Functional Dissection of the Hepatitis C Virus Non-Structural Proteins and miR-122 in Viral Replication and Translation

Hepatitis C Virus (HCV) is a positive stranded RNA virus, grouped into the family of Flaviviridae. The HCV genome encodes a single polyprotein, which is co- and posttranslationally cleaved into ten structural and non-structural (NS) proteins by cellular and viral proteases. The coding sequence is flanked by 5’ and 3’ untranslated regions (UTRs), which contain essential cis-acting elements, regulating translation and RNA synthesis, e.g. an internal ribosome entry site (IRES) for cap-independent translation. HCV RNA replication involves the synthesis of a negative strand replication intermediate, serving as a template for the generation of multiple strands of genomic RNA. This process requires a concerted action of several viral nonstructural proteins, cis-acting replication elements and host factors, and is poorly understood at the molecular level. The first part of this study aimed to characterize the viral non-structural proteins comprising the replicase complex in vitro and their mode of action during (-)-strand RNA synthesis. Since the natural 3’(+)-end is a poor template for the viral polymerase NS5B, supporting roles of the viral protease/helicase NS3 and the phosphoprotein NS5A were hypothesized. Optimal conditions for NS3 activity were established by an in vitro helicase assay. By combining the individual proteins with different RNA templates, it was observed that initiation and processivity of NS5B were stimulated by active NS3, but not by inactive mutants. Inhibition of NS3 helicase activity did not impair the stimulatory effect on NS5B, but led to an altered mode of initiation. Addition of purified NS5A further augmented the effect of NS3. In conclusion, this work demonstrates that NS3 and NS5A can improve RNA dependent RNA polymerase activity on a natural template, thereby providing an experimental model to study the molecular mechanisms governing initiation of RNA synthesis. Liver -specific microRNA (miR)-122 is an important host factor of HCV replication, and recognizes two conserved target sites within the first 45nt of the HCV 5’ UTR, close to the IRES. Previous studies suggested a role of miR-122 in RNA stability, translation, and RNA synthesis. The mechanisms, by which miR-122 exerts these functions, remain enigmatic. Insertion of a heterologous IRES element, allowing for miRindependent translation of the non-structural proteins, was sufficient to enable replication in miR122deficient Hep3B cells, suggesting a substantial role of miR-122 in IRES-dependent translation. The miR122 binding region is engaged in a strong secondary in the complementary negative strand. Additionally, we found that a similar structure was predicted inthe positive strand, which would interfere with IRES formation. We therefore hypothesized that miR-122 binding in this region might prevent such alternative structures, thereby facilitating IRES-mediated translation. Indeed, mutations in the miR-122 binding region, but not the IRES sequence, which were designed to stabilize or destabilize the IRES, enhanced or decreased initial translation, respectively, independent of miR-122. Translation enhancement was independent from RNA stability, but short-lived, suggesting additional roles of miR-122, e.g. recruitment of host proteins facilitating steady state translation. Moreover, structural analysis suggested that the HCV IRES folds into a number of conformers in solution, which can be modified by miR-122 under certain conditions. Apart from the 5’ UTR, HCV also contains several seed-matches for miR-122 in the coding sequence of NS5B, and the 3’ UTR, with unknown functional significance. Two novel sites were identified to be conserved over a number of genotypes. The functional characterization of these miR-122 binding sites was evaluated by insertion of point mutations, abrogating miR-122 binding to single and multiple sites, revealing a previously unappreciated role in virion assembly or release. However, assembly of the mutants could not be rescued by a corresponding mutant miR, suggesting a specific need for wild type miR-122. Conclusively, this study provides evidence for miR-122 involvement in almost every intracellular stage of HCV infection, and defines translation enhancement by suppression of RNA structures interfering with IRES activity as a key function of miR-122

Bile Acids Specifically Increase Hepatitis C Virus RNA-Replication

<div><h3>Background</h3><p>Hepatitis C virus (HCV) patients with high serum levels of bile acids (BAs) respond poorly to IFN therapy. BAs have been shown to increase RNA-replication of genotype 1 but not genotype 2a replicons. Since BAs modulate lipid metabolism including lipoprotein secretion and as HCV depends on lipids and lipoproteins during RNA-replication, virus production and cell entry, BAs may affect multiple steps of the HCV life cycle. Therefore, we analyzed the influence of BAs on individual steps of virus replication.</p> <h3>Methods</h3><p>We measured replication of subgenomic genotype (GT) 1b and 2a RNAs as well as full-length GT2a genomes in the presence of BAs using quantitative RT-PCR and luciferase assays. Cell entry was determined using HCV pseudoparticles (HCVpp). Virus assembly and release were quantified using a core-specific ELISA. Replicon chimeras were employed to characterize genotype-specific modulation of HCV by BAs. Lunet CD81/GFP-NLS-MAVS cells were used to determine infection of Con1 particles.</p> <h3>Results</h3><p>BAs increased RNA-replication of GT1b replicons up to 10-fold but had no effect on subgenomic GT2a replicons both in Huh-7 and HuH6 cells. They did not increase viral RNA translation, virus assembly and release or cell entry. Lowering replication efficiency of GT2a replicons rendered them susceptible to stimulation by BAs. Moreover, replication of full length GT1b with or without replication enhancing mutations and GT2a genomes were also stimulated by BAs.</p> <h3>Conclusions</h3><p>Bile acids specifically enhance RNA-replication. This is not limited to GT1, but also holds true for GT2a full length genomes and subgenomic replicons with low replication capacity. The increase of HCV replication by BAs may influence the efficacy of antiviral treatment in vivo and may improve replication of primary HCV genomes in cell culture.</p> </div

