The Protease Domain Increases the Translocation Stepping Efficiency of the Hepatitis C Virus NS3-4A Helicase

Hepatitis C virus (HCV) NS3 protein has two enzymatic activities of helicase and protease that are essential for viral replication. The helicase separates the strands of DNA and RNA duplexes using the energy from ATP hydrolysis. To understand how ATP hydrolysis is coupled to helicase movement, we measured the single turnover helicase translocation-dissociation kinetics and the pre-steady-state P_i release kinetics on single-stranded RNA and DNA substrates of different lengths. The parameters of stepping were determined from global fitting of the two types of kinetic measurements into a computational model that describes translocation as a sequence of coupled hydrolysis-stepping reactions. Our results show that the HCV helicase moves with a faster rate on single stranded RNA than on DNA. The HCV helicase steps on the RNA or DNA one nucleotide at a time, and due to imperfect coupling, not every ATP hydrolysis event produces a successful step. Comparison of the helicase domain (NS3h) with the protease-helicase (NS3-4A) shows that the most significant contribution of the protease domain is to improve the translocation stepping efficiency of the helicase. Whereas for NS3h, only 20% of the hydrolysis events result in translocation, the coupling for NS3-4A is near-perfect 93%. The presence of the protease domain also significantly reduces the stepping rate, but it doubles the processivity. These effects of the protease domain on the helicase can be explained by an improved allosteric cross-talk between the ATP- and nucleic acid-binding sites achieved by the overall stabilization of the helicase domain structure.     

Hepatitis C Virus Non-structural Protein 3 (HCV NS3): A Multifunctional Antiviral Target

Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protease cleaves the genome-encoded polyprotein and inactivates cellular proteins required for innate immunity. Protease has emerged as an important target for the development of antiviral therapeutics, but drug resistance has turned out to be an obstacle in the clinic. Helicase is required for both genome replication and virus assembly. Mechanistic and structural studies of helicase have hurled this enzyme into a prominent position in the field of helicase enzymology. Nevertheless, studies of helicase as an antiviral target remain in their infancy.     

Dynamics of Hepatitis C Virus (HCV) RNA-dependent RNA Polymerase NS5B in Complex with RNA

The hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuations are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3′ terminus at the enzyme active site.     

New approaches to target RNA binding proteins

RNA binding proteins (RBPs) are a large and diverse class of proteins that regulate all aspects of RNA biology. As RBP dysregulation has been implicated in a number of human disorders, including cancers and neurodegenerative disease, small molecule chemical probes that target individual RBPs represent useful tools for deciphering RBP function and guiding the production of new therapeutics. While RBPs are often thought of as tough-to-drug, the discovery of a number of small molecules that target RBPs has spurred considerable recent interest in new strategies for RBP chemical probe discovery. Here we review current and emerging technologies for high throughput RBP-small molecule screening that we expect will help unlock the full therapeutic potential of this exciting protein class.     

Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

HCV infection is an urgent global health problem that has triggered a drive to discover therapies that specifically target the virus. BMS-791325 is a novel direct antiviral agent specifically targeting HCV NS5B, an RNA-dependent RNA polymerase. Robust viral clearance of HCV was observed in infected patients treated with BMS-791325 in combination with other anti-HCV agents in Phase 2 clinical studies. Biochemical and biophysical studies revealed that BMS-791325 is a time-dependent, non-competitive inhibitor of the polymerase. Binding studies with NS5B genetic variants (WT, L30S, and P495L) exposed a two-step, slow binding mechanism, but details of the binding mechanism differed for each of the polymerase variants. For the clinically relevant resistance variant (P495L), the rate of initial complex formation and dissociation is similar to WT, but the kinetics of the second step is significantly faster, showing that this variant impacts the final tight complex. The resulting shortened residence time translates into the observed decrease in inhibitor potency. The L30S variant has a significantly different profile. The rate of initial complex formation and dissociation is 7–10 times faster for the L30S variant compared with WT; however, the forward and reverse rates to form the final complex are not significantly different. The impact of the L30S variant on the inhibition profile and binding kinetics of BMS-791325 provides experimental evidence for the dynamic interaction of fingers and thumb domains in an environment that supports the formation of active replication complexes and the initiation of RNA synthesis.     

Image-based screening identifies novel roles for IkappaB kinase and glycogen synthase kinase 3 in axonal degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.     

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC₅₀ value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC₅₀ of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a K_D of 0.29 μm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.     

A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)

Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.     

丙型肝炎病毒依赖于RNA的RNA聚合酶(RdRp)研究进展

由于缺乏合适的HCV感染细胞模型,严重制约了HCV复制,特别是HCV复制的关键因子依赖于RNA的RNA聚合酶(RdRp)的研究.对HCV序列比较分析并通过异源表达证明NS5B是HCV复制的RdRp.NS5B C端疏水性氨基酸区域以及NS5B与细胞膜形成复合体等影响NS5B溶解性.在合适的反应条件下NS5B可以多种RNA分子为模板催化RNA复制,特别是能有效复制HCV全长(+)RNA.高浓度GTP激活HCV RdRp活性.NS5B N/C端缺失突变和保守性A、B、C区中的点突变影响RdRp活性,但D区345位精氨酸突变为赖氨酸时RdRp活性明显升高.HCV RdRp的发现及其功能研究为HCV药物研究提供了新型靶标.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.     

