Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees

Abstract

The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo. Hepatitis C virus (HCV) is an enveloped virus with a positivestrand RNA genome (1). Worldwide, more than 100 million people are persistently infected with HCV (2). Infected individuals are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. In a subset of infected individuals it is possible to eliminate the infection by therapy with IFN and ribavirin (3,4). Yet, for the development of vaccines or new therapeutics for HCV, it is of great importance to develop cell culture systems to replicate and propagate HCV. The recent construction of a subgenomic replicon, capable of replication in Huh-7 cells, might represent a first step in achieving this goal (5).The HCV genome (Ϸ9,600 nucleotides) has one ORF, which is flanked by UTRs. Translation produces a polyprotein of Ϸ3,000 amino acids that is cleaved into structural [core (C), viral envelope glycoproteins (E1 and E2)], p7, and nonstructural (NS2-5B) proteins by host or viral proteases (6, 7). The NS2 protein, with the N-terminal third of the NS3 protein, is a protease that mediates NS2͞3 cleavage. The N-terminal part of the NS3 protein, with NS4A as a cofactor, has serine-protease activity and mediates the NS3͞4A, 4A͞4B, 4B͞5A, and 5A͞5B cleavages. The carboxy-terminal part of the NS3 protein contains an NTPase and an RNA-helicase. NS4B is a hydrophobic protein that induces the formation of a cytoplasmic membranous structure where all viral proteins are located and that most likely represents the site of RNA replication (8). The NS5A is a phosphorylated protein believed to be important for viral replication. A short region of the NS5A protein is thought to modulate the host IFN-mediated antiviral response (9). Mutations in this region, the IFN sensitivity-determining region, seems to be associated with the sensitivity of HCV genotype 1b viruses to IFN treatment. The protein ...

Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles

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