therefore supports the previous hypothesis that EspG acts as an enzyme scaffold perturbing cell signalling events, in this case altering recycling endosome function and cell surface receptor levels during infection.
Subject(s)
Endosomes/drug effects , Endosomes/metabolism , Enterohemorrhagic Escherichia coli/physiology , Escherichia coli Proteins/metabolism , Host-Pathogen Interactions , Membrane Proteins/metabolism , Receptors, Transferrin/metabolism , ADP-Ribosylation Factor 1/metabolism , ErbB Receptors/metabolism , GTPase-Activating Proteins/metabolism , HeLa Cells , Humans , Integrin beta1/metabolism , Microtubule-Associated Proteins/metabolism , Protein BindingABSTRACT
Adeno-associated virus/phage (AAVP) is a gene delivery vector constructed as a hybrid between adeno-associated virus and filamentous phage. Tumor targeting following systemic administration has previously been demonstrated in several in vivo cancer models, with tumor specificity achieved through display of an α(v) integrin-targeting ligand on the capsid. However, high titers of AAVP are required for transduction of large numbers of mammalian cells. This study is the first to investigate the mechanisms involved in entry and intracellular trafficking of AAVP. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, we have demonstrated that targeted AAVP endocytosis is both dynamin and clathrin-dependent. Following entry, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway. Finally, we have demonstrated that disruption of this pathway leads to improved transgene expression by AAVP, thus demonstrating that escape from the late endosomes/lysosomes is a critical step for improving gene delivery by AAVP. These findings have important implications for the rational design of improved AAVP and RGD-targeted vectors.
Subject(s)
Clathrin/metabolism , Dependovirus/physiology , Dynamins/metabolism , Endocytosis , Endosomes/metabolism , Lysosomes/metabolism , Virus Internalization , Biological Transport, Active , Endosomes/virology , HEK293 Cells , HeLa Cells , Humans , Lysosomes/virology , TransfectionABSTRACT
Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.
Subject(s)
Bacterial Proteins/metabolism , Flavoproteins/metabolism , Legionella pneumophila/pathogenicity , Moths/microbiology , Animals , Bacterial Secretion Systems , Female , Flagellin/metabolism , Hemocytes/metabolism , Hemocytes/microbiology , Larva/microbiology , Legionella pneumophila/metabolism , Legionellosis/microbiology , Lung/microbiology , Lung/pathology , Male , Mice , Mice, Inbred A , Mice, Inbred C57BL , Models, Animal , Protein Transport , Virulence Factors/metabolismABSTRACT
The host protein SERINC5 inhibits the infectivity of HIV-1 virions in an Env-dependent manner and is counteracted by Nef. The conformation of the Env trimer reportedly correlates with sensitivity to SERINC5. Here, we tested the hypothesis that the "open" conformation of the Env trimer revealed by sensitivity to the V3-loop specific antibody 447-52D directly correlates with sensitivity to SERINC5. Of five Envs tested, SF162 was the most sensitive to neutralization by 447-52D, but it was not the most sensitive to SERINC5; instead the Env of LAI was substantially more sensitive to SERINC5 than all the other Envs. Mutational opening of the trimer by substitution of two tyrosines that mediate interaction between the V2 and V3 loops sensitized the Envs of JRFL and LAI to 447-52D as previously reported, but only BaL was sensitized to SERINC5. These data suggest that trimer "openness" is not sufficient for sensitivity to SERINC5.
Subject(s)
Antibodies, Monoclonal/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , Membrane Proteins/immunology , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/immunology , Antibodies, Neutralizing/immunology , HIV Infections/genetics , HIV Infections/virology , HIV-1/chemistry , HIV-1/genetics , HIV-1/physiology , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , env Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.
ABSTRACT
The plasma membrane is a site of conflict between host defenses and many viruses. One aspect of this conflict is the host's attempt to eliminate infected cells using innate and adaptive cell-mediated immune mechanisms that recognize features of the plasma membrane characteristic of viral infection. Another is the expression of plasma membrane-associated proteins, so-called restriction factors, which inhibit enveloped virions directly. HIV-1 encodes two countermeasures to these host defenses: The membrane-associated accessory proteins Vpu and Nef. In addition to inhibiting cell-mediated immune-surveillance, Vpu and Nef counteract membrane-associated restriction factors. These include BST-2, which traps newly formed virions at the plasma membrane unless counteracted by Vpu, and SERINC5, which decreases the infectivity of virions unless counteracted by Nef. Here we review key features of these two antiviral proteins, and we review Vpu and Nef, which deplete them from the plasma membrane by co-opting specific cellular proteins and pathways of membrane trafficking and protein-degradation. We also discuss other plasma membrane proteins modulated by HIV-1, particularly CD4, which, if not opposed in infected cells by Vpu and Nef, inhibits viral infectivity and increases the sensitivity of the viral envelope glycoprotein to host immunity.
