Article Figures & Data
Figures
- FIG. 1.
Schematic diagram of SHDAg and its mutants. Clones d-2 and d-3 were derived from SHDAg with the C terminus fused with the NoLS of the HIV Rev protein.
- FIG. 2.
Subcellular localization of SHDAg and NoLS-fused mutants. Expression plasmids of SHDAg (clone d) or its NoLS-fused constructs (clones d-2 and d-3) were used to transfect Huh7 cells with or without the expression plasmid of the cDNA dimer of the HDV genome (pCDm2G) or antigenome (pCDm2AG) with a two-base deletion in the SHDAg ORF. Transfected cells were fixed 2 days posttransfection and analyzed with an immunofluorescence assay. Composite, merge of SHDAg and nucleolin images.
- FIG. 3.
A heterokaryon assay demonstrated that subcellular localization of the SHDAg-NoLS mutant was confined to the nucleolus. Huh7 cells seeded on coverslips were cotransfected with 3 μg of pCDm2AG as well as 1 μg of the expression plasmid of SHDAg (d+AG) or clone d-2 (d-2+AG). The transfected cells were incubated and then fused with NIH 3T3 cells (see Materials and Methods). Five hours after heterokaryon formation, cells were fixed and analyzed by immunofluorescence microscopy. Frames I to III in panels A and B are merged images of the immunofluorescence results as follows: frames I, SHDAg and human hnRNP C (C1/C2); frames II, SHDAg and DAPI (4′,6′-diamidino-2-phenylindole) staining; and frames III, human hnRNP C (C1/C2) and DAPI staining. White lines mark cell boundaries.
- FIG. 4.
SHDAg-NoLS mutant could support the initiation of HDV antigenomic RNA synthesis but not that of genomic RNA synthesis. (A) In vitro transcribed mRNAs of SHDAg (clone d) or SHDAg-NoLS mutants (clones d-2 and d-3) were cotransfected with HDV genomic RNA to Huh7 cells. Cellular RNAs and proteins were extracted 6 days posttransfection, and the expression of HDV antigenomic RNA and HDAg was analyzed. (B) The same as the experiment as shown in panel A except that antigenomic RNA was used for transfection. G, genomic; AG, antigenomic; P, positive control; N, untransfected cells; α, anti; WB, Western blotting.
- FIG. 5.
Actinomycin D treatment could induce the export of SHDAg and its mutants from the nucleolus. Expression plasmids for SHDAg (clone d) or its NoLS-fused mutant (clone d-2) were used to transfect Huh7 cells with the expression plasmid of the cDNA dimer of the HDV antigenome (AGm) with a two-base deletion in the SHDAg ORF (pCDm2AG). Transfected cells were treated with 0.1 μg/ml actinomycin D at 42 h posttransfection, fixed at 48 h posttransfection, and then analyzed by immunofluorescence assay. Composite I, merged image of the results of HDAg and nucleolin staining; composite II, merged image of the results of HDAg and DAPI (4′,6′-diamidino-2-phenylindole) staining.
- FIG. 6.
Actinomycin D treatment partially rescued the ability of SHDAg-NoLS to facilitate the initiation of HDV genomic RNA (G RNA) synthesis from antigenomic RNA. In vitro transcribed mRNAs of SHDAg (clone d) or clone d-2 were cotransfected with HDV antigenomic RNAs to Huh7 cells. After overnight incubation, the transfected cells were reseeded and treated with 0.1 μg/ml actinomycin D (ActD) for 8 h. Cellular RNA and proteins were extracted 6 days posttransfection, and the expression levels of HDV antigenomic RNA and HDAg were analyzed. P, positive control; N, protein samples from untransfected cells; WB, Western blotting; α, anti.
- FIG. 7.
Interaction of SHDAg-NoLS mutant and cellular RNA Pol II. (A) Huh7 cells transfected with expression plasmids for SHDAg (clone d) or clone d-2 were harvested at 48 h posttransfection. Cells were lysed with radioimmunoprecipitation assay buffer, and the lysates were immunoprecipitated with anti-HDAg antibodies. The precipitants were analyzed. (B) The same experiment as shown in panel A except that the transfected cells were treated with 0.1 μg/ml actinomycin D (ActD) at 42 h posttransfection. P, positive control; H7, untransfected Huh7 cells; WB, Western blotting; IP, immunoprecipitation; α, anti.
