Adaptive Mutations Producing Efficient Replication of Genotype 1a Hepatitis C Virus RNA in Normal Huh7 Cells

Abstract

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Additionally, three other strains, H77-S (genotype 1a), Con1 and NC1 (genotype 1b), were found capable to produce infectious virions in cell culture [57][58][59]. The H77-S sequence corresponds to the prototype H77 strain with five engineered replication-enhancing mutations [60,61]. The titers yielded by this virus are however moderate (10-100 times lower than JFH-1 virus, with a maximal titer of 10 3 FFU/ml obtained with a particular Huh-7-derived subclone and an extra titer-enhancing mutation [61]).…”

Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III

“…For the quantitative measurement of the HCV RNA replication capacity, Huh-7.5 cells were transfected by electroporation (46), and total cellular RNA was isolated at the time of harvest by using the RNeasy minikit (Qiagen). Real-time quantitative reverse transcription (RT)-PCRs (qRT-PCRs) were carried out with the iQ5 real-time PCR detection system (Bio-Rad) as described previously (27).…”

Hepatitis C Virus NS5A Activates the Mammalian Target of Rapamycin (mTOR) Pathway, Contributing to Cell Survival by Disrupting the Interaction between FK506-binding Protein 38 (FKBP38) and mTOR

“…It has been found that NS5A contained an interferon sensitivity-determining region with the relationship to interferon therapeutic efficacy (9,10). Subsequent researches have proved that NS5A was a pleiotropic and membrane-associated viral phosphoprotein (11) that regulated broad aspects of viral life cycle, such as replication (12)(13)(14)(15), assembly (16,17), and release (18,19). NS5A also modulated various cell events including cell cycle (20), proliferation (21), and antiviral immunity (22), which coordinated host cell environment for virus persistence and pathogenesis.…”