Naive Huh7 Cells Research Articles

GW25-e1704 Fructose Diet Elevates Circulating PCSK9 and LDL-cholesterol Levels and Reduces Liver LDL Receptor Protein Abundance in Hamsters through a Novel Mechanism

The liver secreted serum protein PCSK9 binds to hepatic LDL receptor (LDLR) and triggers its intracellular degradation. Thus, circulating PCSK9 concentrations influence plasma LDL-c levels and impact cardiovascular disease risk. High fructose diet (HFD) induces dyslipidemia and insulin resistance in animals and humans; however, whether PCSK9 has a causal role in the development of dyslipidmia by high fructose consumption remains elusive. In this study, we showed that serum levels of LDL-c were elevated in hamsters fed HFD as compared to animals fed a normal diet (ND). The increase in circulating LDL-c was accompanied by a 50% reduction of liver LDLR protein abundance in HFD group without changes in LDLR mRNA levels. Examinations of serum PCSK9 levels and liver PCSK9 expressions revealed that hepatic PCSK9 mRNA and protein were reduced by HFD, but serum PCSK9 levels in HFD group were 2.2-fold higher than ND group. PCSK9 in hamster serum was detected as two forms of ~60 kDa and ~53 kDa, the latter being the dominant one. It was previously reported that proprotein convertases furin and PC5A cleave human PCSK9 at Arg 218 -Gln 219 to produce PCSK9-ΔN218 (53 kDa) that circulates in blood along with the intact form. To assess the activity of the equivalent hamster 53 kDa PCSK9-ΔN224 in degrading hepatic LDLR, we conducted in vitro experiments in Huh7 cells by exogenous overexpression of hamster PCSK9 alone or with furin/PC5A. Western blotting of culture medium from transfected Huh7 cells with anti-hamster PCSK9 antibody demonstrated that coexpression of hamster PCSK9 with either furin or PC5A increased the 53 kDa form to 82% and 70% of total PCSK9 species respectively, compared to 18% of the PCSK9 species in the absence of furin or PC5A coexpression. Importantly, the corresponding LDLR protein abundances were reduced by similar extents in Huh7 cells expressing hamster PCSK9 without or with furin/PC5A coexpression. Comparable results were obtained by treating naive Huh7 cells with culture medium of HEK293 cells expressing hamster PCSK9 alone or with furin/PC5A. Altogether, these new findings suggest that HFD may reduce serum LDL-c uptake by LDLR in hamster liver through a novel mechanism that elevates circulating PCSK9 concentration without increasing its hepatic synthesis.

Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.

Development of persistent HCV genotype 3a infection cell culture model in huh-7 cell

BackgroundHepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model.MethodsWe inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells.ResultsHCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40th day, while HCV core protein was detected from the second day up to 40th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA.ConclusionFinally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.

Enzymatic activity of Phosphatidyl-inositol-4 kinase III alpha (PI4KIIIα) is critically involved in HCV replication and regulated by HCV NS5A

PI4KIIIα has been identified as important host factor for HCV replication (1). To gain insight into the mechanism of action, we generated cell lines with a stable knock-down of PI4KIIIα, resulting in a significant reduction of HCV replication upon infection with replication competent genomes. Interestingly, upon expression of non-structural proteins NS3–5B the structure of HCV induced membrane alterations was different in stable knock-down cells changing from a dot-like distribution to large membrane clusters. HCV replication as well as the induction of typical membrane alterations could be rescued by expression of wildtype PI4KIIIα but not by an enzymatic inactive point mutant. Since the enzymatic activity of PI4KIIIα is catalyzing the production of phosphatidyl-inositol-4-phosphate (PI4P) from PI, we analyzed the distribution of this lipid. In naive Huh-7 cells PI4P mainly localized on Golgi-derived membranes. In contrast, HCV infection resulted in an increase in PI4P levels and redistribution into dot like compartments partially co-localizing to the HCV non-structural proteins. Quantitation by immunofluorescence and lipid extraction revealed higher PI4P levels in cells harboring replicating virus or subgenomic replicons. Increasing PI4P staining could also be detected in liver cryosections of chronically infected HCV patients. PI4P pattern was detected in the same areas of consecutive sections as NS5A. We were able to identify domain 1 of NS5A to be responsible for the generation of new intracellular PI4P pools as well as for the direct interaction with the kinase. Strikingly purified NS5A was able to stimulate PI4KIIIα activity resulting in higher PI4P synthesis in vitro. Taken together our data indicate that HCV replication requires PI4KIIIα enzymatic activity, resulting in an increase and redistribution of intracellular PI4P pools. NS5A is involved in recruitment and activation of PI4KIIIα, which might play a role in the establishment of the membranous web.

