Measuring Fitness Cost of HCV Drug-resistance Mutations in a Fully Replication-competent Assay
B Lassmann1, V Arumugaswami2, K Chew1, and Martha Lewis*1
1Univ of California, Los Angeles, David Geffen Sch of Med, US and 2Cedars-Sinai Regenerative Med Inst, Los Angeles, CA, US
Background: Knowing hepatitis C virus (HCV) replication capacity is important to assessing the effect of drug-induced mutations on viral fitness. The replicon system, commonly used to assess replication capacity in vitro, is limited to evaluating effect on RNA replication, translation, and protein processing only. To overcome these limitations, a replication assay was developed using full length, replication competent HCV. This system can be used to measure the impact of drug resistance and immune escape mutations in structural and nonstructural HCV proteins on all stages of the viral life cycle.
Methods: A modified version (X8) of the genotype 2a J6/JFH-1 hybrid molecular clone (JFH-FNX) was constructed with a sequence tag allowing the original and modified clones to be simultaneously quantified by real-time PCR using distinct probes. Site-directed mutagenesis introduced the NS3_R155K or NS3_T54A drug resistance mutations into JFH-FNX. An additional JFH-FNX mutant with a deletion in the 3 UTR with known lower replication capacity was used to validate the assay. Mutant and X8 viruses were used to co-infect the same cells at low multiplicity of infection (MOI) and copies of HCV in the supernatant over time were measured by qPCR and expressed on a log scale. A linear regression was performed to obtain slope for each virus (m). Differences between the replication capacity of the reference X8 and mutant viruses were statistically evaluated using a 2-tailed t-test.
Results: Relative viral growth rates were reproducible over independent experiments. There was no difference (p = 0.4) in the growth rate of X8 (m = 1.3, SD 0.2) and JFH-FNX (m = 1.2, SD 0.1). Replication capacity of the virus with the NS3_R155K drug-resistance mutation was equivalent to the replication capacity of the reference strain (1.09±0.1, p = 0.3). Replication capacity of the virus with the NS3_T54A drug-resistance mutation was lower than that of the reference strain (0.88±0.04), but without statistical significance (p = 0.1). Replication capacity of the virus with the deletion in the 3 UTR was significantly lower (0.65±0.13, p = 0.03) than replication capacity of the reference strain.
Conclusions: Given the intense drug discovery and development efforts ongoing for HCV, we anticipate that our assay will play an important role in further assessing the cost of drug resistance and immune escape mutations on all stages of HCV replication.