Showing papers by "Andreas Herrmann published in 2014"

Virus inhibition induced by polyvalent nanoparticles of different sizes

Abstract: The development of antiviral agents is one of the major challenges in medical science. So far, small monovalent molecular drugs that inhibit the late steps in the viral replication cycle, i.e., virus budding, have not worked well which emphasizes the need for alternative approaches. Polyvalently presented viral receptors, however, show potential as good inhibitors of virus–cell binding, which is the first step in the viral infection cycle. By gradually increasing the size of ligand functionalized gold nanoparticles, up to virus-like dimensions, we are now able to quantify the polyvalent enhancement of virus–cell binding inhibition and to identify varying mechanisms of virus inhibition with different efficacies: by employing a new binding assay we found that surface area-normalized polysulfated gold nanoparticles of diameters equal to and larger than the virus diameter (>50 nm) more efficiently inhibit the binding of vesicular stomatitis virus (VSV) to cells than smaller particles. On a per particle basis, larger sized gold nanoparticles were surprisingly shown to inhibit the viral infection up to two orders of magnitude more efficiently than smaller particles, which suggests different mechanisms of virus inhibition. Based on complementary electron microscopic data, we noticed that larger gold nanoparticles act as efficient cross-linkers between virions, whereas smaller gold nanoparticles decorate the surface of individual virus particles. Our systematic study accentuates the need for the design of biodegradable, virus-sized inhibitors capitalizing on polyvalent binding.

Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

Abstract: We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

The cholesterol‐binding motif of the HIV‐1 glycoprotein gp41 regulates lateral sorting and oligomerization

Abstract: Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.

A Histidine Residue of the Influenza Virus Hemagglutinin Controls the pH Dependence of the Conformational Change Mediating Membrane Fusion

Abstract: The conformational change of the influenza virus hemagglutinin (HA) protein mediating the fusion between the virus envelope and the endosomal membrane was hypothesized to be induced by protonation of specific histidine residues since their pK a s match the pHs of late endosomes (pK a of ∼6.0). However, such critical key histidine residues remain to be identified. We investigated the highly conserved His184 at the HA1-HA1 interface and His110 at the HA1-HA2 interface of highly pathogenic H5N1 HA as potential pH sensors. By replacing both histidines with different amino acids and analyzing the effect of these mutations on conformational change and fusion, we found that His184, but not His110, plays an essential role in the pH dependence of the conformational change of HA. Computational modeling of the protonated His184 revealed that His184 is central in a conserved interaction network possibly regulating the pH dependence of conformational change via its pK a . As the propensity of histidine to get protonated largely depends on its local environment, mutation of residues in the vicinity of histidine may affect its pK a . The HA of highly pathogenic H5N1 viruses carries a Glu-to-Arg mutation at position 216 close to His184. By mutation of residue 216 in the highly pathogenic as well as the low pathogenic H5 HA, we observed a significant influence on the pH dependence of conformational change and fusion. These results are in support of a pK a -modulating effect of neighboring residues. IMPORTANCE The main pathogenic determinant of influenza viruses, the hemagglutinin (HA) protein, triggers a key step of the infection process: the fusion of the virus envelope with the endosomal membrane releasing the viral genome. Whereas essential aspects of the fusion-inducing mechanism of HA at low pH are well understood, the molecular trigger of the pH-dependent conformational change inducing fusion has been unclear. We provide evidence that His184 regulates the pH dependence of the HA conformational change via its pK a . Mutations of neighboring residues which may affect the pK a of His184 could play an important role in virus adaptation to a specific host. We suggest that mutation of neighboring residue 216, which is present in all highly pathogenic phenotypes of H5N1 influenza virus strains, contributed to the adaptation of these viruses to the human host via its effect on the pK a of His184.

High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Abstract: Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo) or liquid disordered (ld) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.

Dynamics of the circadian clock protein PERIOD2 in living cells.

Abstract: In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1-PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1 Moreover, PER2 nuclear import is crucially dependent on a specific nuclear-receptor-binding motif in PER2 that also mediates nuclear immobility Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation

Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity

Abstract: Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separa- tion and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and func- tional aspects of three lipid raft model systems of vary- ing degrees of biological relevance - PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol - using 2 H solid- state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the gen- eral behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composi- tion of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras pro- tein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-depend- ent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively sim- ple models of raft membranes are able to reproduce many of their specific properties and functions.

DNA-controlled aggregation of virus like particles – mimicking a tetherin-like mechanism

Abstract: Mimicking cellular processes by functional comparable structures helps to understand their molecular mechanism. We report on an oligonucleotide modified with two α-tocopherol anchors mimicking tetherin, a cellular protein reducing spreading of viruses. The lipophilic DNA was incorporated into vesicles and virus like particles; their aggregation was induced by the complement addition.

STAT3 nuclear egress requires exportin 7 via engaging lysine acetylation

Abstract: Nucleocytoplasmic shuttling of signaling molecules is crucial for regulating their activity. Regulation of signal transducer and activator of transcription 3 (STAT3) is critical for normal physiology while STAT3 dysregulation underlies many diseases such as cancer. However, the mechanism(s) underlying STAT3 nuclear egress remains unclear. Here, we show that exportin 7 is critical for STAT3 nuclear egress. Lysine acetylation at K685, frequently found in tumors and tumor cell lines, mediates STAT3 engagement with exportin 7. Blocking acetylation through drug administration or mutation of lysine K685 disrupted STAT3’s physical interaction with exportin 7, leading to STAT3 nuclear retention. Inhibition of STAT3 lysine acetylation significantly altered the functional localization of exportin 7 from the cell cytoplasm toward the nucleus which can be reversed by treating tumor with a lysine-acetylated peptide spanning STAT3 K685. Taken together, our results have identified exportin 7 as an essential karyopherin for STAT3 nucleocytoplasmic shuttling.

Dynamics of the circadian clock protein PERIOD2 in living cells

Abstract: In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1–PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes. Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms. We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period. Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1. Moreover, PER2 nuclear import is crucially dependent on a specific nuclearreceptor-binding motif in PER2 that also mediates nuclear immobility. Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation.