Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Molecular insights of a CBP/ß-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder.

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/ß-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724. Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly. PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/ß-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.

Phospholipase A and acyltransferase 4/retinoic acid receptor responder 3 at the intersection of tumor suppression and pathogen restriction.

Phospholipase A and acyltransferase (PLAAT) 4 is a class II tumor suppressor with phospholipid metabolizing abilities. It was characterized in late 2000s, and has since been referred to as 'tazarotene-induced gene 3' (TIG3) or 'retinoic acid receptor responder 3' (RARRES3) as a key downstream effector of retinoic acid signaling. Two decades of research have revealed the complexity of its function and regulatory roles in suppressing tumorigenesis. However, more recent findings have also identified PLAAT4 as a key anti-microbial effector enzyme acting downstream of interferon regulatory factor 1 (IRF1) and interferons (IFNs), favoring protection from virus and parasite infections. Unveiling the molecular mechanisms underlying its action may thus open new therapeutic avenues for the treatment of both cancer and infectious diseases. Herein, we aim to summarize a brief history of PLAAT4 discovery, its transcriptional regulation, and the potential mechanisms in tumor prevention and anti-pathogen defense, and discuss potential future directions of PLAAT4 research toward the development of therapeutic approaches targeting this enzyme with pleiotropic functions.

CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.

FADS2-dependent fatty acid desaturation dictates cellular sensitivity to ferroptosis and permissiveness for hepatitis C virus replication.

The metabolic oxidative degradation of cellular lipids severely restricts replication of hepatitis C virus (HCV), a leading cause of chronic liver disease, but little is known about the factors regulating this process in infected cells. Here we show that HCV is restricted by an iron-dependent mechanism resembling the one triggering ferroptosis, an iron-dependent form of non-apoptotic cell death, and mediated by the non-canonical desaturation of oleate to Mead acid and other highly unsaturated fatty acids by fatty acid desaturase 2 (FADS2). Genetic depletion and ectopic expression experiments show FADS2 is a key determinant of cellular sensitivity to ferroptosis. Inhibiting FADS2 markedly enhances HCV replication, whereas the ferroptosis-inducing compound erastin alters conformation of the HCV replicase and sensitizes it to direct-acting antiviral agents targeting the viral protease. Our results identify FADS2 as a rate-limiting factor in ferroptosis, and suggest the possibility of pharmacologically manipulating the ferroptosis pathway to attenuate viral replication.

Longer Poly(U) Stretches in the 3'UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo.

The 3' untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5' and 3' RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5'UTR and 177 nucleotides of the 3'UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3'UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3'-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45-4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.

Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies.

Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA-Gaussia princeps luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models.IMPORTANCE Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.

Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models.

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

Interferon regulatory factor 1 (IRF1) and anti-pathogen innate immune responses.

The eponymous member of the interferon regulatory factor (IRF) family, IRF1, was originally identified as a nuclear factor that binds and activates the promoters of type I interferon genes. However, subsequent studies using genetic knockouts or RNAi-mediated depletion of IRF1 provide a much broader view, linking IRF1 to a wide range of functions in protection against invading pathogens. Conserved throughout vertebrate evolution, IRF1 has been shown in recent years to mediate constitutive as well as inducible host defenses against a variety of viruses. Fine-tuning of these ancient IRF1-mediated host defenses, and countering strategies by pathogens to disarm IRF1, play crucial roles in pathogenesis and determining the outcome of infection.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.

Construction of complete Tupaia belangeri transcriptome database by whole-genome and comprehensive RNA sequencing.

The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org ). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the "type I interferon signaling pathway". Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.

Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses.

