Hepatitis C Virus Translation Research Articles

DNA methyltransferases 1 and 3B are required for hepatitis C virus infection in cell culture

DNA methyltransferases (DNMTs) are responsible for establishing and maintaining DNA methylation, which are dysregulated in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC). In this report, using lentivirus-mediated shRNA interference technology, we identified DNMT1 and DNMT3B as host factors involved in HCV propagation. Our results demonstrated that down-regulation of DNMT1 or DNMT3B expression in Huh7.5.1 cells severely impaired cell culture-produced HCV (HCVcc) infection. Furthermore, knockdown of DNMT1 or DNMT3B did not affect HCV entry and internal ribosome entry site (IRES)-directed translation but did inhibit subgenomic replication. In contrast, knockdown of DNMT3A had no significant effect on HCV infection, entry, translation, or replication, which suggested that DNMT3A did not play a significant role in HCV life cycle. Moreover, we showed that DNMT inhibitors 5-Aza-C and 5-Aza-dC significantly suppressed HCVcc infection, viral RNA replication, and protein expression. These results suggest that DNMTs are critical for HCV replication and may represent potent targets for the treatment of HCV infection.

A novel strategy to inhibit the reproduction and translation of hepatitis C virus

Hepatitis C virus (HCV), a positive single-stranded RNA virus, is a major cause of liver disease in humans. Herein we report a novel strategy to inhibit the reproduction and translation of HCV using a short RNA, named an Additional RNA, to activate the endonuclease activity of Argonaute 2 (Ago2). In the presence of the Additional RNA, the HCV genome RNA has the requisite 12 nucleotides of base-pairing with microRNA-122. This activates the endonuclease activity of Ago2, resulting in cleavage and release of the HCV genome RNA from Ago2 and microRNA-122. The free HCV genome RNA would be susceptible to intracellular degradation, effectively inhibiting its reproduction and translation. This study presents a new method to inhibit HCV that may hold great potential for HCV treatment in the future.

MicroRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation

Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.

MiR-122 acts as a tumor suppressor in hepatocarcinogenesis in vivo

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocel- lular carcinoma (HCC) correlates with metastasis and poor progno- sis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, deliv- ery of miR-122 to a MYC-driven mouse model of HCC strongly inhib- ited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeu- tic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV. http://www.ncbi.nlm.nih.gov/pubmed/22820290 Abstract: MicroRNA-122 (miR-122), which accounts for 70% of the liver's total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We dem- onstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo resto- ration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 tran- script. In addition, Mir122a � /� livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced dis- ease manifestation and tumor incidence in Mir122a � /� mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

The folding of the hepatitis C virus internal ribosome entry site depends on the 3′-end of the viral genome

Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3′-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3′-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3′-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA–RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

Requirements for human Dicer and TRBP in microRNA-122 regulation of HCV translation and RNA abundance

MicroRNA-122 (miR-122) promotes Hepatitis C Virus (HCV) RNA stability, accumulation, and translation through hybridization with the 5′ untranslated region (5′ UTR) of the HCV genome. Depletion of Dicer and TRBP, proteins involved in miRNA biogenesis, reduced HCV RNA accumulation, mature duplex miR-122 abundance, and miR-122 directed mRNA translation suppression, suggesting roles in miR-122 processing. HCV RNA accumulation independent of endogenous mature duplex miR-122 was not affected by Dicer knockdown, suggesting that Dicer is required solely for miR-122 biogenesis, but TRBP knockdown reduced HCV RNA accumulation in this system, suggesting an additional role in supporting HCV RNA accumulation. Mature duplex miR-122 and pre-miR-122 hairpin, but not single-stranded miR-122 (guide or ⁎ strand), augmented HCV RNA accumulation and translation, and Dicer and TRBP were essential for the activity of pre-miR-122 in mouse fibroblasts. Thus, canonical miRNA processing and strand selection is essential for the activity of miR-122 on HCV translation and RNA accumulation.

Attenuation of 40S Ribosomal Subunit Abundance Differentially Affects Host and HCV Translation and Suppresses HCV Replication

For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.

