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Abe K  Ikeda M  Ariumi Y  Dansako H  Kato N 《Virus research》2007,125(2):162-168
HuH-7 is a highly differentiated hepatoma cell line and the only cell line that supports robust RNA replication of the hepatitis C virus (HCV). HuH-7 cells cause cell death in serum-free culture condition. However, the effect is reversed by supplementation with selenium. Serum-free cell cultures are advantageous for vaccine development and experimental reproducibility. However, HCV RNA replication in HuH-7 cells in serum-free medium had not yet been achieved. Therefore, we tried to develop a system for robust HCV RNA replication in a serum-free cell culture. Although HuH-7 cells grew in serum-free medium in the presence of selenium, HuH-7 cells under these conditions did not support HCV RNA replication in long-term culture. Among the supplements tested, serum-free medium with lipid-rich albumin (LRA) was found to yield robust HCV RNA replication. HCV proteins were detected for more than 9 months in serum-free medium supplemented with LRA. This is the first report to demonstrate a long-term, serum-free cell culture that successfully maintained robust HCV RNA replication. This cell culture system is expected to be a useful tool for vaccine development, as well as for further investigation of cellular factors that are essential for HCV RNA replication.  相似文献   

3.
Fascinating progress in the understanding of the molecular biology of hepatitis C virus (HCV) was achieved recently. The replicon system revolutionized the investigation of HCV RNA replication and facilitated drug discovery. Novel systems for functional analyses of the HCV glycoproteins allowed the validation of HCV receptor candidates and the investigation of cell entry mechanisms. Most recently, recombinant infectious HCV could be produced in cell culture, rendering all steps of the viral life cycle, including entry and release of viral particles, amenable to systematic analysis. In this review, we summarize recent advances and discuss future research directions.  相似文献   

4.
Persistent hepatitis C virus (HCV) infection frequently causes hepatocellular carcinoma. However, the mechanisms of HCV-associated hepatocarcinogenesis and disease progression are unclear. Although the human hepatoma cell line, HuH-7, has been widely used as the only cell culture system for robust HCV replication, we recently developed new human hepatoma Li23 cell line-derived OL, OL8, OL11, and OL14 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates. OL, OL8, OL11, and OL14 cells were cultured for more than 2 years. We prepared cured cells from OL8 and OL11 cells by interferon-γ treatment. The cured cells were also cultured for more than 2 years. cDNA microarray and RT-PCR analyses were performed using total RNAs prepared from these cells. We first selected several hundred highly or moderately expressed probes, the expression levels of which were upregulated or downregulated at ratios of more than 2 or less than 0.5 in each set of compared cells (e.g., parent OL8 cells versus OL8 cells cultured for 2 years). From among these probes, we next selected those whose expression levels commonly changed during a 2-year culture of genome-length HCV RNA-replicating cells, but which did not change during a 2-year culture period in cured cells. We further examined the expression levels of the selected candidate genes by RT-PCR analysis using additional specimens from the cells cultured for 3.5 years. Reproducibility of the RT-PCR analysis using specimens from recultured cells was also confirmed. Finally, we identified 5 upregulated genes and 4 downregulated genes, the expression levels of which were irreversibly altered during 3.5-year replication of HCV RNA. These genes may play roles in the optimization of the environment in HCV RNA replication, or may play key roles in the progression of HCV-associated hepatic diseases.  相似文献   

5.
Understanding the pathogenesis of hepatitis C requires the availability of tissue culture models that sustain viral replication and produce infectious particles. We report on the establishment of a culture system of nontransformed human fetal hepatocytes that supports hepatitis C virus (HCV) replication after transfection with full-length in vitro-transcribed genotype 1a HCV RNA without adaptive mutations and infection with patient sera of diverse HCV genotypes. Transfected and infected hepatocytes expressed HCV core protein and HCV negative-strand RNA. For at least 2 months, transfected or infected cultures released HCV into the medium at high levels and usually with a cyclical pattern. Viral replication had some cytotoxic effects on the cells, which produced interferon (IFN)-beta as a component of the antiviral response. Medium from transfected cells was able to infect na?ve cultures in a Transwell system, and the infection was blocked by IFN-alpha and IFN-lambda. Viral particles analyzed by sucrose density centrifugation had a density of 1.17 g/ml. Immunogold labeling with antibody against HCV envelope protein E2 decorated the surface of the viral particles, as visualized by electron microscopy. This culture system may be used to study the responses of nontransformed human hepatocytes to HCV infection, to analyze serum infectivity, and to clone novel HCVs from infected patients.  相似文献   