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons1. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance2. Less attention has been paid to how IRFs maintain basal levels of protection against viruses. Here, we depleted antiviral factors linked to RIG-I-like receptor and Toll-like receptor signalling to map critical host pathways restricting positive-strand RNA virus replication in immortalized hepatocytes and identified an unexpected role for IRF1. We show that constitutively expressed IRF1 acts independently of mitochondrial antiviral signalling (MAVS) protein, IRF3 and signal transducer and activator of transcription 1 (STAT1)-dependent signalling to provide intrinsic antiviral protection in actinomycin D-treated cells. IRF1 localizes to the nucleus, where it maintains the basal transcription of a suite of antiviral genes that protect against multiple pathogenic RNA viruses, including hepatitis A and C viruses, dengue virus and Zika virus. Our findings reveal an unappreciated layer of hepatocyte-intrinsic immunity to these positive-strand RNA viruses and identify previously unrecognized antiviral effector genes.

Stearoyl-CoA desaturase-1 is required for flavivirus RNA replication.

Dengue virus (DENV) is the most prevalent human arthropod-borne virus and causes severe problems worldwide, mainly in tropical and sub-tropical regions. However, there is no specific antiviral drug against DENV infection. We and others recently reported that stearoyl-CoA desaturase-1 (SCD1) inhibitor showed potent suppression of hepatitis C virus replication. In this study, we examined the impact of SCD1 on DENV replication. We found that SCD1 inhibitors (MK8245 and #1716) dramatically suppressed DENV replication in a dose-dependent manner without cytotoxicity. This anti-DENV efficacy was observed against all four DENV serotypes and other flaviviruses, including Zika virus and Japanese encephalitis virus. A subgenomic replicon system of DENV was used to confirm that SCD1 inhibitor suppressed viral RNA replication. Interestingly, exogenous supplementation of unsaturated fatty acids resulted in recovery of the DENV titer even in the presence of SCD1 inhibitor, suggesting that fatty acid biosynthesis contributes to DENV genome replication. These findings indicate that SCD1 is a novel host factor required for DENV replication, and SCD1 inhibitor is a potential candidate for treating dengue fever.

Hepatovirus 3ABC proteases and evolution of mitochondrial antiviral signaling protein (MAVS).

BACKGROUND & AIMS: Unlike other hepatitis viruses that have infected primates for millions of years, hepatitis A virus (HAV) likely entered human populations only 10-12 thousand years ago after jumping from a rodent host. The phylogeny of modern hepatoviruses that infect rodents and bats suggest that multiple similar host shifts have occurred in the past. The factors determining such shifts are unknown, but the capacity to overcome innate antiviral responses in a foreign species is likely key. METHODS: We assessed the capacity of diverse hepatovirus 3ABC proteases to cleave mitochondrial antiviral signaling protein (MAVS) and disrupt antiviral signaling in HEK293 and human hepatocyte-derived cell lines. We also applied maximum-likelihood and Bayesian algorithms to identify sites of diversifying selection in MAVS orthologs from 75 chiropteran, rodent and primate species. RESULTS: 3ABC proteases from bat, but not rodent hepatoviruses efficiently cleaved human MAVS at Glu463/Gly464, disrupting virus activation of the interferon-ß promoter, whereas human HAV 3ABC cleaved at Gln427/Val428. In contrast, MAVS orthologs from rodents and bats were resistant to cleavage by 3ABC proteases of cognate hepatoviruses and in several cases human HAV. A search for diversifying selection among MAVS orthologs from all 3 orders revealed 90 of ∼540 residues to be under positive selection, including residues in chiropteran MAVS that align with the site of cleavage of human MAVS by bat 3ABC proteases. CONCLUSIONS: 3ABC protease cleavage of MAVS is a conserved attribute of hepatoviruses, acting broadly across different mammalian species and associated with evidence of diversifying selection at cleavage sites in rodent and bat MAVS orthologs. The capacity of hepatoviruses to disrupt MAVS-mediated innate immune responses has shaped evolution of both hepatoviruses and their hosts, and facilitates cross-species transmission of hepatitis A. LAY SUMMARY: Hepatitis A virus, a common cause of acute hepatitis globally, is likely to have evolved from a virus that jumped from a rodent species to humans within the last 10-12 thousand years. Here we show that distantly related hepatoviruses, that infect bats and rodents today, express proteases that disrupt innate antiviral responses in human cells. This conserved attribute of hepatoviruses may have contributed to that ancient host species shift.

