Abstract: Despite recent advances in the treatment of hepatitis C , the quest for pan-genotype , effective , and well-tolerated inhibitors continues .
To facilitate these efforts , it is desirable to have in vitro replication systems for all major HCV genotypes .
However , cell culture replication systems exist for only genotypes 1a , 1b and 2a .
In this study , we generated G418-selectable subgenomic replicons for prototype strains of genotype 3a ( S52 ) and 4a ( ED43 ) .
Production of G418-resistant colonies by S52 and ED43 in Huh-7 . 5 cells required amino acid substitutions S2210I and R2882G respectively ; cell culture adaptive mutations originally reported for genotype 1b replicons .
RNA replication was confirmed by quantitative RT-PCR and detection of viral protein .
Sequencing of multiple independent replicon clones revealed the presence of additional non-synonymous mutations .
Interestingly , all potentially adaptive mutations mapped to the NS3 protein .
These mutations , when introduced back into original constructs , substantially increased colony formation efficiency .
To make these replicons useful for high-throughput screening and evaluation of antiviral compounds , they were modified to express a chimeric fusion protein of firefly luciferase and neomycin phosphotransferase to yield stable replicon-expressing cells .
Using these constructs , the inhibitory effects of IFNbeta , an NS3 protease inhibitor and an NS5B nucleoside polymerase inhibitor were readily detected by monitoring luciferase activity .
In conclusion , we have established functional replicons for HCV genotypes 3a and 4a , important new additions to the armamentarium required to develop inhibitors with a pan-genotype activity .
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