丙型肝炎病毒CS,NS3 NS4区抗原融合基因的构建及其在大肠杆菌中的表达

丙型肝炎病毒是与输血有关的非甲非乙肝病毒[1] ;是全世界输血后获得性肝炎的重要病因之一 ,并与急慢性肝炎、肝硬化和肝癌有关。由于主要通过输血的传播 ,会给人民的身体健康尤其输血事业带来极大危害。采用基因工程技术 ,表达含有多段抗原的融合蛋白作为ＨＣＶ检测试剂的抗原 ,可以简化多种抗原的表达及纯化过程 ,并提高了试剂的均一性[2 ] 。研究表明 ,在ＨＣＶ的抗原基因中 ,核心蛋白Ｃｏｒｅ、ＮＳ3区的Ｃ33ｃ抗原、ＮＳ4区基因编码的抗原免疫原性最强 ,相应抗体出现早 ,分布广 ,亲和力强。因此 ,我们构建了含有中国人ＨＣＶ序列的Ｃｏ…     

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel “tyrosine” finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K_i of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.     

Amiloride Docking to Acid-sensing Ion Channel-1

Amiloride is a small molecule diuretic, which has been used to dissect sodium transport pathways in many different systems. This drug is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as sodium/hydrogen antiporters and sodium/calcium exchangers. The exact structural basis for these interactions has not been elucidated as crystal structures of these proteins have been challenging to obtain, though some involved residues and domains have been mapped. This work examines the interaction of amiloride with acid-sensing ion channel-1, a protein whose structure is available using computational and experimental techniques. Using molecular docking software, amiloride and related molecules were docked to model structures of homomeric human ASIC-1 to generate potential interaction sites and predict which analogs would be more or less potent than amiloride. The predictions made were experimentally tested using whole-cell patch clamp. Drugs previously classified as NCX or NHE inhibitors are shown to also inhibit hASIC-1. Potential docking sites were re-examined against experimental data to remove spurious interaction sites. The voltage sensitivity of inhibitors was also examined. Using the aggregated data from these computational and experimental experiments, putative interaction sites for amiloride and hASIC-1 have been defined. Future work will experimentally verify these interaction sites, but at present this should allow for virtual screening of drug libraries at these putative interaction sites.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

In vitro and in vivo anticancer activity of a synthetic glycolipid as Toll-like receptor 4 (TLR4) activator

Activation of Toll-like receptor 4 (TLR4) triggers the innate immune response and leads to the induction of adaptive immunity. TLR4 agonists are known to function as immunostimulants and exhibit promising therapeutic potential for cancer immunotherapy. We have previously developed a synthetic serine-based glycolipid (designated as CCL-34) that can activate TLR4-dependent signaling pathways. In this study, the anticancer immunity of CCL-34 was further demonstrated. CCL-34-activated macrophages induced cancer cell death via the apoptotic pathway, and this cytotoxicity was significantly inhibited by NG-monomethyl-L-arginine (an inducible NOS inhibitor). Notably, conditioned medium collected from CCL-34-treated splenocytes also induced cytotoxicity toward cancer cells. Furthermore, CCL-34 treatment suppressed tumor growth and increased the survival rate in TLR4-functional C3H/HeN mice but not in TLR4-defective C3H/HeJ mice. Increased apoptosis, the induction of cytokines (IFN-γ and IL-12) and chemokines (CXCL9 and CXCL10), and the elevation of leukocyte markers (CD11b, CD11c, CD4, and CD8) were detected at tumor sites in C3H/HeN mice but not in C3H/HeJ mice. Structure-and-activity relationship analysis of CCL-34 and its structural analogs revealed that a sugar moiety is essential for its activity. However, the substitution of the galactose in CCL-34 with glucose or fucose did not reduce its activity. Altogether, this study reveals the anticancer activity of a new synthetic TLR4 agonist and broadens the molecular basis of TLR4-activating glycolipids.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

Heat Shock Protein 72 Is Associated with the Hepatitis C Virus Replicase Complex and Enhances Viral RNA Replication

The NS5A protein of the hepatitis C virus (HCV) is an integral component of the viral replicase. It also modulates cellular signaling and perturbs host interferon responses. The multifunctional characteristics of NS5A are mostly attributed to its ability to interact with various cellular proteins. This study aimed to identify the novel cellular factors that interact with NS5A and decipher the significance of this interaction in viral replication. The NS5A-interacting proteins were purified by the tandem affinity purification (TAP) procedure from cells expressing NS5A and identified by mass spectrometry. The chaperone protein Hsp72 was identified herein. In vivo protein-protein interaction was verified by co-immunoprecipitation and an in situ proximity ligation assay. In addition to NS5A, Hsp72 was also associated with other members of the replicase complex, NS3 and NS5B, suggesting that it might be directly involved in the HCV replication complex. Hsp72 plays a positive regulatory role in HCV RNA replication by increasing levels of the replicase complex, which was attributed either to the increased stability of the viral proteins in the replicase complex or to the enhanced translational activity of the internal ribosome entry site of HCV. The fact that the host chaperone protein Hsp72 is involved in HCV RNA replication may represent a therapeutic target for controlling virus production.     

NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by ¹H and ¹³C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.     

Transient Activation of the PI3K-AKT Pathway by Hepatitis C Virus to Enhance Viral Entry

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.