Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon.

Hepatitis C virus (HCV) genotype (GT) 3 is the second most prevalent of the seven HCV genotypes and exhibits the greatest resistance to the highly potent, direct-acting antivirals (DAAs) that are currently in use. Previously a stable cell line harbouring the S52 GT3 sub-genomic replicon (SGR) was established, but this SGR was unable to robustly replicate transiently. As transient SGRs are a critical tool in the development of DAAs, and in the study of viral resistance, we sought to establish a transient SGR system based on S52. Next-generation sequencing was used to identify putative cultureadaptive substitutions that had arisen during long-term selection of the S52 SGR. A subset of these substitutions was built back into the S52 SGR in the context of a CpG/UpA-low luciferase reporter, with a single point mutation in NS4A conferring the greatest replication capability upon S52. Modification of the innate immune-sensing pathways of Huh7.5 hepatoma cells by expression of the parainfluenza virus type 5 V protein and SEC14L2 resulted in a further enhancement of S52 replication. Furthermore, this transiently replicating SGR showed genotype-specific differences in sensitivity to two clinically relevant NS5A DAAs. In conclusion, we report that a single substitution in NS4A, coupled with host cell modifications, enabled robust levels of transient replication by the GT3 S52 SGR. This system will have beneficial uses in both basic research into the unique aspects of GT3 biology and drug discovery.