Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action - PubMed (original) (raw)
Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action
Jinhong Chang et al. J Virol. 2003 Sep.
Abstract
In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNA-directed mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein. Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.
Figures
FIG. 1.
Representation of three main species of HDV RNA. Also indicated are the genomic and antigenomic ribozymes (cleavage site is shown as a circle) and the open reading frame (ORF) for the δAg (25). At the right is the number of molecules of each RNA per average liver cell for an infected woodchuck and chimpanzee, as previously reported (5). Indicated on the 1,679-nt genomic RNA is the origin for the nucleotide numbering, according to the sequence of Kuo et al. (15).
FIG. 2.
Transfected siRNAs could target HDV mRNA species. Huh7 cells were transfected with one of three plasmids that express an HDV mRNA species. (A) pDL444 (16) expressed an mRNA equivalent to normal HDV mRNA. (B and C) pDL444 was modified to contain 657 nt of extra sequences in the 3′ untranslated region, which led to the transcription of either partial genomic HDV RNA sequences (position 4 to 660) (B) or antigenomic RNA sequences (position 660 to 4) (C). The constructs were cotransfected with a plasmid expressing GFP. After 2 days the total protein was extracted and examined by immunoblotting to detect both delta protein and GFP. Detection and quantitation were with a bioimager (Fuji). The 16 HDV-specific siRNA indicated in the figure were designed and delivered as a cotransfection using Lipofectamine 2000 (Invitrogen). As a negative control (lanes C) we used siRNA against glyceraldehyde-3-phosphate dehydrogenase.
FIG. 3.
Effect of transfected siRNA on the accumulation of replicating HDV RNAs. Cells were transfected with pDL553 (16) to initiate HDV genome replication. siRNA species (at 30 nM) were cotransfected as indicated and as previously described in Fig. 2. At day 2, total RNA was extracted, glyoxalated, and analyzed by electrophoresis in a 1% agarose gel. HDV antigenomic RNA was then detected by Northern assay (A). Similarly, total protein was examined by immunoblotting to detect δAg-S (B). For both panels, bioimager data were subjected to quantitation, expressing the amount of signal detected relative to that obtained for the control transfection. As a negative control (lane C), we used siRNA against endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH); treatment with this siRNA reduced GAPDH mRNA but had no effect on HDV RNA levels (data not shown). Also shown in panel B is the immunoblot assay for expression of GFP, which was cotransfected as a control.
FIG. 4.
Transfected siRNA did not target nonreplicating unit-length HDV genomic RNA. Cells were transfected with pDL542 (16) to achieve the transcription and accumulation of nonreplicating unit-length genomic HDV RNA circles. Cotransfected with this plasmid was either a glyceraldehyde-3-phosphate dehydrogenase control siRNA (lane C) or genomic HDV-specific siRNA (lanes 10 to 13), as indicated at the top of the figure and as previously described for Fig. 2. At day 2, total RNA was extracted, glyoxalated, and analyzed by electrophoresis in a 3% agarose gel. Linear and circular forms of HDV genomic RNA were then detected by Northern assay. After hybridization and quantitation using the bioimager, we deduced the amounts of linear and circular RNAs, as summarized in the histogram.
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