Hepatitis C Virus Infection Alters P-Body Composition but Is Independent of P-Body Granules

Processing bodies (P-bodies) are highly dynamic cytoplasmic granules conserved among eukaryotes. They are present under normal growth conditions and contain translationally repressed mRNAs together with proteins from the mRNA decay and microRNA (miRNA) machineries. We have previously shown that the core P-body components PatL1, LSm1, and DDX6 (Rck/p54) are required for hepatitis C virus (HCV) RNA replication; however, how HCV infection affects P-body granules and whether P-body granules per se influence the HCV life cycle remain unresolved issues. Here we show that HCV infection alters P-body composition by specifically changing the localization pattern of P-body components that are required for HCV replication. This effect was not related to an altered expression level of these components and could be reversed by inhibiting HCV replication with a polymerase inhibitor. Similar observations were obtained with a subgenomic replicon that supports only HCV translation and replication, indicating that these early steps of the HCV life cycle trigger the P-body alterations. Finally, P-body disruption by Rap55 depletion did not affect viral titers or HCV protein levels, demonstrating that the localization of PatL1, LSm1, and DDX6 in P-bodies is not required for their function on HCV. Thus, the HCV-induced changes on P-bodies are mechanistically linked to the function of specific P-body components in HCV RNA translation and replication; however, the formation of P-body granules is not required for HCV infection.

Stimulation of Hepatitis C Virus RNA translation by microRNA-122 occurs under different conditions in vivo and in vitro

Translation of the Hepatitis C Virus (HCV) positive strand RNA genome is directed by an internal ribosome entry site (IRES) in the viral RNA's 5′-untranslated region (5′-UTR). HCV propagates preferentially in the liver, and HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) acting on two target sites in the 5′-UTR. This stimulation is effective in living cells containing miR-122 and also in the rabbit reticulocyte lysate in vitro-translation system after addition of miR-122. Another RNA sequence located in the Core protein coding sequence can base-pair in a long-range RNA–RNA interaction to the HCV 5′-UTR, overlapping with the miR-122 target sites and the short spacer between them, and thereby inhibits HCV translation. Here we show genetic evidence that in reticulocyte lysate single-stranded miR-122 interferes with this inhibitory long-range RNA–RNA interaction and thereby contributes to enhanced HCV translation, involving not only the 5′-seed sequence of miR-122 but also sequences at its 3′-end. Also RNA oligonucleotides shorter than a typical microRNA stimulate HCV translation, confirming that in the reticulocyte lysate the stimulation of HCV translation functions by displacement of the inhibitory long-range interaction by miR-122. In contrast, in transfected HuH-7 hepatoma cells and in HeLa cells this interference of miR-122 with the inhibitory long-range RNA–RNA interaction plays not a major role, but only duplex miR-122 RNAs of the correct length stimulate HCV translation. These results suggest that: (1) the processing of the microRNA precursors and (2) the events occurring at the HCV RNA differ between cells and reticulocyte lysate.

Regulation of hepatitis C virus translation initiation by iron: Role of eIF3 and La protein

Eukaryotic initiation factors (eIFs) are required for encoding polyprotein of hepatitis C virus (HCV) which is mediated by an internal ribosome-entry site (IRES). Iron overload, a common finding among HCV patients, may be correlated with HCV pathology, but the underlying molecular mechanisms are poorly understood. In this study, we investigated the possible relationship among iron status, eIFs and HCV IRES-mediated translation in vitro. Using bicistronic reporter gene constructs carrying HCV IRES sequence, we found that the levels of intracellular iron were positively associated with the HCV IRES-dependent translation initiation in Huh-7 cells. RT-PCR method showed that iron treatment specifically increased the levels of eIF3A mRNA and La mRNA, whereas iron chelation reduced them. Western blots also confirmed that iron-dependent changes in eIF3A mRNA and La mRNA affected the expression of their proteins. Moreover, antisense phosphorothioate oligodeoxynucleotides to eIF3A and La successfully suppressed the levels of eIF3A and La protein and significantly reduced iron-dependent HCV translation. Taken together, our results suggest that iron promotes the translation initiation of HCV by stimulating the expression of eIF3A and La proteins. Inhibition of eIF3A and La proteins substantially repressed iron-dependent HCV translation, a beneficial effect that may have significant clinical implications.

Let-7b is a novel regulator of hepatitis C virus replication.