Subject(s)
Antigens, CD/metabolism , HIV Infections/immunology , HIV-1/pathogenicity , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Animals , GPI-Linked Proteins/metabolism , HIV Infections/virology , HIV-1/metabolism , Host-Pathogen Interactions , HumansABSTRACT
Two flavonol tetraglycosides comprising a trisaccharide at C-3 and a monosaccharide at C-7 were isolated from the leaves of Styphnolobium japonicum (L.) Schott and characterised as the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranosides of quercetin and kaempferol. The 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside of kaempferol, the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranosides of kaempferol and quercetin and the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside of kaempferol were also obtained from this species for the first time. Some or all of these flavonol tetra- and triglycosides were detected in 17 of 18 specimens of S. japonicum examined from living and herbarium material, although the most abundant flavonoid in the leaves was generally quercetin 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside (rutin). The triglycosides, but not the tetraglycosides, were detected in herbarium specimens of Styphnolobium burseroides M. Sousa, Rudd & Medrano and Styphnolobium monteviridis M. Sousa & Rudd, but specimens of Styphnolobium affine (Torrey & A. Gray) Walp. contained a different profile of flavonol glycosides. The flavonol tetra- and triglycosides of S. japonicum were also present in leaves of Cladrastis kentukea (Dum. Cours.) Rudd, a representative of a genus placed close to Styphnolobium in current molecular phylogenies. An additional constituent obtained from leaves of Styphnolobium japonicum was identified as the maltol derivative, 3-hydroxy-2-methyl-4H-pyran-4-one 3-O-(4'-O-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-beta-glucopyranoside.
Subject(s)
Fabaceae/chemistry , Flavonols/chemistry , Glycosides/chemistry , Kaempferols/chemistry , Quercetin/analogs & derivatives , Quercetin/chemistry , Chemical Fractionation , Flavonols/isolation & purification , Glycosides/isolation & purification , Kaempferols/isolation & purification , Plant Extracts/chemistry , Plant Leaves/chemistryABSTRACT
Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.
Subject(s)
Alkynes/analysis , Chromatography, High Pressure Liquid/methods , Fatty Alcohols/analysis , Horse Diseases/diagnosis , Oenanthe/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Alkynes/chemistry , Alkynes/poisoning , Animals , Enediynes , Fatty Alcohols/chemistry , Fatty Alcohols/poisoning , Hazardous Substances/analysis , Hazardous Substances/poisoning , Horse Diseases/chemically induced , Horses , Magnetic Resonance Spectroscopy , Methanol/chemistry , Molecular Structure , Plant Roots/chemistry , Reproducibility of Results , Sensitivity and Specificity , StereoisomerismABSTRACT
UNLABELLED: Legionella pneumophila, the causative agent of Legionnaires' disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to the Legionella-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing the Escherichia coli biotin ligase BirA and infected the cells with L. pneumophila expressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni(2+) nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity. IMPORTANCE: The respiratory pathogen Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate more than 300 effector proteins into host cells. The function of most effectors in infection remains unknown. One of the bottlenecks for their characterization is the identification of target proteins. Frequently used in vitro approaches are not applicable to all effectors and suffer from high rates of false positives or missed interactions, as they are not performed in the context of an infection. Here, we determine key functional domains of the effector PieE and describe a new method to identify host cell targets under physiological infection conditions. Our approach, which is applicable to other pathogens, uncovered the interaction of PieE with several proteins involved in membrane trafficking, in particular Rab GTPases, revealing new details of the Legionella infection strategy and demonstrating the potential of this method to greatly advance our understanding of the molecular basis of infection.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chromatography, Affinity/methods , Legionella pneumophila/metabolism , Membrane Proteins/metabolism , Vacuoles/microbiology , rab GTP-Binding Proteins/metabolism , Animals , Bacterial Proteins/genetics , Cell Line, Tumor , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , HeLa Cells , Histidine , Host-Pathogen Interactions , Humans , Intracellular Membranes/metabolism , Legionella pneumophila/genetics , Mass Spectrometry , Membrane Proteins/chemistry , Membrane Proteins/genetics , Microscopy, Electron, Transmission , Models, Molecular , Oligopeptides , Protein Transport , Vacuoles/metabolismABSTRACT
Bacteriophage (phage), which are viruses that infect bacteria only, have shown promise as vehicles for targeted cancer gene therapy, albeit with poor efficiency. Recently, we generated an improved version of phage vectors by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) efficiently delivered systemically administered therapeutic genes to various tumor targets by displaying an integrin tumor-targeting ligand on the phage capsid. However, inherent limitations in bacteriophage mean that these AAVP vectors still need to be improved. One of the limitations of AAVP in mammalian cells may be its susceptibility to proteasomal degradation. The proteasome is upregulated in cancer and it is known that it constitutes a barrier to gene delivery by certain eukaryotic viruses. We report here that inhibition of proteasome improved targeted reporter gene delivery by AAVP in cancer cells in vitro and in tumors in vivo after intravenous vector administration to tumor-bearing mice. We also show enhanced targeted tumor cell killing by AAVP upon proteasome inhibition. The AAVP particles persisted significantly in cancer cells in vitro and in tumors in vivo after systemic administration, and accumulated polyubiquitinated coat proteins. Our results suggest that the proteasome is indeed a barrier to tumor targeting by AAVP and indicate that a combination of proteasome-inhibiting drugs and AAVP should be considered for clinical anticancer therapy.
Subject(s)
Bacteriophages/metabolism , Dependovirus/metabolism , Proteasome Endopeptidase Complex/metabolism , Animals , Cell Line , Cell Line, Tumor , Fluorescent Antibody Technique , Humans , Leupeptins/pharmacology , MiceABSTRACT
Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.