Cell-intrinsic genomic reassortment of pandemic H1N1 2009 and Eurasian avian-like swine influenza viruses results in potentially zoonotic variants.

Influenza A viruses (IAV) cause annual epidemics and occasional pandemics in humans. The most recent pandemic outbreak occurred in 2009 with H1N1pdm09. This virus, which most likely reassorted in swine before its transmission to humans, was reintroduced into the swine population and continues circulating ever since. In order to assess its potential to cause reassortants on a cellular level, human origin H1N1pdm09 and a recent Eurasian avian-like H1N1 swine IAV were (co-)passaged in the newly generated swine lung cell line C22. Co-infection with both viruses gave rise to numerous reassortants that additionally carry different mutations which can partially be found in nature as well. Reassortment most frequently affected the PB1, PA and NA segments with the swine IAV as recipient. These reassortants reached higher titers in swine lung cells and were able to replicate in genuine human lung tissue explants ex vivo, suggesting a possible zoonotic potential. Interestingly, reassortment and mutations in the viral ribonucleoprotein complex influence the viral polymerase activity in a cell type and species-specific manner. In summary, we demonstrate reassortment promiscuity of these viruses in a novel swine lung cell model and indicate a possible zoonotic potential of the reassortants.

Phosphorylation of JIP4 at S730 Presents Antiviral Properties against Influenza A Virus Infection.

Influenza A virus (IAV) is the causative agent of flu disease that results in annual epidemics and occasional pandemics. IAV alters several signaling pathways of the cellular host response in order to promote its replication. Therefore, some of these pathways can serve as targets for novel antiviral agents. Here, we show that c-Jun NH2-terminal kinase (JNK)-interacting protein 4 (JIP4) is dynamically phosphorylated in IAV infection. The lack of JIP4 resulted in higher virus titers, with significant differences in viral protein and mRNA accumulation as early as within the first replication cycle. In accordance, decreased IAV titers and protein accumulation were observed during the overexpression of JIP4. Strikingly, the antiviral function of JIP4 does not originate from modulation of JNK or p38 mitogen-activated protein kinase (MAPK) pathways or from altered expression of interferons or interferon-stimulated genes but rather originates from a direct reduction of viral polymerase activity. Furthermore, the interference of JIP4 with IAV replication seems to be linked to the phosphorylation of the serine at position 730 that is sufficient to impede the viral polymerase. Collectively, we provide evidence that JIP4, a host protein modulated in IAV infection, exhibits antiviral properties that are dynamically controlled by its phosphorylation at S730. IMPORTANCE Influenza A virus (IAV) infection is a world health concern, and current treatment options encounter high rates of resistance. Our group investigates host pathways modified in IAV infection as promising new targets. The host protein JIP4 is dynamically phosphorylated in IAV infection. JIP4 absence resulted in higher virus titers and viral protein and mRNA accumulation within the first replication cycle. Accordingly, decreased IAV titers and protein accumulation were observed during JIP4 overexpression. Strikingly, the antiviral function of JIP4 does not originate from modulation of JNK or p38 MAPK pathways or from altered expression of interferons or interferon-stimulated genes but rather originates from a reduction in viral polymerase activity. The interference of JIP4 with IAV replication is linked to the phosphorylation of serine 730. We provide evidence that JIP4, a host protein modulated in IAV infection, exhibits antiviral properties that are dynamically controlled by its phosphorylation at S730.

Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4.

Coordination of energy metabolism is essential for homeostasis of stem cells, whereas an imbalance in energy homeostasis causes disease and accelerated aging. Here we show that deletion or enzymatic inactivation of lysine-specific demethylase 1 (Lsd1) triggers senescence in trophoblast stem cells (TSCs). Genome-wide transcriptional profiling of TSCs following Lsd1 inhibition shows gene set enrichment of aging and metabolic pathways. Consistently, global metabolomic and phenotypic analyses disclose an unbalanced redox status, decreased glutamine anaplerosis and mitochondrial function. Loss of homeostasis is caused by increased expression of sirtuin 4 (Sirt4), a Lsd1-repressed direct target gene. Accordingly, Sirt4 overexpression in wild-type TSCs recapitulates the senescence phenotype initiated by Lsd1 deletion or inhibition. Inversely, absence of Lsd1 enzymatic activity concomitant with knockdown of Sirt4 reestablishes normal glutamine anaplerosis, redox balance and mitochondrial function. In conclusion, by repression of Sirt4, Lsd1 directs the epigenetic control of TSC immortality via maintenance of metabolic flexibility.

Electron transfer kinetics of cytochrome c probed by time-resolved surface-enhanced resonance Raman spectroscopy.

