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1.
Nat Commun ; 11(1): 4355, 2020 08 28.
Article in English | MEDLINE | ID: mdl-32859915

ABSTRACT

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9 × 105 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.


Subject(s)
Influenza A virus/genetics , Influenza A virus/physiology , RNA, Viral/genetics , Virus Assembly/genetics , Virus Assembly/physiology , A549 Cells , Epithelial Cells/virology , Evolution, Molecular , Humans , In Situ Hybridization , Influenza A Virus, H3N2 Subtype , Influenza, Human/virology , Lung , Models, Theoretical , Ribonucleoproteins/metabolism
2.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: mdl-32350075

ABSTRACT

Viruses from the family Hantaviridae are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates of up to 50%. Here, we comprehensively investigated entry of the Old World hantavirus Puumala virus (PUUV) into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are greatly reduced. We demonstrate that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, decreases PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV particles and show colocalization of stained virions and markers of macropinosomes. Finally, we report a significant increase in the fluid-phase uptake of cells infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis.IMPORTANCE The family Hantaviridae comprises a diverse group of virus species and is considered an emerging global public health threat. Individual hantavirus species differ considerably in terms of their pathogenicity but also in their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV into mammalian cells. We show that both clathrin-mediated endocytosis and macropinocytosis are cellular pathways exploited by the virus to establish productive infections and demonstrate that pharmacological inhibition of macropinocytosis or a targeted knockdown using RNA interference significantly reduced viral infections. We also found indications of an increase of macropinocytic uptake upon PUUV infection, suggesting that the virus triggers specific cellular mechanisms in order to stimulate its own internalization, thus facilitating infection.


Subject(s)
Clathrin/metabolism , Hemorrhagic Fever with Renal Syndrome/metabolism , Pinocytosis , Puumala virus/metabolism , Virus Internalization , Animals , Chlorocebus aethiops , Hemorrhagic Fever with Renal Syndrome/pathology , Vero Cells
3.
Sci Rep ; 9(1): 707, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30679542

ABSTRACT

Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.


Subject(s)
Glycoproteins/metabolism , Hantavirus Infections/metabolism , Hemorrhagic Fever with Renal Syndrome/metabolism , Protein Multimerization , Puumala virus/physiology , Subcellular Fractions/metabolism , Viral Envelope Proteins/metabolism , Animals , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Glycoproteins/chemistry , Golgi Apparatus/metabolism , Golgi Apparatus/virology , HEK293 Cells , Hantavirus Infections/virology , Hemorrhagic Fever with Renal Syndrome/virology , Humans , Luminescent Proteins/metabolism , Subcellular Fractions/virology , Vero Cells , Viral Envelope Proteins/chemistry
4.
Chembiochem ; 18(16): 1589-1592, 2017 08 17.
Article in English | MEDLINE | ID: mdl-28557173

ABSTRACT

The influenza A virus (IAV) genome is segmented into eight viral ribonucleoproteins, each expressing a negatively oriented viral RNA (vRNA). Along the infection cycle, highly abundant single-stranded small viral RNAs (svRNA) are transcribed in a segment-specific manner. The sequences of svRNAs and of the vRNA 5'-ends are identical and highly conserved among all IAV strains. Here, we demonstrate that these sequences can be used as a target for a pan-selective sensor of IAV infection. To this end, we used a complementary fluorescent forced-intercalation RNA (IAV QB-FIT) probe with a single locked nucleic acid substitution to increase brightness. We demonstrated by fluorescence in situ hybridization (FISH) that this probe is suitable and easy to use to detect infection of different cell types by a broad variety of avian, porcine, and human IAV strains, but not by other influenza virus types. IAV QB-FIT also provides a useful tool to characterize different infection states of the host cell.


