Tetherin has negligible activity in restricting hepatitis C virus in hepatocytes

We investigated the ability of tetherin, a recently identified antiviral factor, in restricting hepatitis C virus (HCV) in the Japanese fulminant hepatitis-1 (JFH-1) infectious cell culture system. Human hepatocytes (Huh7, Huh7.5.1) expressedlow levels of endogenous tetherin, which could be induced by IFN-α. However, tetherin contributes little to IFN-α-mediated anti-HCV JFH-1 activity. Although tetherin could inhibit Vpu-deleted HIV-1 release, it had negligible activity in restricting HCV JFH-1 release from hepatocytes, which was evidenced by unaffected levels of intracellular/extracellular HCV RNA and infectious virus. The failure of tetherin's anti-HCV activity could not be related to the counteraction of HCV, as HCV infection of hepatocytes affected neither tetherin expression nor anti-HIV function of tetherin. These observations imply that tetherin has negligible activity in the restriction of HCV JFH-1 in human hepatocytes.     

Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles

The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (approximately 65 to 70 nm) but different in buoyant density (approximately 1.15 to 1.20 g/ml) from extracellular particles (approximately 1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.     

Modeling hepatitis C virus dynamics: liver regeneration and critical drug efficacy

Mathematical models for hepatitis C viral (HCV) RNA kinetics have provided a means of evaluating the antiviral effectiveness of therapy, of estimating parameters such as the rate of HCV RNA clearance, and they have suggested mechanism of action against HCV for both interferon and ribavirin. Nevertheless, the model that was originally formulated by Neumann et al. [1998. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282 (5386), 103-107] is unable to explain all of the observed HCV RNA profiles under treatment e.g., a triphasic viral decay and a viral rebound to baseline values after the cessation of therapy. Further, the half-life of productively HCV-infected cells, estimated from the second phase HCV RNA decline slope, is very variable and sometimes zero with no clear understanding of why. We show that extending the original model by including hepatocyte proliferation yields a more realistic model without any of these deficiencies. Further, we define and characterize a critical drug efficacy, such that for efficacies above the critical value HCV is ultimately cleared, while for efficacies below it, a new chronically infected viral steady-state level is reached.     

Innate host response in primary human hepatocytes with hepatitis C virus infection

Background and Aim

The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.

Methods

An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.

Results

Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.

Conclusion

Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection.     

丙型肝炎病毒感染过程中其NS3/4A蛋白酶切割线粒体抗病毒信号蛋白的动态过程

已知丙型肝炎病毒(hepatitis C virus,HCV)可通过其蛋白酶NS3/4A切割线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)来逃逸天然免疫识别,但尚不清楚其切割动力学及切割在抑制干扰素中的作用。本研究旨在细胞模型中探讨HCV感染过程中病毒复制建立及病毒NS3/4A切割MAVS的动态过程,探究NS3/4A切割MAVS对病毒逃逸宿主天然免疫建立感染的贡献。首先构建基于绿色荧光蛋白(green fluorescent protein,GFP)的MAVS切割报告系统(GFP-NLS-MAVS-TM462),用 HCV Jc1-Gluc 感染Huh7.5/GFP-NLS-MAVS-TM462细胞。结果显示,病毒复制早期MAVS切割效率较低;NS3/4A高效切割MAVS发生于HCV复制晚期,且其切割效率与NS3蛋白水平相关。利用带有GFP ypet的HCV报告病毒Jc1-378-1感染Huh7.5/RFP-NLS-MAVS-TM462细胞,在单细胞水平观察HCV感染阳性细胞中MAVS被切割情况,发现HCV复制细胞中仅部分细胞MAVS被切割。进一步研究发现,不同基因型NS3/4A切割MAVS的效率仅与NS3表达水平相关。以上结果提示,HCV蛋白酶NS3/4A切割MAVS依赖NS3/4A蛋白在病毒复制过程中的累积,对在病毒复制早期逃逸宿主天然免疫建立感染可能无显著贡献。     

Adenovirus-mediated gene transfer of interferon alpha inhibits hepatitis C virus replication in hepatocytes

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases.     

Trade-offs in resource allocation in the intracellular life-cycle of hepatitis C virus

Positive sense single-stranded RNA viruses undergo three mutually exclusive processes to replicate within a cell. These are translation to produce proteins, replication to produce RNA viral genomes, and packaging to form virions. The allocation of newly synthesised viral genomes to these processes, which can be regarded as life-history traits, may be subject to natural selection for efficient reproduction. Here, we develop a mathematical model of the process of intracellular viral replication to study alternative strategies for the allocation and reallocation of viral genomes to these processes. We explore four cases of the model: (1) Free Movement, in which viral genomes can freely be allocated and reallocated among translation, replication and packaging; (2) Unidirectional Reallocation, in which allocation occurs freely but reallocation can only proceed from translation to replication to packaging; (3) Conveyor Belt, in which viral genomes are first allocated to translation, then passed on to replication and finally to packaging; and (4) Permanent Allocation in which new genomes are allocated to the three processes but not reallocated between them. We apply this model to hepatitis C virus and study changes in the production of virus as the rates of allocation and reallocation are varied. We find that high viral production occurs when allocation and reallocation of the genome are weighted towards the translation and replication processes. The replication process in particular is favoured. The most productive strategy is a form of the Free Movement model in which genomes are allocated entirely to the replication-translation cycle while allowing some genomes to be packaged through reallocation.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Trafficking of hepatitis C virus core protein during virus particle assembly

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.     

Properties of intracellular virus-specific ribonucleoproteins containing negative and positive hepatitis A virus RNA]

The characteristics of the intracellular virus-specific nucleocapsids containing either a negative or a positive RNA strand were studied. The immunosorption of nucleocapsids by the monoclonal antibodies against the three epitopes of NP protein failed to reveal any antigenic difference between the negative strand or positive strand-containing nucleocapsids. On the other hand, the sensitivity of virus-specific RNA in the nucleocapsids to digestion by the pancreatic ribonuclease proved to be lower for the positive strand-containing nucleocapsids.     

Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs

Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector-derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5′ untranslated region (5′ UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5′ UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5′ UTR at which ~80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA-based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA-based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

Scavenger receptor class B type I and hepatitis C virus infection of primary tupaia hepatocytes

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.     

Metabolomic profile of hepatitis C virus-infected hepatocytes

Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 5'-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes.     

Elimination of hepatitis C virus from hepatocytes by a selective activation of therapeutic molecules

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNβ promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNβ, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.