1 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 112
2 Department of Life Science, National Yang-Ming University, Taipei, Taiwan 112
3 Department of Life Science, School of Medicine, Chang Gung University, TaoYun, Taiwan 333
Correspondence
Szecheng J. Lo
losj{at}mail.cgu.edu.tw
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ABSTRACT |
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Present address: Department of Radiological Technology, Yuanpei University of Science and Technology, Hsinchu, Taiwan 300.
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INTRODUCTION |
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Two forms of HDAg, small and large (SDAg and LDAg, respectively), are encoded by a single open reading frame (ORF) of antigenomic RNA (Lai, 1995). The SDAg contains 195 amino acids while the LDAg contains 19 additional amino acids at its C terminus (Taylor, 1990
). The SDAg trans-activates HDV RNA replication while the LDAg suppresses this process and interacts with HBsAgs to assemble mature virions (Chao et al., 1990
; Chen et al., 1993
; Ryu et al., 1992
; for a review see Lai, 1995
, and references therein). The sequential appearance of SDAg and LDAg is achieved via an RNA editing event in which the amber stop codon (UAG) for the SDAg ORF is converted to a tryptophan codon (UGG) (Casey & Gerin, 1995
; Polson et al., 1996
, 1998
). Thus, RNA editing is essential to the HDV life cycle since without it there would be no LDAg production and subsequently no mature HDV formation.
HDV RNA editing occurs on the rod-structured antigenomic RNA (Casey & Gerin, 1995) by host enzymes called ADARs (adenosine deaminases that act on double-stranded RNA) (Wong & Lazinski, 2002
). Three members of the ADAR gene family have been identified and characterized in recent years (Bass, 2002
; Gott & Emeson, 2000
). ADAR1 and ADAR2 are capable of editing adenosines in double-stranded RNA of mammalian cells (Melcher et al., 1996
; Seeburg et al., 1998
), while ADAR3 was found to play a regulatory role in RNA editing (Chen et al., 2000
). The ADAR1 homologues from Xenopus laevis, HeLa nuclear extract and Drosophila melanogaster embryo nuclear extract have been demonstrated to edit HDV RNA efficiently (Polson et al., 1996
; Casey & Gerin, 1995
). Recently, overexpression of human ADAR1 and ADAR2 was shown to increase the editing efficiency of a reporter mRNA in transfected HEK293 cells and to inhibit HDV RNA replication in HuH-7 cells (Sato et al., 2001
; Jayan & Casey, 2002
).
Although the host enzymes and the RNA substrate structure of HDV RNA editing have been well studied (Wong et al., 2001; Sato et al., 2001
), the involvement of other host factors and the nuclear locations of the event remain largely unknown. Previously, we demonstrated that GFP fusion proteins are powerful tools in understanding the locations and the functions of the components involved (Shih & Lo, 2001
). In this study, we generated three new GFP fusion proteins containing various lengths of HDAg and demonstrated that one of these fusion proteins, designated D(188)-GFP, inhibited HDV RNA editing. This fusion protein allowed us to investigate the locations of HDV RNA editing.
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METHODS |
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Cell culture and transfection of HuH-7 and HeLa cells.
Two human cell lines were used for plasmid transfection in this study, HuH-7, a well-differentiated human hepatoma cell line, and HeLa, an epitheloid carcinoma cell line. Both cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % foetal bovine serum, penicillin (100 IU ml-1), streptomycin (100 µg ml-1), Fungizone (50 µg ml-1) and 2 mM L-glutamine and grown at 37 °C under 5 % CO2. Plasmids in a supercoiled form were obtained using the Qiagen Plasmid Maxi Kit and used for transfection. HeLa and HuH-7 cells were grown in 10 cm Petri dishes to 60 % confluence and transfected with 20 µg of the indicated plasmids by the calcium phosphate/DNA precipitation method (Graham & van der Eb, 1973). To obtain cells that permanently expressed the plasmids, the transfected HeLa cells were scraped off, replated into 10 cm Petri dishes and selected with G418. Single colonies were generated by limiting dilution cloning as described previously (Shih & Lo, 2001
). HeLa cells expressing various GFP fusion proteins were designated HeLa-D(188)-GFP, HeLa-D(1163)-GFP, HeLa-SD-GFP and HeLa-GFP-LD(31214).
Fluorescent microscopy.
