Construction and characterization of a new hepatitis C virus genotype 6a subgenomic replicon that is prone to render the sofosbuvir resistance

Abstract

Hepatitis C virus (HCV) infection remains a challenge to human public health despite the development of highly effective direct‐acting antivirals (DAAs). Sofosbuvir (SOF), a key component in most DAA‐based anti‐HCV cocktail regimens, is a potent viral RNA polymerase (NS5B) inhibitor with a high barrier to drug resistance. The serine‐to‐threonine mutation at NS5B 282 (S282T) confers the SOF resistance, but severely impairs viral replication in most HCV genotypes (GTs) and cannot be stably maintained after the termination of the SOF‐based therapies. In this study, we first developed a new HCV GT‐6a subgenomic replicon PR58D6. Next, we selected SOF‐resistant PR58D6 variants by culturing the replicon cells in the presence of SOF. Interestingly, unlike many other HCV replicons which require additional mutations to compensate for the S282T‐inducing fitness loss, S282T alone in PR58D6 is genetically stable and confers the SOF resistance without significantly impairing viral replication. Furthermore, we showed that amino acid residue at NS5B 74 (R74) and 556 (D556) which are conserved in GT 6a HCV contribute to efficient replication of PR58D6 containing S282T. Finally, we showed that the G556D mutation in NS5B could rescue the replication deficiency of the S282T in JFH1, a GT‐2a replicon. In conclusion, we showed that a novel GT‐6a HCV replicon may easily render SOF resistance, which may call for attention to potential drug resistance during DAA therapies of HCV GT‐6a patients.

Identification and characterization of Sofosbuvir‐resistant mutations of hepatitis C virus genotype 3a replicon

“…Previous studies on in vitro SOF resistance selection in HCV GT1a, 1b, 2a, and 3a reported S282T as the primary induced substitution which is genetically unstable and impairs the polymerase activity of NS5B but can persist for long if additional compensatory mutations within the NS5B are acquired, such as K74R or A74R which is located on an outside of helix of the finger domain 12 as well as G556D which is in close proximity to S282T 13 . In this study, we identified a novel pair of mutations, S282T in NS5B and Q606R in nontarget NS3, which together confer the SOF resistance in a GT3a replicon.…”

“…Interestingly, several studies showed that S282T may be stably maintained in the viral genome if additional compensatory mutations in NS5B are acquired. For example, we previously showed that the replication defect of S282T can be rescued by K/A74R 12 or G556D 13 in NS5B. Another study showed that SOF treatment induces the emergence of S282T together with other mutations in NS5B such as M289L, but the rescuing role of these additional mutations has not been established by reverse genetics 9 …”