Subject(s)
Fluorescent Dyes/chemistry , Influenza A virus/genetics , Intercalating Agents/chemistry , Orthomyxoviridae Infections/diagnostic imaging , RNA Probes/chemistry , A549 Cells , Animals , Base Sequence , Dogs , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Influenza A virus/chemistry , Madin Darby Canine Kidney Cells , Oligodeoxyribonucleotides/chemistry , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , Puumala virus/genetics , Quinolinium Compounds/chemistry , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Viral/chemistry , RNA, Viral/genetics
5.
J Virol ; 89(23): 11899-908, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26378176

ABSTRACT

UNLABELLED: Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, cocultivation assays were performed. Only EHV-1 was transferred from PBMC to EC, and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to that of cells infected with parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taking the results together, we conclude that systemic spread and EC infection by EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer. IMPORTANCE: EHV-1 is widespread throughout the world and causes substantial economic losses through outbreaks of respiratory disease, abortion, and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed, and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus, and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication but have significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines.


Subject(s)
Endothelial Cells/virology , Herpesviridae Infections/physiopathology , Herpesvirus 1, Equid/physiology , Herpesvirus 4, Equid/physiology , Leukocytes, Mononuclear/virology , Protein Serine-Threonine Kinases/metabolism , Viral Envelope Proteins/metabolism , Analysis of Variance , Animals , Cell Aggregation , Cells, Cultured , Fluorescence , Horses , In Vitro Techniques , Statistics, Nonparametric , Virus Internalization
6.
Adv Colloid Interface Sci ; 208: 235-51, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24650567

ABSTRACT

Lipophilic nucleic acids have become a versatile tool for structuring and functionalization of lipid bilayers and biological membranes as well as cargo vehicles to transport and deliver bioactive compounds, like interference RNA, into cells by taking advantage of reversible hybridization with complementary strands. This contribution reviews the different types of conjugates of lipophilic nucleic acids, and their physicochemical and self-assembly properties. Strategies for choosing a nucleic acid, lipophilic modification, and linker are discussed. Interaction with lipid membranes and its stability, dynamic structure and assembly of lipophilic nucleic acids upon embedding into biological membranes are specific points of the review. A large diversity of conjugates including lipophilic peptide nucleic acid and siRNA provides tailored solutions for specific applications in bio- and nanotechnology as well as in cell biology and medicine, as illustrated through some selected examples.


Subject(s)
Biophysics/methods , Membranes, Artificial , Models, Biological , Nucleic Acids/chemistry , Biophysical Phenomena , Biophysics/trends , Elasticity , Elasticity Imaging Techniques , Hydrophobic and Hydrophilic Interactions , Surface Properties
8.
J Am Chem Soc ; 134(50): 20490-7, 2012 Dec 19.
Article in English | MEDLINE | ID: mdl-23163619

ABSTRACT

Lateral partitioning of lipid-modified molecules between liquid-disordered (ld) and liquid-ordered (lo) domains depends on the type of lipid modification, presence of a spacer, membrane composition, and temperature. Here, we show that the lo domain partitioning of the palmitoylated peptide nucleic acid (PNA) can be influenced by formation of a four-component complex with the ld domain partitioning tocopherol-modified DNA: the PNA-DNA complex partitioned into the ld domains. Enzymatic cleavage of the DNA linker led to the disruption of the complex and restored the initial distribution of the lipophilic nucleic acids into the respective domains. This modular system offers strategies for dynamic functionalization of biomimetic surfaces, for example, in nanostructuring and regulation of enzyme catalysis, and it provides a tool to study the molecular basis of controlled reorganization of lipid-modified proteins in membranes, for example, during signal transduction.


Subject(s)
DNA/chemistry , Enzymes/chemistry , Nucleic Acid Hybridization , Peptide Nucleic Acids/chemistry , Biocatalysis , Biomimetics , Tocopherols/chemistry
9.
Beilstein J Org Chem ; 7: 1261-77, 2011.
Article in English | MEDLINE | ID: mdl-21977211

ABSTRACT

In this review we summarize the most important procedures for the preparation of functionalized organzinc and organomagnesium reagents. In addition, new methods for the preparation of polyfunctional aryl- and heteroaryl zinc- and magnesium compounds, as well as new Pd-catalyzed cross-coupling reactions, are reported herein. Experimental details are given for the most important reactions in the Supporting Information File 1 of this article.

