Predicted secondary structures of wild-type and mutant antigenomic RNA. (A) The wild type and 6×His and 6×His+compensatory (6×His+comp.) mutations. Nine substitutions were made to obtain the 6×His mutant, and seven additional mutations are present in the 6×His+compensatory mutant. (B) Mutants with a stabilized secondary structure upstream and downstream of the amber/W site shown in comparison to the wild-type predicted structure. The mutated nucleotides are shown in shaded letters, and the amber/W codon is boxed. Deleted and inserted nucleotides are indicated by Δ and *, respectively.

Editing of nonreplicating amber/W reporters in HEK293 cells. (A) Western blot analysis with wild-type reporters that express functional HDAg. (B) Western blot analysis with reporters that express RNA-binding-deficient HDAg. In this construct, the natural initiator codon has been mutated to alanine, and position 39 has been mutated to methionine. (C) RT-PCR analysis followed by StyI digestion of reporters that do not express HDAg. Lanes: 1, wild-type reporter; 2, 6×His reporter; 3, 6×His+compensatory reporter; 4, upstream reporter; 5, downstream reporter; 6, upstream-downstream combination reporter. The 479-nucleotide unedited RT-PCR product is indicated by a •, while the 393-nucleotide edited product is indicated by *. The percentage of editing was calculated as described in Materials and Methods. All abbreviations are as described in the legend to Fig. 2.

Replication and HDAg expression of the 6×His and 6×His+compensatory (6×His+comp.) mutants. Northern blot analysis probing for genomic RNA (A) and antigenomic RNA (B) and Western blot analysis for HDAg (C) with samples obtained from HuH7 cells transiently transfected with cDNA that expressed circular genomic RNA and harvested at 4, 8, and 12 days posttransfection is shown. Normalized expression values of genomic RNA, antigenomic RNA, and HDAg (% relative genome, % relative antigenome, and % relative total HDAg, respectively), where the highest value among the wild-type samples in each set is defined as 100%, are shown. The percentage of HDAg-L was calculated by dividing the HDAg-L signal by the total HDAg signal and multiplying the value by 100. Levels too low to quantify are indicated by −. PT, posttransfection; L, HDAg-L; S, HDAg-S.

Time course analysis of HDV replication, amber/W editing, and HDAg-L expression, with wild-type HDV and the downstream mutant, in the presence and absence of the Q214Stop mutation. HuH7 cells transiently transfected with cDNA that expresses circular genomic RNA were harvested at days 2, 3, 4, 5, 6, and 9, and then the total RNA and protein were analyzed by Northern blot probing for genomic RNA (A), Northern blot probing for antigenomic RNA (B), Western blot probing for HDAg (C), StyI digestion of RT-PCR products that resulted from reverse transcription initiated with a primer complementary to genomic RNA (D), and StyI digestion of RT-PCR products that resulted from reverse transcription initiated with a primer complementary to antigenomic RNA (E). All calculations and abbreviations are as described in the legends to Fig. 2 and 3. Quantification of RNA and protein samples is reported to three significant figures to reflect the precision of the phosphoimager or software used to calculate these values. The amount of total RNA loaded for the Northern blots (A and B) were quantified from ethidium bromide staining of the agarose gels and were as follows (samples are from day 2 to day 9, respectively; the highest sample was given a value of 10). (A) Wild type, 8.72, 9.53, 8.31, 8.98, 8.90, and 7.19; downstream (down) mutant, 4.29, 8.01, 8.42, 8.56, 9.78, and 10; wild-type Q214Stop, 4.33, 5.98, 7.24, 8.53, 7.83, and 8.34; downstream Q214Stop mutant, 5.23, 8.69, 8.52, 8.07, 9.88, and 9.67. (B) Wild type, 7.80, 9.32, 7.47, 8.57, 9.12, and 6.67; downstream mutant, 3.95, 7.32, 6.83, 6.40, 7.73, and 8.59; wild-type Q214Stop, 4.08, 5.57, 5.95, 7.65, 7.36, and 8.72; downstream Q214Stop mutant, 4.99, 9.60, 7.73, 8.41, 9.24, and 10. The amount of total protein loaded into each lane for the Western blots (C) were quantified after Ponceau S staining and were as follows (samples are from day 2 to day 9, respectively; the highest sample was given a value of 10): wild type, 6.18, 6.51, 6.64, 7.76, 8.68, and 9.67; downstream mutant, 7.89, 9.34, 9.21, 10, 9.57, and 8.49; wild-type Q214Stop, 4.95, 6.53, 7.24, 8.09, 8.68, and 9.28; downstream Q214Stop mutant, 7.63, 7.83, 7.57, 8.49, 8.22, and 6.84.

