FIG. 4.

Concentration-dependent effect of HDAg expression on editing at the amber/W site. Human Huh-7 hepatoma cells were transfected with 5 μg of the nonreplicating HDV RNA expression construct pHDVΔx1-NR plus different amounts of the HDAg expression construct pCMV-AgS (NR; lanes 1 to 6) or 5 μg of the replicating HDV RNA expression construct pHDVx1.2-R (R; lanes 7). The amounts of pCMV-AgS cotransfected were as follows: lanes 1, 0.2 μg; lanes 2, 0.05 μg; lanes 3, 0.01 μg; lanes 4, 0.002 μg; and lanes 5, 0.0005 μg. All cells were harvested 5 days posttransfection and analyzed for editing at the amber/W site as described for Fig. 3 (A) as well as for HDAg expression levels (B). (A) ³²P-labeled RT-PCR products, uncut (−) or cut (+) with StyI. Lanes 1 to 6, PCR amplification with primers 7646 and 7647; lanes 7, primers 5414 and 5415. PCR products shown in lanes 1 to 6 contained an additional StyI site that was not affected by editing (Fig. 1 and 3). (B) SDS-PAGE-immunoblot analysis of HDAg expression (see Materials and Methods). The relative HDAg expression levels were determined by radioanalytic imaging with a Packard InstantImager. The numerical value indicated for lane 6 was obtained from a duplicate lane in the same gel that was not next to the strong signal in lane 7.