Transiently or permanently transfected cells that expressed GFP-SD or other variants were cultured on 22x22 mm coverslips. Cells were fixed using 4 % paraformaldehyde in PBS for 30 min at room temperature, probed with anti-HA (human influenza virus haemagglutinin), anti-SC-35 (a splicing factor) or anti-PML (premyelocytic leukaemia antigen) antibodies and visualized with a secondary goat anti-mouse rhodamine-conjugated antibody. Finally, cells were stained with Hoechst 33258, mounted on glass slides using mounting solution and visualized using a fluorescent microscope (Olympus B-Max 60) or a confocal microscope (Leitze).
Western blotting.
To identify the presence of various antigens, protein samples obtained from whole-cell extracts or sucrose-gradient fractions were subjected to Western blot analyses. For sucrose-gradient fractionation, nuclear extracts were obtained from cells expressing various GFP fusion proteins. Samples were layered on to a 1050 % (w/v) discontinuous sucrose gradient and subjected to centrifugation at 38 000 r.p.m. in an SW41 rotor for 16 h at 4 °C. The gradients were fractionated into 18 tubes, each containing 0·6 ml. The density of each fraction was determined from the refractive index using a refractometer. Protein samples were separated by SDS-PAGE and electrotransferred on to PVDF membranes (Immobilon-P; Millipore) as described by Towbin et al. (1979). The membranes were incubated with 5 % non-fat milk followed by human anti-HDAg sera (Yeh et al., 1996
), anti-GFP or anti-HA primary antibodies. After incubation with horseradish peroxidase-conjugated anti-human or anti-mouse secondary antibody, the blot was developed by enhanced chemiluminescence (ECL) using a commercial kit (Amersham Japan) or developed using 4-choloro-1-naphthol as the substrate. The intensity of protein bands was quantified by ImageQant TL software (Amersham Biosciences).
Analysis of RNA editing efficiency by RT-PCR.
Total RNA was prepared from wild-type or GFP-expressing HeLa cells that had been transfected with pCMVDag2 using a REzol C&T RNA extraction kit (PROtech Technologies). RNA samples were treated with DNase and either treated with or without RNase. Samples were then subjected to RT-PCR using PCR primers 877 (5'-GAGGTGGAGATGCC-3', genomic strand) and T37A (5'-CCTCCGGAAGACAAA-3', antigenomic strand). The effectiveness of DNase treatment was confirmed by the absence of PCR products after PCR amplification in the absence of reverse transcription. The PCR products were analysed by NcoI restriction enzyme digestion, which generated two DNA fragments (509 and 134 bp). The RT-PCR products from the pMTLD-transfected cells served as the standard index because pMTLD contains the NcoI site and can express LDAg without RNA editing (Hu et al., 1996).
In situ hybridization.
HeLa cells transfected with pCMVDag2 were grown directly on coverslips to 60 % confluency, washed once in PBS and fixed for 20 min at room temperature in 4 % formaldehyde. After penetration with ice-cold acetone for 3 min, cells were dehydrated using increasing concentrations of ethanol for 5 min at room temperature in 2x SSC with 50 % formamide. Cells were hybridized overnight at 37 °C in hybridization buffer (10 % dextran sulfate, 2 mM vanadyl-ribonucleoside complex, 50 µg salmon sperm DNA, 2x SSC, 50 % formamide) with 20 ng HDV DNA probe labelled with digoxigenin or biotin. After hybridization, cells were washed twice at 45 °C for 30 min in 2x SSC, 50 % formamide and once in 0·1x SSC, 0·1 % Tween-20. Digoxigenin-labelled probes were then detected with mouse anti-digoxigenin primary antibody and goat anti-mouse secondary antibody conjugated to rhodamine. Biotin-labelled probes were detected directly with avidin conjugated to rhodamine. For the fluorescence detection, cells were visualized with the Olympus B-Max 60 fluorescent microscope or the Leitze confocal microscope and photographed.
