TABLE 1.

NS5A adaptive mutations of Rep1bNeoa

CloneReplicon RNA (106 ge/μg of cell RNA)bAmino acidcColony-forming efficiency (103 colonies/μg of replicon RNA)d
PositionChange
21.72177D→H500
4210.12197S→F79
4511.92197S→F87
389.52199A→T167
409.22200S→R53
137.42202S→L94
391.82202S→L154
435.32202S→L52
3513.12204S→I72
372.62204S→I147
  • a Huh7 cells were transfected with Rep1bNeo, and G418-resistant colonies were established as cell lines.

  • b The replicon copy number was determined by TaqMan RT-PCR and expressed as ge per microgram of cellular RNA. To determine the number of ge per microgram of RNA, cells were plated at a low density and harvested 48 h later, when the cultures were still subconfluent.

  • c Regions of NS3, NS5A, and NS5B were amplified and sequenced to determine adaptive mutations. The amino acids are given in the single-letter code.

  • d To determine the colony-forming efficiency of the adapted replicons, Huh7 cells were transfected with 10 μg of total cell RNA isolated from each cell line, and the colony-forming efficiency in the presence of G418 was determined. The colony-forming efficiency is presented as the number of colonies per microgram of transfected replication RNA that was extrapolated from the estimated number of replicon ge per microgram of cell RNA.