FIG 3.

Replication capacities of and infectious-virus production by NS3 variants. Mutations such as Q80K, Q80R, Q80R, R155K, Q80K+R155K, Q80R+R155K, D168A, and D168V were introduced into H77S.3/GLuc2A, and the HCV RNAs were transfected into Huh-7.5 cells. The cell culture media were collected and replaced 8 h after transfection and then collected and replaced every 24 h thereafter until 72 h. The GLuc activity in the media collected was measured. (A) Replication capacity. Results for each variant were normalized to the 8-h GLuc activity, represent the means of data for triplicate samples, and are representative of data from multiple experiments. (B) Yield of infectious virus. The media collected 72 h after HCV RNA transfection were used immediately to infect naive Huh-7.5 cells. Seventy-two hours after infection, the numbers of infectious-virus foci were determined by an FFU assay. Data shown here represent the mean FFU per milliliter ± standard deviations from three independent experiments. (C) Impact of each mutation on the replication capacity and yield of infectious virus. The GLuc activity secreted by RNA-transfected cells at 72 h versus 8 h, as shown in panel A, was normalized to that of the wild type and plotted adjacent to the yield of infectious virus from each variant, similarly normalized to that of the wt. The difference between the relative capacities to replicate as RNA and to produce infectious virus is significant by Student's t test. ND, not determined; *, P < 0.05; ***, P < 0.001.