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.

661 COMPARISON OF HCV-ASSOCIATED GENE EXPRESSION AND CELL SIGNALING PATHWAYS IN CELLS WITH OR WITHOUT HCV REPLICON AND IN REPLICON-CURED CELLS

Background Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naive Huh7 cells.

Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Phenotypic and Structural Analyses of Hepatitis C Virus NS3 Protease Arg155 Variants: SENSITIVITY TO TELAPREVIR (VX-950) AND INTERFERON α

Cell culture and infection system for hepatitis C virus

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro-transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.

Expressional screening of interferon‐stimulated genes for antiviral activity against hepatitis C virus replication

Type-I interferons (IFNs) and the interferon-stimulated genes (ISGs) play a major role in antivirus responses against hepatitis C virus (HCV) infection. In this study, we studied expression profiles of ISGs in cells supporting subgenomic HCV replication (Huh7/Rep), and screened their activities to suppress HCV replication. Real-time PCR analyses showed that the expression levels of 23 ISGs were significantly lower in Huh7/Rep than naive Huh7 cells due to transcriptional suppression of the interferon-stimulated response element (ISRE). Furthermore, the expression level of ISGs was also decreased in the cured Huh7 cells in which replicon had been eliminated (cHuh7), indicating adaptation of the cells to support HCV replication by downregulating ISGs. On the other hand, expression of HCV replicon was significantly suppressed by overexpression of several ISGs including PKR, MxA, IRF-9, GBP-1, IFI-6-16, IFI-27, 25OAS and IRF-1. Knock down of GBP-1, IFI-6-16 and IFI-27 by short hairpin RNA resulted in increase of HCV replication. Thus, we conclude that downregulation of ISG expression is required in the host cells supporting HCV replication and that several ISGs directly suppress HCV replication. The search for ISGs that regulate HCV replication may help to elucidate the cellular antiviral defence mechanisms against HCV infection.

A milestone for hepatitis C virus research: A virus generated in cell culture is fully viable in vivo

Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease in the United States, and the Centers for Disease Control has predicted that ≈10,000 Americans die annually from this disease. Since the discovery of HCV in 1989, basic research on this important pathogen has been difficult because of the lack of a robust cell-culture system. Thus, the recent development of a cell culture resulting in production of significant levels of infectious virus particles (1–3) will without doubt lead to rapid progress in our understanding of the HCV viral life cycle and, it is hoped, development of better therapies. The development of molecular clones of strain H77 (genotype 1a) infectious for chimpanzees in 1997 was an important initial advance, but these clones and subsequent infectious clones of other genotype strains did not produce viruses in cell culture (4–7). Another critical advance was the development of subgenomic replicons in 1999 that permitted studies of RNA replication (8). Efficient replication, however, depended on adaptive mutations (9, 10). In 2003, Kato et al. (11) observed that the WT JFH1 strain of HCV, belonging to genotype 2a, replicated to high levels in the replicon system. Subsequently, in 2005, Wakita and coworkers (2) demonstrated that RNA transcripts from the full-length JFH1 genome could produce viruses in a human liver hepatoma cell line (Huh-7 cells), and it was demonstrated by Chisari and coworkers (3) that this system could be developed to generate relatively high titers of HCV. In a different approach, Rice and coworkers (1) developed a chimeric genome in which the structural genes (C, E1, and E2), p7, and NS2 from an infectious clone of another genotype 2a strain (strain HC-J6) (6) were inserted into the subgenomic replicon of the JFH1 strain and demonstrated that RNA transcripts from this full-length chimeric genome could produce relatively high titers of HCV. In the current issue of PNAS, a study by Lindenbach et al. (12) demonstrated that chimeric J6/JFH1 viruses generated in vitro were fully viable also in vivo, as tested in chimpanzees and in SCID-uPA mice with human liver grafts, and that virus recovered from infected animals efficiently infected naive Huh-7 cells. An overview of this important study is shown in Fig. 1. Therefore, it appears that not only has a cell-culture system been developed for HCV, but this system produces viruses that are biologically relevant.