6.
Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as well as cellular factors. Recently, a cellular protein, synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was found to bind HCV RNA and enhance HCV IRES-dependent translation. We investigate whether this protein is also involved in the HCV RNA replication. We found that SYNCRIP was associated with detergent-resistant membrane fractions and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP by siRNA significantly decreased the amount of HCV RNA in the cells containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was directly involved in HCV RNA replication. These findings indicate that SYNCRIP has dual functions, participating in both RNA replication and translation in HCV life cycle.  相似文献   

7.
Co-infection of hepatitis C virus (HCV) with other blood-borne pathogens such as human T cell leukemia virus (HTLV) is common in highly endemic areas. Clinical evidence showing a correlation between HTLV-I co-infection and rapid progression of HCV-associated liver disease promoted us to investigate the effect of HTLV-I-encoded Tax protein on HCV replication. Reporter assay showed that HCV replicon-encoded luciferase expression was significantly augmented by co-transfection of the Tax-expressing plasmid. Further, HCV RNA replication in replicon cells was increased either by co-culture with cells stably expressing Tax protein (Huhtax) or by culture in the presence of Huhtax-conditioned medium, indicating that Tax could also modulate HCV replication of adjacent cells in a paracrine manner. Additionally, HCV replication in Huhtax exhibited a reduced responsiveness to interferon-alpha-induced antiviral activity. This study demonstrates the facilitation of HCV replication by Tax protein, which may partially account for severer clinical consequences of HCV-related disease in HCV/HTLV co-infected individuals.  相似文献   

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Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).  相似文献   

10.
Grassmann CW  Yu H  Isken O  Behrens SE 《Virology》2005,333(2):349-366
The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.  相似文献   

11.
The cellular models used usually to study hepatitis C virus replication involve coupling between translation and replication. Because this linkage makes detailed analyses difficult a new cellular model was developed where replication is rendered independent of translation. The RNA replication was studied using RNA minigenomes where the reporter gene was flanked by the two untranslated regions of HCV. It was shown that these RNA minigenomes could be stably replicated into Huh7 cells expressing the HCV replication complex. This was obtained either by constitutively expressing the non-structural proteins into Huh7 hepatoma cells or by using Huh7 cells harboring replicons.  相似文献   

12.
Based on an infectious cDNA clone of classical swine fever virus (CSFV) strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) a full-length cDNA was constructed harbouring a nonviral 44 base insertion in the internal ribosome entry site (IRES) within the 5' nontranslated region (5'NTR) of the genome. Genome size RNA transcribed in vitro served as a positive control in routine RT-PCR used to detect CSFV RNA in diagnostic material. Unexpectedly this RNA proved to be infectious upon transfection into susceptible cells. The replication kinetics of the resulting virus vA187-Ins44 were characterized and found to be indistinguishable from its parent virus. However, a deletion mutant with 29 of the 44 inserted bases missing was detected after multiple cell culture passages. RNA secondary structure analysis of the 5'NTR showed that the 44 base insertion destroyed a stem-loop structure and a pseudoknot previously described to be essential for virus replication, demonstrating that insertions within this functionally essential IRES element are tolerated by CSFV.  相似文献   

13.
To identify the host factors implicated in the regulation of hepatitis C virus (HCV) genome replication, we performed comparative proteome analyses of HCV replication complex (RC)-rich membrane fractions prepared from cells harboring genome-length bicistronic HCV RNA at the exponential and stationary growth phases. We found that the eukaryotic chaperonin T-complex polypeptide 1 (TCP1)-ring complex/chaperonin-containing TCP1 (TRiC/CCT) plays a role in the replication possibly through an interaction between subunit CCT5 and the viral RNA polymerase NS5B. siRNA-mediated knockdown of CCT5 suppressed RNA replication and production of the infectious virus. Gain-of-function activity was shown following co-transfection with whole eight TRiC/CCT subunits. HCV RNA synthesis was inhibited by an anti-CCT5 antibody in a cell-free assay. These suggest that recruitment of the chaperonin by the viral nonstructural proteins to the RC, which potentially facilitate folding of the RC component(s) into the mature active form, may be important for efficient replication of the HCV genome.  相似文献   