10.
Chemistry ; 17(15): 4232-7, 2011 Apr 04.
Article in English | MEDLINE | ID: mdl-21384450

ABSTRACT

Readily available 1,1-dichloro-2-alkynes are versatile starting materials for the synthesis of polyfunctionalized allenes. They can be prepared from commercially available terminal alkynes in a two-step procedure. The Cu-mediated reaction of several 1,1-propargylic derivatives with functionalized alkyl, benzylic, or allylic zinc reagents proceeds exclusively with S(N)2' selectivity and allows a rapid and efficient synthesis of functionalized chloroallenes. These chloroallenes undergo a novel Cu(I) -catalyzed substitution reaction with functionalized arylmagnesium reagents with excellent S(N)2 selectivity to give trisubstituted polyfunctionalized allenes. The functional group tolerance is excellent and functionalities, such as cyano, keto, ester, phosphate, trifluoromethyl, and halogens, are well tolerated.

11.
J Org Chem ; 75(20): 6848-57, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20836538

ABSTRACT

Organic iodides bearing a cationic quaternary ammonium group at a remote position react with zerovalent Pd complexes, Zn, or In leading to a C-I bond insertion. The resulting charge-tagged organometallics can be detected by electrospray-ionization mass spectrometry, which provides detailed information on their stoichiometry, oxidation state, and coordination sphere. The properties of the observed organopalladium and -zinc intermediates largely agree with previous findings, whereas the organoindium species show a surprisingly high tendency to form ate complexes. Magnesium also undergoes insertion into the C-I bond of the charge-tagged organic iodides, but instead of the expected organomagnesium intermediates only the corresponding hydrolysis products could be detected in the diluted solutions. Electrospray-ionization mass spectrometry can also be used to study the reactivity of the charge-tagged species, as was demonstrated for a Pd-catalyzed Negishi cross-coupling reaction. The presented approach permits a straightforward identification of the rate-limiting step and the determination of the corresponding second-order rate constant.


Subject(s)
Iodobenzenes/chemistry , Organometallic Compounds/chemistry , Palladium/chemistry , Quaternary Ammonium Compounds/chemistry , Molecular Structure , Organometallic Compounds/chemical synthesis , Spectrometry, Mass, Electrospray Ionization
12.
Org Lett ; 12(16): 3648-50, 2010 Aug 20.
Article in English | MEDLINE | ID: mdl-20704410

ABSTRACT

Organozinc halides, which are prepared either by direct zinc insertion or halogen-magnesium exchange and subsequent transmetalation with ZnCl(2), react smoothly with commercially available trichloroacetyl isocyanate to give, after hydrolysis, the corresponding primary amides. This method is compatible with a variety of functional groups such as an ester or a cyano group. Also heterocyclic-, alkenyl, and acetylenic zinc reagents are converted to the corresponding primary amides under these conditions.


Subject(s)
Amides/chemical synthesis , Hydrocarbons, Halogenated/chemistry , Zinc/chemistry , Amides/chemistry , Catalysis , Combinatorial Chemistry Techniques , Molecular Structure
13.
Chemistry ; 15(29): 7192-202, 2009 Jul 20.
Article in English | MEDLINE | ID: mdl-19544505

ABSTRACT

The presence of LiCl considerably facilitates the insertion of magnesium into various aromatic and heterocyclic bromides. Several functional groups, such as -OBoc, -OTs, -Cl, -F, -CF(3), -OMe, -NMe(2), and -N(2)NR(2), are well tolerated. The presence of a cyano group leads in some cases to competitive reduction of the organic halide to the corresponding ArH compound. The presence of sensitive groups such as methyl or ethyl ester is tolerated upon in situ trapping of the intermediate magnesium reagent with ZnCl(2). This method can also be applied to the preparation of functionalized benzylic zinc reagents from benzylic chlorides. In the case of di- or tribromoaryl derivatives, directing groups such as -OPiv, -OTs, -N(2)NR(2), or -OAc orient the zinc insertion (Zn/LiCl) to the ortho-position, while the reaction with Mg/LiCl or Mg/LiCl/ZnCl(2) leads to regioselective insertion into the para-carbon-bromine bond. Large-scale experiments (20-100 mmol) for all of the metalation procedures are described.