The non-coding microRNA (miRNA) is involved in the regulation of hepatitis C virus (HCV) infection and offers an alternative target for developing anti-HCV agent. In this study, we aim to identify novel cellular miRNAs that directly target the HCV genome with anti-HCV therapeutic potential. Bioinformatic analyses were performed to unveil liver-abundant miRNAs with predicted target sequences on HCV genome. Various cell-based systems confirmed that let-7b plays a negative role in HCV expression. In particular, let-7b suppressed HCV replicon activity and down-regulated HCV accumulation leading to reduced infectivity of HCVcc. Mutational analysis identified let-7b binding sites at the coding sequences of NS5B and 5'-UTR of HCV genome that were conserved among various HCV genotypes. We further demonstrated that the underlying mechanism for let-7b-mediated suppression of HCV RNA accumulation was not dependent on inhibition of HCV translation. Let-7b and IFNα-2a also elicited a synergistic inhibitory effect on HCV infection. Together, let-7b represents a novel cellular miRNA that targets the HCV genome and elicits anti-HCV activity. This study thereby sheds new insight into understanding the role of host miRNAs in HCV pathogenesis and to developing a potential anti-HCV therapeutic strategy.

Inhibition of translation initiation factors might be the potential therapeutic targets for HCV patients with hepatic iron overload

Standard therapy, interferon-alpha (IFN-α) and ribavirin, remains the only available option for treatment of patients with hepatitis C virus (HCV) infection. However, iron overload, a common finding among HCV patients, have a poor response to treatment with current therapy. These data suggest that both host and viral factors are involved in the determination of the outcome of the therapy. Currently, novel antiviral compounds focus on the development of indirect antiviral drugs. The process of the viral translation is considered as the potential therapeutic targets. Coincidentally, study has found that hepatic iron load enhances the levels of eukaryotic initiation factor 3 (eIF3), which is essential for HCV translation. Reversely, iron chelation could reduce eIF3 p170 translation. Our hypothesis is that iron overload may specifically enhance cellular eIFs. As a result, the cellular mechanisms, in patients with iron overload, are utilized for translating viral mRNA into protein. Thus, treatment strategies that target eIFs should be an exceptionally good candidate therapeutic method for HCV patients with hepatic iron overload.

Multiple effects of honokiol on the life cycle of hepatitis C virus

Honokiol, a small active molecular compound extracted from magnolia, has recently been shown to inhibit hepatitis C virus (HCV) infection in vitro. This study further characterized aspects of the HCV lifecycle affected by the antiviral functions of honokiol. The influence of honokiol on HCV infection, entry, translation and replication was assessed in Huh-7.5.1 cells using cell culture-derived HCV (HCVcc), HCV pseudo-type (HCVpp) and sub-genomic replicons. Honokiol had strong antiviral effect against HCVcc infection at non-toxic concentrations. Combined with interferon-α, its inhibitory effect on HCVcc was more profound than that of ribavirin. Honokiol inhibited the cell entry of lentiviral particles pseudo-typed with glycoproteins from HCV genotypes 1a, 1b, and 2a, but not of the vesicular stomatitis virus. It had inefficient activity on HCV internal ribosome entry site (IRES)-translation at concentrations with significant anti-HCVcc effects. The expression levels of components of replication complex, NS3, NS5A and NS5B, were down-regulated by honokiol in a dose-dependent manner. It also inhibited HCV replication dose dependently in both genotypes 1b and 2a sub-genomic replicons. Honokiol inhibits HCV infection by targeting cell entry and replication and, only at a concentration >30μM, IRES-mediated translation of HCV life cycle. Based on its high therapeutic index (LD(50) /EC(90) =5.4), honokiol may be a promising drug for the treatment of HCV infection.

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5'-end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs.

MiR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3′-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5′-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5′-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5′-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3′ region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122–HCV interaction, and the broader consequences of miRNAs binding to 5′-UTR sites.

Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5′ Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome

Sequences in the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. The translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5'UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of the 5'UTR and different sets of RNA-binding proteins. Here, we showed that the 5'-most 157 nucleotides of HCV RNA is the minimum 5'UTR for RNA replication, and it partially overlaps with the IRES. Stem-loops 1 and 2 of the 5'UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of the 5'UTR and associated with detergent-resistant membrane fractions, which are the sites of the HCV replication complex. The knockdown of PCBP2 by short hairpin RNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5'- and 3'UTR RNA fragments to form an RNA-protein complex and induces the circularization of HCV RNA, as revealed by electron microscopy. This study thus demonstrates the mechanism of the participation of PCBP2 in HCV translation and replication and provides physical evidence for HCV RNA circularization through 5'- and 3'UTR interaction.

