Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remains relatively unexplored as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient transfection format. In the current study, we have evaluated effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCV of different genotypes and evaluated the resulting assay formats for susceptibility measurements to the DAA, Sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and 2a (JFH-1) achieved between 10-100-fold enhancement of replication over wild type post transfection. Removal of CpG/UpA-high neomycin genes in replicons of genotype 3a (S52) and 4a (ED43) enhanced replication but phenotypic effects on altering luciferase gene composition were minimal. Further ten-fold replication enhancement of replicons from all four genotypes was achieved using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect of HCV replication that was reversible by siRNA knockdown of gene expression. Combining these strategies, the 100 to 1000-fold enhancement of replication allowed susceptibility to the RNA polymerase inhibitor, Sofosbuvir, in a transient transfection assay format to be robustly determined for all four genotypes. These methods of replication enhancement provide new tools for the monitoring of susceptibility and resistance of a wide range of HCV genotypes to DAAs.