Membrane Remodeling by the Double-Barrel Scaffolding Protein of Poxvirus

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 A ˚ crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxviru

microRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site

International audienceThe liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5' end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5' terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures

A microRNA Encoded by Kaposi Sarcoma-Associated Herpesvirus Promotes B-Cell Expansion <em>In Vivo</em>

<div><p>The human gammaherpesvirus Kaposi sarcoma-associated herpesvirus is strongly linked to neoplasms of endothelial and B-cell origin. The majority of tumor cells in these malignancies are latently infected, and latency genes are consequently thought to play a critical role in virus-induced tumorigenesis. One such factor is kshv-miR-K12-11, a viral microRNA that is constitutively expressed in cell lines derived from KSHV-associated tumors, and that shares perfect homology of its seed sequence with the cellular miR-155. Since miR-155 is overexpressed in a number of human tumors, it is conceivable that mimicry of miR-155 by miR-K12-11 may contribute to cellular transformation in KSHV-associated disease. Here, we have performed a side-by-side study of phenotypic alterations associated with constitutive expression of either human miR-155 or viral miR-K12-11 in bone marrow-derived hematopoietic stem cells. We demonstrate that retroviral-mediated gene transfer and hematopoietic progenitor cell transplantation into C57BL/6 mice leads to increased B-cell fractions in lymphoid organs, as well as to enhanced germinal center formation in both microRNA-expressing mouse cohorts. We furthermore identify Jarid2, a component of Polycomb repressive complex 2, as a novel validated target of miR-K12-11, and confirm its downregulation in miR-K12-11 as well as miR-155 expressing bone marrow cells. Our findings confirm and extend previous observations made in other mouse models, and underscore the notion that miR-K12-11 may have arisen to mimic miR-155 functions in KSHV-infected B-cells. The expression of miR-K12-11 may represent one mechanism by which KSHV presumably aims to reprogram naïve B-cells towards supporting long-term latency, which at the same time is likely to pre-dispose infected lymphocytes to malignant transformation.</p> </div

Influence of bile acids on HCV whole life cycle.

<p><b>A:</b> Experimental setup and schematic drawing of the Luc-Jc1 reporter virus genome carrying a firefly luciferase gene and of the gaussia luciferase construct. Lunet G-luc cells were transfected with the chimeric full-length reporter virus genome and seeded on a 96-well plate. 4 h post-electroporation the medium was removed and new medium containing bile acids was added. After 48 h, RNA-replication in the transfected cells was determined by firefly luciferase assays. At the same time, culture fluid of the cells was collected to determine cell viability through G-luc activity and to inoculate naïve Lunet G-luc cells. 48 h later efficiency of virus production and infection was determined by measuring firefly luciferase in the inoculated cells. <b>B:</b> RNA-replication (left) and virus production/infection (right) in the presence or absence of given doses of BAs determined in Lunet G-luc cells. Replication and whole life cycle data were normalized to DMSO control. The symbol † designates concentrations with a cell viability of less than 50% of the DMSO control. <b>C:</b> Analysis of the influence of given BA doses on Luc-Jc1 replication in HuH6 cells (left) and on the infectivity of secreted particles upon inoculation of Lunet G-luc cells. Means values of triplicates and the standard deviation is given.</p

Influence of BAs on HCV particle production and infectivity.

<p>Cells were transfected, seeded and treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036029#pone-0036029-g002" target="_blank">Fig. 2</a>. Release of core protein as a measure of viral particles in the culture fluid was determined by a commercial core-specific ELISA (<b>A</b>). Infectivity of release particles was assessed by inoculation of naïve Lunet G-luc cells (<b>B</b>). Mean values of duplicates and the standard deviation are shown.</p

Genotype and cell type dependent influence of bile acids on HCV RNA-replication.

<p>Lunet G-luc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036029#pone.0036029-Gentzsch1" target="_blank">[19]</a> were transfected with either SG-Con1/ET (<b>A</b>) or SG-JFH1 replicons (<b>B</b>) and seeded on a 96-well plate. After 4 h the medium was changed and different BAs (CA = cholic acid; CDCA = chenodeoxycholic acid; DCA = deoxycholic acid; LCA = lithocholic acid; UDCA = ursodeoxycholic acid) in concentrations ranging from 25 µM–400 µM were added. 48 h later cell viability was measured by gaussia luciferase assays. The symbol † designates concentrations with a cell viability of less than 50% of the DMSO control. 72 h after electroporation cells were lysed and replication was determined using the firefly luciferase assay. Data were normalized to DMSO control. Con1-derived genome segments are depicted in white, JFH1-derived sequences in black, and non-HCV elements are depicted in grey (PI, polio IRES; EI, encephalomyocarditis virus IRES; luc, firefly luciferase). (<b>C</b>) HuH6 cells were transfected with JFH1 replicon RNA and seeded on a 96-well plate. After 4 h, BAs were added and after 72 h cells were lysed and replication was measured using the firefly-luciferase assay. <b>D:</b> Lunet G-luc cells were transfected with Con1/ET (left panel) or Con1/GND (right panel) replicons and seeded on a 12-well plate. Bile acids or DMSO were added 4 h after electroporation. Cells were lysed at given time points; luciferase activity was determined and normalized for the 4 h value. In each case mean values of triplicates and the standard deviation is given.</p