Down-regulation of IRES containing 5'UTR of HCV genotype 3a using siRNAs

BackgroundHepatitis C virus (HCV) is a major causative agent of liver associated diseases leading to the development of hepatocellular carcinoma (HCC) all over the world and genotype-3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current chemotherapy of interferon-α (IFN-α) and ribavirin against HCV infection alternative options are desperately needed out of which the recently discovered RNAi represent a powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process to silence virus infection or replication. HCV translation is mediated by a highly conserved internal ribosome entry site (IRES) within the 5'UTR region making it a relevant target for new drug development.Materials and methodsThe present study was proposed to assess and explore the possibility of HCV silencing using siRNA targeting 5'UTR. For this analysis full length HCV 5'UTR of HCV-3a (pCR3.1/5'UTR) was tagged with GFP protein for in vitro analysis in Huh-7 cells. siRNA targeting 5'UTR were designed, and tested against constructed vector in Huh-7 cell line both at RNA and Protein levels. Furthermore, the effect of these siRNAs was confirmed in HCV-3a serum infected Huh-7 cell line.ResultsThe expression of 5'UTR-GFP was dramatically reduced both at mRNA and protein levels as compared with Mock transfected and control siRNAs treated cells using siRNAs against IRES of HCV-3a genotype. The potential of siRNAs specificity to inhibit HCV-3a replication in serum-infected Huh-7 cells was also investigated; upon treatment with siRNAs a significant decrease in HCV viral copy number and protein expression was observed.ConclusionsOverall, the present work of siRNAs against HCV 5'UTR inhibits HCV-3a expression and represents effective future therapeutic opportunities against HCV-3a genotype.

A Biophysical Study of the Hepatitis C Virus 5’Untranslated Region Interactions with MIR-122

The liver-specific microRNA, miR-122 has been shown to be involved in positively regulating Hepatitis C Virus (HCV), which infects on average 3.2 million Americans. Emerging evidence in the literature suggests that miR-122 binds to two seed sequences (S1 and S2) located within the 5’ untranslated region (UTR) of HCV genomic RNA. Both sites S1 and S2 are important for the effect of miR122 in promoting an upregulation of HCV translation, whereas the S1 site plays a more crucial role in increasing the effect of miR122 upon the viral replication. In this study, we used biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy and native gel electrophoresis to analyze in vitro the direct interactions between miR-122 and the HCV 5’UTR RNA.

Human Ago2 is required for efficient microRNA 122 regulation of hepatitis C virus RNA accumulation and translation.

MicroRNA 122 (miR-122) increases the accumulation and translation of hepatitis C virus (HCV) RNA in infected cells through direct interactions with homologous sequences in the 5' untranslated region (UTR) of the HCV genome. Argonaute 2 (Ago2) is a component of the RNA-induced silencing complex (RISC) and mediates small interfering RNA (siRNA)-directed mRNA cleavage and microRNA translational suppression. We investigated the function of Ago2 in HCV replication to determine whether it plays a role in enhancing the synthesis and translation of HCV RNA that is associated with miR-122. siRNA-mediated depletion of Ago2 in human hepatoma cells reduced HCV RNA accumulation in transient HCV replication assays. The treatment did not adversely affect cell viability, as assessed by cell proliferation, capped translation, and interferon assays. These data are consistent with complementary roles for Ago2 and miR-122 in enhancing HCV RNA amplification. By using a transient HCV replication assay that is dependent on an exogenously provided mutant miR-122, we determined that Ago2 depletion still reduced luciferase expression and HCV RNA accumulation, independently of miR-122 biogenesis. miR-122 has previously been found to stimulate HCV translation. Similarly, Ago2 knockdown also reduced HCV translation, and its depletion reduced the ability of miR-122 to stimulate viral translation. These data suggest a direct role for Ago2 in miR-122-mediated translation. Finally, Ago2 was also necessary for efficient miR-122 enhancement of HCV RNA accumulation. These data support a model in which miR-122 functions within an Ago2-containing protein complex to augment both HCV RNA accumulation and translation.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.