Gamma-aminobutyric acid fermentation with date residue by a lactic acid bacterium, Lactobacillus brevis.

Dates are commercially consumed as semi-dried fruit or processed into juice and puree for further food production. However, the date residue after juice and puree production is not used, although it appears to be nutrient enriched. Here, date residue was fermented by a lactic acid bacterium, Lactobacillus brevis, which has been generally recognized as safe. Through degradation of sodium glutamate added to the residue during the fermentation, γ-aminobutyric acid (GABA), which reduces neuronal excitability, was produced at the conversion rate of 80-90% from glutamate. In order to achieve this GABA production level, pretreatment of the date residue with carbohydrate-degrading enzymes, i.e., cellulase and pectinase, was necessary. All ingredients used for this GABA fermentation were known as being edible. These results provide us with a solution for the increasing commercial demand for GABA in food industry with the use of date residue that has been often discarded.

NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR.

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains.

Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOS HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPOR HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOS H77S.3 and the LPOR H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOS and LPOR viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.

Selective inhibitor of Wnt/ß-catenin/CBP signaling ameliorates hepatitis C virus-induced liver fibrosis in mouse model.

Chronic hepatitis C virus (HCV) infection is one of the major causes of serious liver diseases, including liver cirrhosis. There are no anti-fibrotic drugs with efficacy against liver cirrhosis. Wnt/ß-catenin signaling has been implicated in the pathogenesis of a variety of tissue fibrosis. In the present study, we investigated the effects of a ß-catenin/CBP (cyclic AMP response element binding protein) inhibitor on liver fibrosis. The anti-fibrotic activity of PRI-724, a selective inhibitor of ß-catenin/CBP, was assessed in HCV GT1b transgenic mice at 18 months after HCV genome expression. PRI-724 was injected intraperitoneally or subcutaneously in these mice for 6 weeks. PRI-724 reduced liver fibrosis, which was indicated by silver stain, Sirius Red staining, and hepatic hydroxyproline levels, in HCV mice while attenuating αSMA induction. PRI-724 led to increased levels of matrix metalloproteinase (MMP)-8 mRNA in the liver, along with elevated levels of intrahepatic neutrophils and macrophages/monocytes. The induced intrahepatic neutrophils and macrophages/monocytes were identified as the source of MMP-8. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. We hypothesize that inhibition of hepatic stellate cells activation and induction of fibrolytic cells expressing MMP-8 contribute to the anti-fibrotic effects of PRI-724. PRI-724 is a drug candidate which possesses anti-fibrotic effect.

Differential hepatitis C virus RNA target site selection and host factor activities of naturally occurring miR-122 3΄ variants.

In addition to suppressing cellular gene expression, certain miRNAs potently facilitate replication of specific positive-strand RNA viruses. miR-122, a pro-viral hepatitis C virus (HCV) host factor, binds and recruits Ago2 to tandem sites (S1 and S2) near the 5΄ end of the HCV genome, stabilizing it and promoting its synthesis. HCV target site selection follows canonical miRNA rules, but how non-templated 3΄ miR-122 modifications impact this unconventional miRNA action is unknown. High-throughput sequencing revealed that a 22 nt miRNA with 3΄G ('22-3΄G') comprised <63% of total miR-122 in human liver, whereas other variants (23-3΄A, 23-3΄U, 21-3΄U) represented 11-17%. All loaded equivalently into Ago2, and when tested individually functioned comparably in suppressing gene expression. In contrast, 23-3΄A and 23-3΄U were more active than 22-3΄G in stabilizing HCV RNA and promoting its replication, whereas 21-3΄U was almost completely inactive. This lack of 21-3΄U HCV host factor activity correlated with reduced recruitment of Ago2 to the HCV S1 site. Additional experiments demonstrated strong preference for guanosine at nt 22 of miR-122. Our findings reveal the importance of non-templated 3΄ miR-122 modifications to its HCV host factor activity, and identify unexpected differences in miRNA requirements for host gene suppression versus RNA virus replication.

Structural and functional insights into thermally stable cytochrome c' from a thermophile.

Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability. BRIEF SUMMARY: We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c.