14.
J Org Chem ; 73(21): 8422-36, 2008 Nov 07.
Article in English | MEDLINE | ID: mdl-18834176

ABSTRACT

A wide range of polyfunctional aryl, heteroaryl, alkyl, and benzylic zinc reagents were coupled with unsaturated aryl halides bearing an acidic NH or OH proton, using Pd(OAc)2 (1 mol %) and S-Phos (2 mol %) as catalyst without the need of protecting groups. A similar nickel-catalyzed reaction is described. The relative kinetic basicity of organozinc compounds as well as their stability toward acidic protons is also described.


Subject(s)
Cross-Linking Reagents/chemistry , Halogens/chemistry , Nickel , Palladium , Catalysis , Organometallic Compounds , Zinc
15.
Chem Commun (Camb) ; (26): 3046-8, 2008 Jul 14.
Article in English | MEDLINE | ID: mdl-18688343

ABSTRACT

Benzylic zinc reagents prepared by direct insertion of zinc to benzylic chlorides in the presence of LiCl undergo smooth cross-coupling reactions with aromatic chlorides, bromides and tosylates using Ni(acac)(2) and PPh(3) as a catalyst system.


Subject(s)
Benzyl Compounds/chemistry , Bromides/chemistry , Chlorides/chemistry , Nickel/chemistry , Tosyl Compounds/chemistry , Zinc/chemistry , Catalysis
16.
Chem Asian J ; 3(8-9): 1678-91, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18604829

ABSTRACT

A new method for the preparation of highly functionalized benzylic zinc chlorides by the direct insertion of zinc dust into the corresponding benzylic chlorides in the presence of LiCl is described without the formation of homocoupling products (<5 %). Various reactions of these benzylic zinc reagents with a broad range of electrophiles, which lead to polyfunctionalized products, are reported. In particular, the cross-coupling reactions of the benzylic zinc chlorides with aromatic chlorides, bromides, and tosylates in the presence of [Ni(acac)(2)] (acac=acetylacetonate) and PPh(3) proceeded in good to excellent yields to give the corresponding diaryl and heterodiaryl methanes.

17.
Org Lett ; 10(13): 2765-8, 2008 Jul 03.
Article in English | MEDLINE | ID: mdl-18529011

ABSTRACT

A wide range of polyfunctional aryl, heteroaryl, alkyl, and benzylic zinc reagents were coupled with unsaturated halides bearing an acidic NH or OH function, using Pd(OAc)(2) (1 mol %) and S-Phos (2 mol %) as catalyst without the need of protecting groups.

18.
Org Lett ; 10(6): 1107-10, 2008 Mar 20.
Article in English | MEDLINE | ID: mdl-18288850

ABSTRACT

In the presence of zinc dust (1.5-2.0 equiv) and LiCl (1.5-2.0 equiv), various benzylic chlorides bearing functional groups (iodide, cyanide, ester, ketone) are smoothly converted at 25 degrees C to the corresponding zinc reagents without homo-coupling (<5%). The utility of these benzylic zinc reagents is demonstrated by a short synthesis of papaverine.

19.
Org Lett ; 8(4): 725-8, 2006 Feb 16.
Article in English | MEDLINE | ID: mdl-16468752

ABSTRACT

[reaction: see text] Organocopper compounds prepared by the transmetalation of functionalized arylmagnesium halides with CuCN.2LiCl undergo smooth cross-coupling reactions with aryl fluorides and tosylates bearing a carbonyl function in the ortho position in the presence of Co(acac)(2) (7.5 mol %), Bu(4)NI (1 equiv), and 4-fluorostyrene (20 mol %) as promoters in DME/THF/DMPU leading to polyfunctional aromatics or heterocycles.

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