Identification of a Key Determinant of Hepatitis C Virus Cell Culture Adaptation in Domain II of NS3 Helicase

Abstract

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates. The hepatitis C virus (HCV)1 is a major human pathogen for which both vaccines and broadly effective therapeutics are not available (1-3). HCV has a positive-sense RNA genome of ϳ9600 bases and expresses a polyprotein of ϳ3000 amino acids (4) that is processed by host and viral proteases to yield 10 mature proteins (5, 6). Subgenomic HCV RNAs from which the structural proteins have been excised but which express nonstructural proteins and cis-encoded RNA elements essential for replication (replicons) (7-10) are capable of autonomous replication in cell culture (11-13).An important feature of HCV is its high level of genetic variability, which is believed to be a consequence of the low fidelity of the viral polymerase. This variability is underscored by the identification of six major HCV genotypes (designated 1-6), more than 50 subtypes, and numerous quasi-species within each subtype (14). Several clones that are infectious in chimpanzees have been described previously (15-22). In contrast, replicons derived from only the HCV-con1 and HCV-N isolates have been shown to replicate robustly in cell culture thus far (11-13). Efficient replication in cell culture has been invariably associated with adaptive mutations that dramatically increase the frequency with which replication is established (12,(23)(24)(25). Adaptive mutations in the HCV-con1 replicon have been localized to various non-structural genes, although substitutions in NS5A, for example S232I, appear to be the most effective (23). Similarly a 4-amino acid insertion in NS5A th...

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

“…Genotype 1a viruses are the most-prevalent types of HCV in the United States, and like genotype 1 b virus they are relatively refractory to treatment with interferon (9,22). Thus far, a detectable level of genotype 1a RNA replication has been reported only in specially isolated, highly permissive Huh7 human hepatoma cell sublines (e.g., Huh-7.5 cells) generated by eliminating the replication of genotype 1b RNA replicons from established replicon cell lines using alpha interferon in vitro (4,11). These previously described genotype 1a RNAs possess cell culture-adaptive mutations that enhance their replication in these special cells, including those selected during the isolation of antibiotic-resistant cell lines containing these 1a replicons (4,11).…”

“…These RNAs also contained a purposefully introduced mutation in the NS5A protein (S2204I), which had been shown previously to enhance replication of 1b replicons. In a second report, Grobler et al (11) described a systematic mutational approach leading to a similar conclusion, i.e., that both P1496L and S2204I are necessary for efficient replication of genotype 1a RNA in a highly permissive Huh7 subline which was generated independently, but in a manner similar to the Huh-7.5 cell line (11). Both groups identified other adaptive mutations within the helicase domain of NS3 (S1222T and A1226D), but genotype 1a RNAs containing any of these adaptive mutations were not capable of replication in regular Huh7 cells, indicating a relatively limited range of cellular permisiveness.…”

Cell Culture Systems for Hepatitis C Virus

“…Likely as a consequence, most of the changes adapting a given non-JFH1 strain to the JFH1-derived NS3 to NS5B replicase in full length chimeras are strictly chimera-specific and cannot be trans ferred to other chimeras. In contrast, there is a certain degree of flex ibility with REMs which can be successfully transferred from Con1 to H77 and even to genotype 2a replicons (Grobler, Markel et al 2003;Kato, Sugiyama et al 2003;Maekawa, Enomoto et al 2004;Ikeda, Abe et al 2005;Abe, Ikeda et al 2007;Mori, Abe et al 2008). Cell culture adaptations of JFH1-chimeras were mostly conducted in the highly permissive cell line Huh7.5 and are based on the passage of JFH1 infected cells or by serial passages of viral supernatants.…”

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

“…[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. Amongst others, bioinformatics was used to study protein structure and function as an alternative approach.…”