Correlation between NS5A dimerization and hepatitis C virus replication

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl) phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn2+ but not by Mg2+ or Mn2+. Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance

Combined transcriptomic-(1)H NMR metabonomic study reveals yhat monoethylhexyl phthalate stimulates adipogenesis and glyceroneogenesis in human adipocytes

Adipose tissue is a major storage site for lipophilic environmental contaminants. The environmental metabolic disruptor hypothesis postulates that some pollutants can promote obesity or metabolic disorders by activating nuclear receptors involved in the control of energetic homeostasis. In this context, monoethylhexyl phthalate (MEHP) is of particular concern since it was shown to activate the peroxisome proliferator-activated receptor γ (PPARγ) in 3T3-L1 murine preadipocytes. In the present work, we used an untargeted, combined transcriptomic-(1)H NMR-based metabonomic approach to describe the overall effect of MEHP on primary cultures of human subcutaneous adipocytes differentiated in vitro. MEHP stimulated rapidly and selectively the expression of genes involved in glyceroneogenesis, enhanced the expression of the cytosolic phosphoenolpyruvate carboxykinase, and reduced fatty acid release. These results demonstrate that MEHP increased glyceroneogenesis and fatty acid reesterification in human adipocytes. A longer treatment with MEHP induced the expression of genes involved in triglycerides uptake, synthesis, and storage; decreased intracellular lactate, glutamine, and other amino acids; increased aspartate and NAD, and resulted in a global increase in triglycerides. Altogether, these results indicate that MEHP promoted the differentiation of human preadipocytes to adipocytes. These mechanisms might contribute to the suspected obesogenic effect of MEHP

An NLO QCD analysis of inclusive cross-section and jet-production data from the ZEUS experiment

The ZEUS inclusive differential cross-section data from HERA, for charged and neutral current processes taken with e+ and e- beams, together with differential cross-section data on inclusive jet production in e+ p scattering and dijet production in \gamma p scattering, have been used in a new NLO QCD analysis to extract the parton distribution functions of the proton. The input of jet data constrains the gluon and allows an accurate extraction of \alpha_s(M_Z) at NLO; \alpha_s(M_Z) = 0.1183 \pm 0.0028(exp.) \pm 0.0008(model) An additional uncertainty from the choice of scales is estimated as \pm 0.005. This is the first extraction of \alpha_s(M_Z) from HERA data alone.Comment: 37 pages, 14 figures, to be submitted to EPJC. PDFs available at http://durpdg.dur.ac.uk/hepdata in LHAPDFv

Search for lepton-flavor violation at HERA

A search for lepton-flavor-violating interactions $e p \to \mu X$ and $e p\to \tau X$ has been performed with the ZEUS detector using the entire HERA I data sample, corresponding to an integrated luminosity of 130 pb^{-1}. The data were taken at center-of-mass energies, $\sqrt{s}$, of 300 and 318 GeV. No evidence of lepton-flavor violation was found, and constraints were derived on leptoquarks (LQs) that could mediate such interactions. For LQ masses below $\sqrt{s}$, limits were set on $\lambda_{eq_1} \sqrt{\beta_{\ell q}}$, where $\lambda_{eq_1}$ is the coupling of the LQ to an electron and a first-generation quark $q_1$, and $\beta_{\ell q}$ is the branching ratio of the LQ to the final-state lepton $\ell$ ($\mu$ or $\tau$) and a quark $q$. For LQ masses much larger than $\sqrt{s}$, limits were set on the four-fermion interaction term $\lambda_{e q_\alpha} \lambda_{\ell q_\beta} / M_{\mathrm{LQ}}^2$ for LQs that couple to an electron and a quark $q_\alpha$ and to a lepton $\ell$ and a quark $q_\beta$, where $\alpha$ and $\beta$ are quark generation indices. Some of the limits are also applicable to lepton-flavor-violating processes mediated by squarks in $R$-Parity-violating supersymmetric models. In some cases, especially when a higher-generation quark is involved and for the process $e p\to \tau X$, the ZEUS limits are the most stringent to date.Comment: 37 pages, 10 figures, Accepted by EPJC. References and 1 figure (Fig. 6) adde

Quantitative Analysis of Lipid Droplet Fusion: Inefficient Steady State Fusion but Rapid Stimulation by Chemical Fusogens

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types

High-E_T dijet photoproduction at HERA

The cross section for high-E_T dijet production in photoproduction has been measured with the ZEUS detector at HERA using an integrated luminosity of 81.8 pb-1. The events were required to have a virtuality of the incoming photon, Q^2, of less than 1 GeV^2 and a photon-proton centre-of-mass energy in the range 142 < W < 293 GeV. Events were selected if at least two jets satisfied the transverse-energy requirements of E_T(jet1) > 20 GeV and E_T(jet2) > 15 GeV and pseudorapidity requirements of -1 < eta(jet1,2) < 3, with at least one of the jets satisfying -1 < eta(jet) < 2.5. The measurements show sensitivity to the parton distributions in the photon and proton and effects beyond next-to-leading order in QCD. Hence these data can be used to constrain further the parton densities in the proton and photon.Comment: 36 pages, 13 figures, 20 tables, including minor revisions from referees. Accepted by Phys. Rev.

Multijet production in neutral current deep inelastic scattering at HERA and determination of alpha_s

Multijet production rates in neutral current deep inelastic scattering have been measured in the range of exchanged boson virtualities 10 < Q2 < 5000 GeV2. The data were taken at the ep collider HERA with centre-of-mass energy sqrt(s) = 318 GeV using the ZEUS detector and correspond to an integrated luminosity of 82.2 pb-1. Jets were identified in the Breit frame using the k_T cluster algorithm in the longitudinally invariant inclusive mode. Measurements of differential dijet and trijet cross sections are presented as functions of jet transverse energy E_{T,B}{jet}, pseudorapidity eta_{LAB}{jet} and Q2 with E_{T,B}{jet} > 5 GeV and -1 < eta_{LAB}{jet} < 2.5. Next-to-leading-order QCD calculations describe the data well. The value of the strong coupling constant alpha_s(M_Z), determined from the ratio of the trijet to dijet cross sections, is alpha_s(M_Z) = 0.1179 pm 0.0013(stat.) {+0.0028}_{-0.0046}(exp.) {+0.0064}_{-0.0046}(th.)Comment: 22 pages, 5 figure

Inclusive jet cross sections and dijet correlations in D∗±D^{*\pm} photoproduction at HERA

Inclusive jet cross sections in photoproduction for events containing a $D^*$ meson have been measured with the ZEUS detector at HERA using an integrated luminosity of $78.6 {\rm pb}^{-1}$. The events were required to have a virtuality of the incoming photon, $Q^2$, of less than 1 GeV$^2$, and a photon-proton centre-of-mass energy in the range $130<W_{\gamma p}<280 {\rm GeV}$. The measurements are compared with next-to-leading-order (NLO) QCD calculations. Good agreement is found with the NLO calculations over most of the measured kinematic region. Requiring a second jet in the event allowed a more detailed comparison with QCD calculations. The measured dijet cross sections are also compared to Monte Carlo (MC) models which incorporate leading-order matrix elements followed by parton showers and hadronisation. The NLO QCD predictions are in general agreement with the data although differences have been isolated to regions where contributions from higher orders are expected to be significant. The MC models give a better description than the NLO predictions of the shape of the measured cross sections.Comment: 43 pages, 12 figures, charm jets ZEU