14.
15.
Chronic hepatitis C virus (HCV) infection is a well-recognized risk factor for hepatocellular carcinoma (HCC). As a co-risk factor, the role of tobacco use in HCV-driven carcinogenesis and relevant underlying mechanisms remain largely unclear. The latest discoveries about HCV replication have shown that HCV RNA hijacks cellular miRNA-122 by forming an Ago2-HCV-miR-122 complex that stabilizes the HCV genome and enhances HCV replication. Our previous work has demonstrated that aqueous tobacco smoke extract (TSE) is a potent activator of HIV replication via TSE-mediated viral protection from oxidative stress and activation of a set of genes that can promote viral replication. Since HCV is, like HIV, an enveloped virus that should be equally susceptible to lipid peroxidation, and since one of the TSE-upregulated genes, the DDX3 helicase, is known to facilitate HCV replication, we hypothesize that (1) tobacco use can similarly enhance HCV viability and replication, and promote HCC progression by up-regulation of DDX3, and (2) by competing for binding with miR-122 as a competing endogenous RNA (ceRNA), HCV replication can liberate miR-122’s direct target, oncogenic gene cyclin G1 (CCNG1); furthermore, simultaneous tobacco use can synergistically enhance this competing effect via HCV upregulation. Our hypotheses may lay a foundation for better understanding of carcinogenesis in HCV-driven HCC and the potential role of tobacco as a cofactor. Disrupting the HCV ceRNA effect may provide a new strategy for designing anti HCV/HCC drugs.  相似文献   

16.
Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.  相似文献   

17.
A chimeric cDNA genome was constructed in which the core, E1 and E2 genes of hepatitis C virus (HCV) replaced the core, E(rns), E1 and E2 genes of bovine viral diarrhea virus (BVDV). High levels of HCV structural proteins were expressed in a small number of human or bovine cells following transfection with chimeric RNA. However, in one cell line, bovine embryonic trachea cells [EBTr(A)], the number of cells expressing HCV proteins increased to greater than 70% following serial passage of culture medium. These cells were persistently infected with a non-cytopathogenic BVDV helper virus. In these cells, the chimeric genome was packaged into infectious particles that accumulated in the culture medium at a titer as high as 10(7)-10(9) genome equivalents per ml. The virus particles were pseudotypes, because they were neutralized by anti-BVDV but not by anti-HCV.  相似文献   

18.
Li X  Kuang E  Dai W  Zhou B  Yang F 《Virus research》2005,114(1-2):126-132
Although it has been suggested that hepatitis delta virus (HDV) can be used as a vector to deliver biologically active RNAs into hepatocytes, modified HDV as a specific transporting and replicating vector in anti-viral research has not been investigated. In this study, we focused on the development of HDV as a replicative vector to deliver hammerhead ribozyme into hepatocytes and the study of the roles of delivered hammerhead ribozyme on the replication of hepatitis B virus (HBV). To investigate the effects of ribozyme delivered by HDV on HBV replication, we designed two hammerhead ribozymes that specifically target the hepatitis B virus genome. These two ribozymes were then inserted into the genome of hepatitis delta virus. Results showed that transfection of cells with tandem modified HDV cDNA resulted in the production of monomer form of sense and anti-sense genomic RNA indicating the recombinant HDV-ribozyme could replicate effectively. Our data also indicated that ribozymes delivered by the modified HDV had higher level of inhibition activity against HBV replication than that of ribozyme alone. This system provides a new approach for the study of mechanisms of HBV replication as well as for the potential treatment of HBV infection.  相似文献   

19.
We have previously reported hepatitis C virus (HCV) replication using a novel binary expression system in which mammalian cells were transfected with a T7 polymerase-driven full-length genotype 1a HCV cDNA plasmid (pT7-flHCV-Rz) and infected with vaccinia-T7 polymerase. We hypothesized that the use of replication-defective adenoviral vectors expressing T7 (Ad-T7pol) or cell lines stably transfected with T7 (Huh-T7) would alleviate cell toxicity and allow for more sustained HCV replication. CV-1, Huh7, and Huh-T7 cells were transfected with pT7-flHCV-Rz and treated with Ad-T7pol (CV-1 and Huh7 only). Protein and RNA were harvested from cells on days 1, 2, 3, 5, 7, and 9 post-infection. No cytotoxicity was observed at 9 days post-infection in any cell type. HCV positive- and negative-strand RNA expression were strongest during days 1-3 post-infection; however, HCV RNA remained detectable throughout the 9-day observation period. Furthermore, transfection with a replication-incompetent plasmid suggested that efficient HCV replication is dependent upon NS5B gene expression. Finally, after 1-2 days of IFN treatment, HCV positive-strand levels decreased significantly compared to HCV-infected but untreated samples (p<0.05). In conclusion, these refined binary systems offer more durable and authentic models for identification of host cellular processes critical to HCV replication and will permit longer-term analysis of virus-host interactions critical to HCV pathogenesis and the treatment of genotype 1 infections.  相似文献   

20.
Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5′ end of the 5′ untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA′s 5′ end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5′-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.  相似文献   

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