Hepatitis C virus comes full circle

Hepatitis C virus comes full circle

Ｃ型肝炎ウイルス培養細胞感染系の確立

Hepatitis C virus (HCV) is a major public health problem, infecting an estimated 170 million people worldwide. Current therapy for HCV-related chronic hepatitis is based on the use of interferon. However, virus clearance rates are insufficient. Investigations to develop the anti-viral therapy or to understand the life cycle of this virus have been hampered by the lack of viral culture systems. We isolated the JFH-1 strain from a patient with fulminant hepatitis, and the JFH-1 subgenomic replicon could replicate efficiently in culture cell without adaptive mutation. Recently, we developed the HCV infection system in culture cells with this JFH-1 strain. The full-length JFH-1 RNA was transfected into Huh7 cells. Subsequently, viral RNA efficiently replicated in transfected cells and viral particles were secreted. Furthermore, secreted virus displayed infectivity for naive Huh7 cells. This system provides a powerful tool for studying the viral life cycle and constructing anti-viral strategies.

Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs.

Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5' nontranslated region (5' NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5' NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5' NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon

Background & Aims: Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis. Methods: A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency. Results: The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/μg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/μg RNA ( P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay. Conclusions: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.

RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells.

RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects >270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both a messenger RNA and replication template, making it an attractive target for the study of RNA interference. Double-stranded small interfering RNA (siRNA) molecules designed to target the HCV genome were introduced through electroporation into a human hepatoma cell line (Huh-7) that contained an HCV subgenomic replicon. Two siRNAs dramatically reduced virus-specific protein expression and RNA synthesis to levels that were 90% less than those seen in cells treated with negative control siRNAs. These same siRNAs protected naive Huh-7 cells from challenge with HCV replicon RNA. Treatment of cells with synthetic siRNA was effective >72 h, but the duration of RNA interference could be extended beyond 3 weeks through stable expression of complementary strands of the interfering RNA by using a bicistronic expression vector. These results suggest that a gene-therapeutic approach with siRNA could ultimately be used to treat HCV.

Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations.

Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV.

T7 RNA polymerase transcripts of a putative full-length cDNA clone of hepatitis C virus type 1 (HCV-1) were used to transfect a differentiated human hepatoma cell line, Huh7. The transfected genome replicated in cells, as evidenced by the appearance of progeny HCV RNA, detection of negative-strand viral RNA, and incorporation of [3H]uridine into the viral genome. Incubation of naive Huh7 cells with conditioned medium from transfected cells resulted in a new HCV infection, suggesting the production of biologically active virus in the inoculum. Maintenance of the transfected cells under serum-free culture conditions resulted in the selection of persistently infected cells which displayed a distinctive cellular morphology. This is the first demonstration that HCV RNA produced from cloned HCV cDNA is infectious and replication competent. This approach should provide a valuable system for studying HCV replication, persistence, and pathogenicity.