Time course analysis of HDV replication, amber/W editing, and HDAg-L expression, with wild-type HDV and the downstream mutant, in the presence and absence of FTI (+FTI and −FTI, respectively). HuH7 cells transiently transfected with cDNA that expresses circular genomic RNA were harvested at days 2, 3, 4, 5, 6, and 9, and then the total RNA and protein were analyzed by Northern blot probing for genomic RNA (A), Northern blot probing for antigenomic RNA (B), Western blot probing for HDAg (C), StyI digestion of RT-PCR products that resulted from reverse transcription initiated with a primer complementary to genomic RNA (D), and StyI digestion of RT-PCR products that resulted from reverse transcription initiated with a primer complementary to antigenomic RNA (E). All calculations and abbreviations are as described in the legends to Fig. 2 and 3. Quantification of RNA and protein samples is reported to three significant figures to reflect the precision of the phosphoimager or software used to calculate these values. The amount of total RNA loaded and transferred for the Northern assay (A and B) were quantified from the blots on which ethidium bromide-stained samples were transferred and were as follows (samples were from day 2 to day 9, respectively; the highest sample was given a value of 10). (A) Wild type (−FTI), 1.62, 3.74, 4.51, 1.78, 5.72, and 2.75; downstream (down) mutant (−FTI), 1.18, 4.56, 10, 5.00, 2.86, and 3.16; wild type (+FTI), 4.31, 5.30, 4.16, 5.57, 4.44, and 4.51; downstream (+FTI) mutant, 2.36, 8.24, 7.87, 5.69, 2.69, and 3.33. (B) Wild type (−FTI), 1.15, 2.05, 4.12, 3.94, 6.30, and 4.63; downstream (−FTI), 2.16, 7.28, 10, 6.46, 3.64, and 4.95; wild type (+FTI), 1.27, 9.48, 8.62, 4.25, 2.40, and 5.34; downstream (+FTI) mutant, 1.00, 4.97, 7.35, 4.45, 3.24, and 4.25 (B). The amounts of total protein loaded into each lane for the Western blots (C) were quantified after Ponceau S staining and are as follows (samples are from day 2 to day 9, respectively; the highest sample was given a value of 10): wild type (−FTI), 5.31, 5.57, 7.59, 8.00, 8.55, and 9.08; downstream (−FTI) mutant, 4.28, 5.66, 7.15, 8.77, 10, and 9.52; wild type (+FTI), 3.91, 5.09, 6.67, 7.94, 7.59, 8.20; downstream (+FTI) mutant, 3.14, 4.65, 5.26, 6.75, 7.76, 7.15.

Time course analysis of HDV replication, amber/W editing, and HDAg-L expression, in the presence and absence of the Q214Stop mutation, with and without exogenous ADAR1-S expression. HuH7 cells were transiently transfected with cDNA expressing either wild-type or Q214Stop mutant circular genomic RNA, and the total RNA and protein were analyzed following harvest at days 2, 3, 4, 5, 6, and 9. (A) Northern analysis of genomic RNA. (B) Northern analysis of antigenomic genomic RNA. (C) Western analysis of HDAg. (D) Western analysis of ADAR1-S-HA. (E) StyI digestion of RT-PCR products that resulted from reverse transcription initiated with a primer complementary to genomic RNA. (F) StyI digestion of RT-PCR products that resulted from reverse transcription initiated with a primer complementary to antigenomic RNA. All calculations and abbreviations are as described in the legends to Fig. 2 and 3. Quantification of RNA and protein samples is reported to three significant figures to reflect the precision of the phosphoimager or software used to calculate these values. The amount of total RNA loaded and transferred for the Northern assay (A and B) was quantified from the blots on which ethidium bromide-stained samples were transferred and were as follows (samples are from day 2 to day 9, respectively; the highest sample was given a value of 10). (A) Wild type without ADAR1-S-HA (−ADAR1-S-HA), 1.96, 1.67, 5.93, 4.74, 5.51, and 6.93; wild type with ADAR1-S-HA (+ADAR1-S-HA), 6.61, 3.28, 5.70, 7.11, 4.91, and 7.53; wild- type Q214Stop without ADAR1-S-HA (−ADAR1-S-HA), 3.53, 4.69, 7.26, 8.72, 7.86, and 8.16; wild-type Q214Stop with ADAR1-S-HA (+ADAR1-S-HA), 4.56, 5.88, 6.84, 8.79, 10, and 8.83. (B) Wild type ADAR1-S-HA (−ADAR1-S-HA), 3.51, 2.98, 10, 5.36, 7.35, and 8.27; wild type with ADAR1-S-HA (+ADAR1-S-HA), 5.47, 3.85, 8.51, 7.89, 3.84, and 8.43; wild-type Q214Stop without ADAR1-S-HA (−ADAR-S-HA), 5.33, 6.18, 6.31, 7.40, 7.61, and 6.68; wild-type Q214Stop with ADAR1-S-HA (+ADAR1-S-HA), 3.84, 4.34, 6.41, 5.56, 8.50, and 8.90. The amounts of total protein loaded in each lane for the Western blots (C) were quantified after Ponceau S staining and are as follows (samples are from day 2 to day9; the highest sample was given a value of 10). (A) Wild type without ADAR1-S-HA (−ADAR1-S-HA), 3.02, 2.68, 3.30, 3.86, 4.47, and 6.15; wild type with ADAR1-S-HA (+ADAR1-S-HA), 5.03, 6.27, 7.82, 8.39, 8.09, and 8.72; wild-type Q214Stop without ADAR1-S-HA, 5.46, 6.29, 7.28, 8.35, 8.69, and 10; wild-type Q214Stop with ADAR1-S-HA, 6.10, 6.57, 7.28, 7.43, 7.00, and 7.88.