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RESULTS |
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HDV RNA editing is inhibited by the D(188)-GFP fusion protein
To examine possible correlation between the specific localization of GFP fusion proteins and their biological activities, we transfected pSVL-d2g into HeLa-SD-GFP, HeLa-D(1163)-GFP or HeLa-D(188)-GFP cells. Because pSVL-d2g expresses the replication-competent 1·7 kb HDV cDNA dimer from the SV40 late promoter, HDV replication could be assessed. Similar experiments were performed in cells expressing GFP-LD, GFP-LDM and GFP-LD(31214). At 4 days post-transfection, cells were harvested and lysed for analysis of HDV RNA and HDAg. Northern blot results showed that HDV RNA replication was completely inhibited in HeLa-SD-GFP, HeLa-D(1163)-GFP, HeLa-GFP-LD and HeLa-GFP-LDM cells, but not in cells expressing D(188)-GFP or GFP-LD(31214) (data not shown). To determine whether HDV replication was related to the presence of GFP fusion proteins, Western blots were performed, (Fig. 3A). The gel migration patterns of GFP-LD(31214), SD-GFP and D(188)-GFP were as expected. The inhibition of HDV RNA replication by SD-GFP appeared to result from the inhibition of authentic HDAg expression as detected by Western blot (Fig. 3B
, lane 3). This result is similar to the previous finding that cells expressing GFP-LD and GFP-LDM allow neither HDAg expression nor HDV RNA replication (Shih & Lo, 2001
). As expected, both SDAg and LDAg were detected in parental HeLa cells and in HeLa-GFP-LD(31214) cells transfected with the pSVL-d2g plasmid, in which HDV RNA replication occurred (Fig. 3B
, lanes 1 and 4). Although HDV RNA replication also occurred in the HeLa-D(188)-GFP cells transfected with pSVL-d2g, only SDAg was detected (Fig. 3B
, lane 2). With normal levels of SDAg but no LDAg expression in the HeLa-D(188)-GFP cells transfected with pSVL-d2g, we hypothesized that D(188)-GFP might have the ability to inhibit HDV RNA editing.
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DISCUSSION |
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Previously, we showed that a GFP fusion of LDAg allowed study of LDAg movement inside the nucleus (Shih & Lo, 2001). In this study, we further demonstrated the usefulness of GFP fusion proteins in localization, as well as in inhibiting HDV RNA replication. Unlike the authentic SDAg, which can promote HDV replication, a full-length SDAg fused to GFP, SD-GFP, suppressed both SDAg and LDAg expression and resulted in no HDV replication (Fig. 3
, lane 3). This was possible through an interaction between SDAg and SD-GFP to antagonize the transactivation ability of SDAg for replication, similar to a dominant-negative effect (Chao et al., 1990
; Dridi et al., 2003
). Another possibility is that SD-GFP occupied the co-factors required for SDAg function in the nucleolus. This supposition was supported by the finding that three GFP fusion proteins that retain the coiled-coil domain, D(1163)-GFP, GFP-LD and GFP-LDM, localized to the nucleolus and inhibited HDV replication (Fig. 2A and F
ig. 3; Fig. 1
and Fig. 5
in Shih & Lo, 2001
). In contrast, GFP-LD(31214), which lacks the coiled-coil domain, was dispersed in the nucleoplasm and could not suppress HDAg production or HDV replication (Fig. 3
, lane 4). The observation that D(188)-GFP localizes in speckles suggests that D(188)-GFP is unlikely to form a complex with SDAg in the nucleolus, thus allowing HDV RNA replication to occur, although it retains the coiled-coil domain.
Nevertheless, the location and function of D(188)-GFP allow us to speculate about the site of HDV RNA editing. Based on the observation that ADAR is found in the nucleolus (Fig. 6), one could hypothesize that HDV RNA editing takes place in the nucleolus. If this is true, HDV RNA editing requires other nuclear factors in addition to ADAR and these factors may be trapped by D(188)-GFP in the speckles. Alternatively, HDV RNA editing could take place in D(188)-GFP-occupied speckles, but ADAR is not able to contact its substrate. The evidence that HDV RNA and D(188)-GFP co-localize in speckles (Fig. 8
) supports this hypothesis. Although we favour the second hypothesis, one could still argue against the hypothesis that ADAR is located solely in the nucleolus. To correlate with the fact that the small amount of HDV RNA is edited, one might suggest that an undetectable amount of ADAR could shuttle between speckles and the nucleolus, allowing HDV RNA editing to occur. Using the photobleaching technique, a recent study has demonstrated that ADAR2 can shuttle between the nucleolus and nucleoplasm and, furthermore, has suggested that the nucleoplasm is the site of RNA editing (Sansam et al., 2003
). Although their demonstration supports our hypothesis that SC-35 is the site of HDV RNA editing, this hypothesis requires further study. Compared with the previous finding that HDV RNA editing takes place in the nucleus (Wong & Lazinski, 2002
), we have narrowed the possible locations to either the nucleolus or speckles.
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ACKNOWLEDGEMENTS |
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Received 17 September 2003;
accepted 24 November 2003.