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. 1998 Apr;18(4):1919-26.
doi: 10.1128/MCB.18.4.1919.

Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen

A G Polson  1 , H L Ley 3rd, B L Bass, J L Casey
Affiliations

Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen

A G Polson et al. Mol Cell Biol. 1998 Apr.

Abstract

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.

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Figures

FIG. 1
FIG. 1
(A) Schematic diagram of HDV cDNA constructs, RNAs, and regions amplified by the PCR. Left: thick straight bar, HDV cDNA; thin line, plasmid sequences; dashed line pHDVΔx1-NR, sequences deleted between ApaI sites (9). Direction of transcription initiated by the CMV promoter is indicated by arrows. Right: oval shapes with heavy lines, expected HDV RNA species; (+), antigenomic sense; (−), genomic sense. The location of the amber/W site is indicated by an asterisk; the genomic and antigenomic ribozyme cleavage sites (38, 41) are indicated by solid and open triangles, respectively. Small open boxes indicate locations of wild-type (pHDVx1.2-R) and mutated (pHDVΔx1-NR) polyadenylation sites. Solid bars indicate expected PCR products; arrows mark primers A (5415), B (5414), C (7646), and D (7647). Primers 7646 and 7647 correspond to sequences present only in the RNA derived from the plasmid pHDVΔx1-NR. There is a single StyI site (indicated in parentheses) in cDNAs amplified with primers 5414 and 5415 and derived from edited RNA. Primers 7646 and 7647 yield cDNAs with the same editing-sensitive site, plus an existing site that is unaffected by editing. Nonreplicating RNA is produced in cells transfected with the deletion construct pHDVΔx1-NR, which contains an ∼514-nt internal deletion and a site-directed mutation at the polyadenylation signal site. The region to be deleted is indicated by an open segment for construct pHDVx1.2-R and its derived RNAs and by a dashed line for the construct pHDVΔx1-NR. Drawings are not necessarily to scale. Horizontal gray arrows indicate transcription of RNAs from plasmid DNA templates; vertical gray arrows indicate RNA template-driven transcription occurring during HDV RNA replication. (B) Sequence of the 358-nt region analyzed by cloning and sequencing after amplification with primers 5414 and 5415. Sequence shown is antigenomic sense; numbering corresponds to the genomic RNA (39). The amber/W site is indicated by an asterisk.
FIG. 2
FIG. 2
The percentage of HDV cDNA clones with A→G transitions at specific sites within the region from nt 907 to 1264 is plotted for cDNA populations derived from replicating or nonreplicating HDV RNAs. cDNA populations were derived from replicating HDV RNA harvested 13 days after Huh-7 cells were transfected with plasmid pHDVx1.2-R (84 clones) (A) and nonreplicating HDV RNA harvested 6 days after Huh-7 cells were transfected with the nonreplicating HDV RNA expression plasmid pHDVΔx1-NR (97 clones) (B). Sequence numbering refers to the genomic strand (39). Numbers in italics indicate sequence positions that exhibited A→G transitions.
FIG. 3
FIG. 3
Effect of HDAg expression on editing at the amber/W site. Human Huh-7 hepatoma cells were transfected with the nonreplicating HDV RNA expression construct pHDVΔx1-NR plus equivalent amounts of the following constructs: pCMV-MCS3, the expression vector alone; pCMV-AgS, which expresses HDAg-S; pCMV-AgS(fs), which expresses HDAg mRNA with a stop codon and frameshift at codon 7; and pCMV-AgSΔStuSma, which expresses HDAg containing an internal deletion. All cells were harvested 5 days posttransfection and analyzed for editing at the amber/W site by the appearance of a StyI restriction site (9, 32). The autoradiogram shows 32P-labeled RT-PCR products, amplified with primers 7646 and 7647, uncut (−) or cut (+) with StyI. PCR products contained an additional StyI site that was not affected by editing (Fig. 1).
FIG. 4
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.
FIG. 5
FIG. 5
Changes in editing associated with altered HDAg levels do not correlate with altered RNA levels. Cells were cotransfected with various amounts of the HDAg expression construct pCMV-AgS and either 5 μg (columns A) or 0.5 μg (columns B) of the nonreplicating HDV RNA expression construct pHDVΔx1-NR. Numbers 1 to 6 refer to the same amounts of pCMV-AgS transfected as for Fig. 3. For each cotransfection, levels of amber/W editing were determined (bar height). In addition, cellular levels of HDV RNA were determined by radioanalytic imaging with a Packard InstantImager of Northern blots hybridized with genomic-sense HDV RNA; the number above each bar indicates the amount of HDV antigenomic RNA relative to that in column 1A. Cell transfection, RNA harvesting, and editing analysis were as for Fig. 3.
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