Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

Abstract

Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.The Hepatitis C virus (HCV) is a leading cause of chronic liver disease (37). Most infections are inapparent or initially associated with mild symptoms, but the virus persists in ϳ80% of all patients, leading to a high risk of developing severe liver damage such as liver cirrhosis or hepatocellular carcinoma.HCV is an enveloped positive-strand RNA virus belonging to the genus Hepacivirus in the family Flaviviridae (33). The genome of HCV encompasses a single ϳ9,600-nucleotide (nt) RNA molecule carrying one large open reading frame that is flanked by nontranslated regions (NTRs). The 5Ј NTR contains an internal ribosome entry site (IRES) that directs translation of the open reading frame (43, 45). In addition, the 5Ј NTR is required for RNA replication as is the case with the 3Ј NTR (11, 12, 27, 47). All HCV proteins are generated from a polyprotein precursor that is cleaved by cellular and viral proteases into at least 10 different products (for reviews, see references 2 and 38). The structural proteins core, E1, and E2 are located in the amino terminus of the polyprotein (19), followed by p7, a hydrophobic pepti...

TCR-Redirected Human T Cells Inhibit Hepatitis C Virus Replication: Hepatotoxic Potential Is Linked to Antigen Specificity and Functional Avidity

“…Hepatoma Huh-7-Lunet cells designated Lunet-HlaA2-luc/neoET (Huh7 A2 HCV Rep) , Lunet-blr/luc/neo ET (Huh7HCV Rep ), or Lunet-HlaA2 (Huh7 A2 ) were generated by similar protocol as described previously (20). The LunetHlaA2-luc/neoET has ectopic HLA-A2 expression and a selectable HCV subgenomic RNA replicon of genotype 1b, harboring replication-enhancing mutations in nonstructural protein (NS) 3 and NS4B (Con1-ET) (21) and expressing the firefly luciferase gene fused to the selectable marker by ubiquitin (22). The control replicon cell line Huh7HCV Rep is the same as above but transduced with an empty viral vector without the HLA-A2 gene.…”

“…All medium and supplements were purchased from Invitrogen (Carlsbad, CA). [18][19][20][21][22][23][24][25][26][27] were from ProImmune (Oxford, U.K.). For intracellular cytokine staining (ICS) multicolor FACS staining, TCR-transduced PBMCs were incubated overnight with indicated stimuli.…”

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.…”

“…22 This has been further facilitated by the creation of subgenomic replicons containing reporter genes such as firefly luciferase or fluorescent proteins, which allows their use in high-throughput screening assays to determine the efficacy of replication inhibitors. 17,23,24 More recently, monocistronic replicons have been developed by inserting green fluorescent protein in the NS5A coding region, which allows direct visualization of replication in living cells without affecting replication. 24 Cell Culture-Derived Infectious HCV.…”

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

“…[3][4][5] The HCV protein NS4B is a key player in the viral replication cycle. [6][7][8] Adaptive amino acid mutations within NS4B have been shown to enhance replication efficacy in the HCV replicon system. 6,[8][9][10][11] NS4B is highly hydrophobic, 12 which makes experimental structure determination a difficult task.…”