An improved setup including a measuring cell was designed for time-resolved surface-enhanced resonance Raman (SERR) spectroscopy. The cell is based on a rotating disk electrode (RDE) made from electrochemically roughened Ag. Cytochrome c (cc) adsorbed on a monolayer of mercaptoethanol is investigated with respect to heterogeneous electron transfer. Cyclic voltammograms and potential-dependent static SERR spectra indicate cc to be electroactive on the Ag electrode. The standard redox potential was found to be 234 mV. Time-resolved SERR spectra were then measured triggered by periodic potential pulses changing the protein between the oxidized and reduced state at a frequency of 10 Hz. Monoexponential functions obtained from the intensity of the band at 1361 cm-1 plotted versus time yielded the rate constants of heterogeneous electron transfer to be k(ox) = 46 +/- 7 s(-1) and k(red) = 84 +/- 20 s(-1). These relatively low rates are in line with the orientation of cc on the mercaptoethanol-modified Ag electrode. In this case the heme cleft pointed away from the surface thus hampering electron transfer.

Modeling ion transport in tethered bilayer lipid membranes. 1. Passive ion permeation.

Ion transport across tethered bilayer lipid membranes (tBLMs) is modeled using a hybrid network description which combines potential-dependent rate equations with passive electrical elements. Passive permeation of ions is described by the integrated Nernst-Planck equation. Simulations based on this model are performed with the network simulation program SPICE (simulation program with integrated circuit emphasis). Electrochemical impedance spectra of tBLMs are simulated with this algorithm and challenged by spectra measured with tBLMs submersed in 0.1 M KCl solution and subjected to various potential differences. It is found that the simulated spectra can only satisfactorily represent the experimental data if the permeability coefficients of the ions are dependent on the membrane potential. It is concluded that the mechanism of passive ion transport across the tBLM seems to follow the transient pore model rather than the solubility-diffusion model. This algorithm can be easily extended to include ion transport processes due to channels, carriers, or pumps incorporated into the tBLM.

In situ monitoring of the catalytic activity of cytochrome C oxidase in a biomimetic architecture.

Cytochrome c oxidase (CcO) from Paracoccus denitrificans was immobilized in a strict orientation via a his-tag attached to subunit I on a gold film and reconstituted in situ into a protein-tethered bilayer lipid membrane. In this orientation, the cytochrome c (cyt c) binding site is directed away from the electrode pointing to the outer side of the protein-tethered bilayer lipid membrane architecture. The CcO can thus be activated by cyt c under aerobic conditions. Catalytic activity was monitored by impedance spectroscopy, as well as cyclic voltammetry. Cathodic and anodic currents of the CcO with cyt c added to the bulk solution were shown to increase under aerobic compared to anaerobic conditions. Catalytic activity was considered in terms of repeated electrochemical oxidation/reduction of the CcO/cyt c complex in the presence of oxygen. The communication of cyt c bound to the CcO with the electrode is discussed in terms of a hopping mechanism through the redox sites of the enzyme. Simulations supporting this hypothesis are included.

Activity of membrane proteins immobilized on surfaces as a function of packing density.

A systematic study of the influence of the packing density of proteins on their activity is performed with cytochrome c oxidase (CcO) from R. sphaeroides as an example. The protein was incorporated into a protein-tethered bilayer lipid membrane and CcO was genetically engineered with a histidine-tag, attached to Subunit II, and then tethered by an interaction with functionalized thiol compounds bound to a gold electrode. The packing density was varied by diluting the functionalized thiol with a nonfunctionalized thiol that does not bind to the enzyme. After attaching the CcO to the gold surface, a lipid bilayer was formed to incorporate the tethered proteins. The reconstituted protein-lipid bilayer was characterized by surface enhanced infrared reflection absorption spectroscopy (SEIRAS), electrochemical impedance spectroscopy, surface plasmon resonance, and atomic force microscopy. The activity of the proteins within the reconstituted bilayer was probed by direct electrochemical electron injection and was shown to be very sensitive to the packing density of protein molecules. At low surface density of CcO, the bilayer did not effectively form, and protein aggregates were observed, whereas at very high surface density, very little lipid is able to intrude between the closely packed proteins. In both of these cases, redox activity, measured by the efficiency to accept electrons, is low. Redox activity of the enzyme is preserved in the biomimetic structure but only at a moderate surface coverage in which a continuous lipid bilayer is present and the proteins are not forced to aggregate. Electrostatic and other interaction forces between protein molecules are held responsible for these effects.

Electronic wiring of a multi-redox site membrane protein in a biomimetic surface architecture.

Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltammetry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins.

Active site structure and redox processes of cytochrome c oxidase immobilised in a novel biomimetic lipid membrane on an electrode.

Membrane-bound cytochrome c oxidase was attached to an electrode via a His-tag linker and studied by surface enhanced resonance Raman spectroscopy, demonstrating intact redox site structures and electron transfer between the electrode and the immobilized enzyme.

The protein-tethered lipid bilayer: a novel mimic of the biological membrane.

A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (CcO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.