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

Abstract

Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatmentresistant 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 fulllength 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.Persistent infection with hepatitis C virus (HCV) is one of the primary causes of chronic liver disease. Progression to chronic active hepatitis with cirrhosis occurs in ϳ20 to 30% of infected individuals, and HCV-associated liver disease is now the leading cause of liver transplantation in the United States (7). Genotypes 1a and 1b, the most prevalent worldwide, have the poorest rates of response to the present treatment regimen, a combination of pegylated alfa interferon 2b with ribavirin (4, 5, 18).HCV, a member of the family Flaviviridae, is a small enveloped virus whose genome is a 9.6-kb single-stranded RNA with positive polarity consisting of a 5Ј nontranslated region (NTR), a large open reading frame encoding the virus-specific proteins, and a 3Ј NTR (reviewed in references 1, 15, and 21). The 5Ј NTR contains an internal ribosome entry site (IRES) mediating translation of a single polyprotein of ϳ3,000 amino acids with the structural proteins (C, E1, and E2) located in the N terminus and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) encoded in the remainder. The NS3-5B coding region is sufficient for RNA replication in cell culture (17), and these proteins are presumed to function as components of the HCV replicase. The NS3 protein possesses serine protease and nucleoside triphosphatase-helicase activities, NS4A is a cofactor for the NS3 serine protease, and NS5B is the RNA-dependent RNA polymerase. The functions of NS4B and NS5A remain elusive, although NS5A, a phosphorylated protein, has been a target for adaptive mutation, allowing efficient initiation of HCV replication in vitro (2, 9, 13). Amino acid substitutions in the NS3, NS4B, and NS5B proteins can also enhance replication to various degrees (9, 13, 16).Initially, only the genotype 1b Con1 RNA sequence was replication competent in the human hepatoma cell line Hu...

Unravelling hepatitis C virus replication from genome to function

“…Although full-length genomes harbouring adaptive mutations replicated efficiently in Huh-7 cells and expressed the structural proteins, infectious particles were not released 61,93,94 . This led to the idea that Huh-7 cells might be unable to support HCV particle assembly or release.…”

“…Furthermore, adaptive mutations in NS3 and NS5B map to surface residues distant from the enzyme active sites, suggesting that these changes are likely to affect interactions between NS proteins and/or cellular factors. A striking feature of highly adaptive mutations is their tendency to decrease NS5A hyperphosphorylation, and many adaptive changes map to NS5A residues implicated in this process [60][61][62] . Similarly, increased hyperphosphorylation of NS5A correlates with lower replication levels 42,63,64 and decreased interaction between NS5A and a cellular factor, human vesicle-associated membrane-protein associated protein A (hVAP-A) 63 .…”

Synergistic Effects of Jerusalem Artichoke in Combination with Pegylated Interferon Alfa-2a and Ribavirin Against Hepatic Fibrosis in Rats

“…It is still unclear what other roles are played by p53. Importantly, it was shown before that p53 is involved in the molecular mechanisms leading to liver injuries (Blight et al, 2003;Schafer et al, 2003). Indeed, patients with various inflammatory liver diseases were found to accumulate p53 protein (Akyol et al, 1999;Panasiuk et al, 2006;Attallah et al, 2009).…”

Experimental models for hepatitis C viral infection

“…Introduction of these specific mutations in the wild-type consensus sequence significantly enhanced viral replication in vitro. [16][17][18][19][20] Mutational hot spots were found clustered primarily in the NS3, NS4B, and NS5A regions. The mechanisms behind the enhanced replication caused by these tissue-culture-adaptive mutations are still largely unknown, and the interesting fact that these mutations are not commonly found in patients suggests that these may have a toll on the viral fitness.…”