TABLE 1.

NS5A adaptive mutations of Rep1bNeo^a

Clone	Replicon RNA (106 ge/μg of cell RNA)b	Amino acidc		Colony-forming efficiency (103 colonies/μg of replicon RNA)d
		Position	Change
2	1.7	2177	D→H	500
42	10.1	2197	S→F	79
45	11.9	2197	S→F	87
38	9.5	2199	A→T	167
40	9.2	2200	S→R	53
13	7.4	2202	S→L	94
39	1.8	2202	S→L	154
43	5.3	2202	S→L	52
35	13.1	2204	S→I	72
37	2.6	2204	S→I	147

^aHuh7 cells were transfected with Rep1bNeo, and G418-resistant colonies were established as cell lines.

^bThe 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.

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

^dTo 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.