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. 2006 Jul;80(13):6478–6486. doi: 10.1128/JVI.02650-05

FIG. 7.

FIG. 7.

In vitro replication assay. (A and B) Quantification of HDV antigenomic and genomic RNAs after in vitro replication. In vitro replication was performed using genomic RNA from the disrupted HDV particles as a template in the HeLa nuclear lysates. After reaction, cDNAs of antigenomic or genomic RNA (after reverse transcription using primers specific for either antigenomic RNA [A] or genomic RNA [B]) were amplified and detected by real-time quantitative PCR. The RNA was normalized with β-actin mRNA. The same amount of viral particles was incubated with either HeLa nuclear extract or PBS (as a negative control) to carry out the in vitro replication assay. The increase of the HDV RNA amounts after the reaction was determined by the reduction of the PCR cycle numbers as indicated by the arrow between the solid and dotted lines. (C) Effects of α-amanitin or antibodies on in vitro RNA synthesis. The in vitro reactions were carried out using purified HDV particles and HeLa nuclear lysates as in panels A and B. α-Amanitin and antibodies against SL1, polymerase II, PML, SC35, or HDAg were added in the nuclear extracts as indicated and incubated as described above. The amounts of antigenomic and genomic RNA products in each reaction were determined by reverse transcription-PCR as shown in panels A and B. The relative amounts of RNA products in each reaction are presented as the ratios of the products relative to those in the standard reactions. Error bars indicate standard deviations. RNA [B]) were amplified and detected by real-time quantitative PCR. The RNA was normalized with β-actin mRNA. The same amount of viral particles was incubated with either HeLa nuclear extract or PBS (as a negative control) to carry out the in vitro replication assay. The increase of the HDV RNA amounts after the reaction was determined by the reduction of the PCR cycle numbers as indicated by the arrow between the solid and dotted lines. (C) Effects of α-amanitin or antibodies on in vitro RNA synthesis. The in vitro reactions were carried out using purified HDV particles and HeLa nuclear lysates as in panels A and B. α-Amanitin and antibodies against SL1, polymerase II, PML, SC35, or HDAg were added in the nuclear extracts as indicated and incubated as described above. The amounts of antigenomic and genomic RNA products in each reaction were determined by reverse transcription-PCR as shown in panels A and B. The relative amounts of RNA products in each reaction are presented as the ratios of the products relative to those in the standard reactions. Error bars indicate standard deviations.