Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10021, USA.
Journal of Virology (Impact Factor: 4.44). 04/2003; 77(5):3181-90. DOI: 10.1128/JVI.77.5.3181-3190.2003
Source: PubMed


Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

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Available from: Joseph Marcotrigiano, Oct 04, 2015
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    • "The H56A and D79A mutations in HPgV NS3/4AB-HA were made using site-directed mutagenesis. The coding regions for HCV NS3/4A (nt 3419–5473) were amplified from the HCV 1a strain H77 clone (kind gift of Dr. Charles Rice) (Blight et al., 2003) using HiFi DNA polymerase (Roche) and inserted into pCDNA3.1/Zeoþ expression vector (Invitrogen). "
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    ABSTRACT: We previously found that human pegivirus (HPgV; formerly GBV-C) NS3 protease activity inhibits Human Immunodeficiency Virus (HIV) replication in a CD4+ T cell line. Given the protease׳s similarity to the Hepatitis C virus (HCV) NS3 protease, we characterized HPgV protease activity and asked whether it affects the type I interferon response or is inhibited by HCV protease antagonists. We characterized the activity of proteases with mutations in the catalytic triad and demonstrated that the HCV protease inhibitors Telaprevir, Boceprevir, and Danoprevir do not affect HPgV protease activity. HPgV NS3 protease cleaved MAVS but not TRIF, and it inhibited interferon responses sufficiently to enhance growth of an interferon-sensitive virus. Therefore, HPgV׳s inhibition of the interferon response could help promote HPgV persistence, which is associated with clinical benefits in HIV-infected patients. Our results also imply that HCV protease inhibitors should not interfere with the beneficial effects of HPgV in HPgV/HCV/HIV infected patients.
    Virology 05/2014; s 456–457(1):300–309. DOI:10.1016/j.virol.2014.03.018 · 3.32 Impact Factor
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    • "This system only depends on the ability to replicate in cells. HCV-RMT was the first genotype 1a clone that could be established in authentic replicon cells without artificially introduced adaptive mutations that are required by H77 [30], [31], although the three spontaneously occurring mutations (E1056V, E1202G, and A2199T) are not novel [11], [31]. Among the mutants with single mutations or a combination of these three adaptive mutations, the amounts of HCV genome and viral proteins did not reflect the colony-forming abilities (Figure 1D, E). "
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    ABSTRACT: Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.
    PLoS ONE 12/2013; 8(12):e82527. DOI:10.1371/journal.pone.0082527 · 3.23 Impact Factor
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    • "Construction and selection of a subgenomic genotype 3a replicon A subgenomic genotype 3a replicon was constructed as previously described by Lohmann et al. (Lohmann et al., 1999) and based on the consensus sequence (GenBank accession #GU814263) of the genotype 3a S52 strain (Fig. 1A) (Gottwein et al., 2010). To enhance the basal level of replication, the NS5A mutation S232I (equivalent to the major adaptive mutation S2204I in genotype 1 polyprotein) was incorporated (Blight et al., 2003). "
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    ABSTRACT: Infection with genotype 3 hepatitis C virus (HCV) is common throughout the world, however no direct-acting antiviral (DAA) has been approved to treat this genotype. We therefore attempted to develop novel genotype 3 replicons to facilitate the discovery and development of new HCV therapies. A novel Huh-7-derived cell line 1C but not Lunet cells enabled the selection of a few stable colonies of a genotype 3a subgenomic replicon (strain S52). Genotypic analysis revealed a mutation of P89L in the viral NS3 protease domain, which was confirmed to enhance genotype 3a RNA replication and enable the establishment of highly replicating luciferase-encoding replicons. Secondary adaptive mutations that further enhanced RNA replication were identified in the viral NS3 and NS4A proteins. In addition, cell lines that were cured of genotype 3a replicons demonstrated higher permissiveness specifically to genotype 3a HCV replication. These novel replicons and cell lines were then used to study the activity of approved and experimental HCV inhibitors. NS3 protease and non-nucleoside NS5B polymerase inhibitors often demonstrated substantially less antiviral activity against genotype 3a compared to genotype 1b. In contrast, nucleoside analog NS5B inhibitors and host-targeting HCV inhibitors showed comparable antiviral activity between genotypes 3a and 1b. Overall, the establishment of this novel genotype 3a replicon system, in conjunction with those derived from other genotypes, will aid the development of treatment regimens for all genotypes of HCV.
    Antiviral research 09/2013; 100(2). DOI:10.1016/j.antiviral.2013.08.018 · 3.94 Impact Factor
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