Evolution and Persistence of Resistance-associated Substitutions of Hepatitis C Virus After Direct-acting Antiviral Treatment Failures

Y. Jeong; B. Jin; H. W. Lee; H. J. Park; J. Y. Park; D. Y. Kim; K.-H. Han; S. H. Ahn; S. Kim


J Viral Hepat. 2018;25(11):1251-1259. 

In This Article

Abstract and Introduction


Daclatasvir plus asunaprevir (DCV+ASV) treatment is an all-oral direct-acting antiviral (DAA) therapy for the genotype 1b HCV-infected patients. In this study, we investigated how resistance-associated substitutions (RASs) evolved after treatment failures and assessed the effect of those substitutions on viral fitness. Sequencing of NS5A and NS3 revealed typical RASs after treatment failures. Interestingly, the RASs of NS3 reverted to the wild-type amino acid within 1 year after treatment failures. However, the RASs of NS5A were stable and did not change. The effect of NS5A and NS3 RASs on viral RNA replication was assessed after mutagenic substitution in the genotype 1b HCV RNA. Among single substitutions, the effect of D168V was more substantial than the others and the effect of the triple mutant combination (D168V+L31V+Y93H) was the most severe. The RAS at NS5A Y93 affected both viral RNA replication and virus production. Finally, the effect of trans–complementation of NS5A was demonstrated in our co-transfection experiments and these results suggest that such a trans–complementation effect of NS5A may help maintain the NS5A RASs for a long time even after cessation of the DAA treatment. In conclusion, the results from this investigation would help understand the emergence and persistence of RASs.


Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, cirrhosis and hepatocellular carcinoma. The virus has a positive-sense, single-strand RNA as the viral genome and belongs to the Hepacivirus genus within the Flaviviridae family.[1] The size of the viral genome is approximately 9.6 kb and this encodes a single polyprotein in the cytoplasm, which is cleaved co– and post-translationally by the host and viral proteases to generate a total of 10 viral proteins. The proteins at the N terminus (C, E1, E2) are the structural proteins and are involved in making infectious viral particles as constituents of virions while the remaining proteins at the C terminus are the nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) and their roles in the viral life cycle are diverse including viral RNA replication, viral particle assembly and evasion in innate immune response. Among these viral proteins, NS3 protease, NS5A and NS5B RNA-dependent RNA polymerase are the main targets of direct-acting antivirals (DAAs) that have been developed to treat patients with chronic hepatitis C.[2,3] Most of the DAAs targeting NS3 and NS5B inhibit enzymatic activities of these nonstructural proteins. However, NS5A does not have any known enzymatic activity. Since the NS5A protein is involved in both viral RNA replication and virus assembly, the DAAs against NS5A (eg, daclatasvir, ledipasvir, velpatasvir, etc.) are thought to affect both processes.[4]

Currently, most of all-oral DAA regimens for patients with chronic hepatitis C are the combination of DAAs targeting at least two viral proteins among NS3, NS5A and NS5B. One of such treatment options is daclatasvir (DCV) plus asunaprevir (ASV) dual therapy[5] and this therapy is the first all-oral DAA regimen for patients with chronic hepatitis C in some countries including Japan and Korea.[6] Daclatasvir is a potent NS5A inhibitor[7] and asunaprevir is a NS3 protease inhibitor.[8] The combination of these two DAAs is effective in the treatment of patients with chronic hepatitis C, specifically those infected with genotype 1b virus. However, achieving SVR12 (sustained virologic response at 12 weeks after treatment) using this combination therapy substantially depends on the presence of NS5A baseline polymorphisms. While the SVR12 without NS5A baseline polymorphisms was more than 90%, the SVR12 in the presence of NS5A baseline polymorphisms at L31 and/or Y93 was below 50%.[5]

Development of resistance-associated substitutions (RASs) in the viral genome is one of the main concerns when treatment options for patients with chronic hepatitis C are considered. The RASs in NS3, NS5A and NS5B have been well characterized[2,3] and identification of RASs after treatment failures has become important in the selection of the next retreatment options. The RASs are different from each other regarding resistance to DAA, genetic barrier, viral fitness cost, etc. Sometimes, the presence of specific RASs affects substantially the treatment outcomes as we have already mentioned for the DCV+ASV treatment.

In this study, we analysed the HCV from the patients who failed in the DCV+ASV treatment. We determined the sequences of both NS5A and NS3 after treatment failures and also chased the evolution patterns of both NS5A and NS3 resistance-associated substitutions. And finally, we addressed the question why some RASs are much more stable than the others by investigating the effects of the RASs and NS5A trans–complementation on viral fitness.