1 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 112
2 Department of Life Science, School of Medicine, Chang Gung University, TaoYun, Taiwan 333
Correspondence
Szecheng 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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Because both SDAg and LDAg lack any enzyme activity, HDV genome and antigenome replication require host RNA polymerases (Modahl et al., 2000; Chang & Taylor, 2002
; MacNaughton et al., 2002
). HDV genome and antigenome replication occur via a rolling-circle method, which produces multiple copies of HDV RNA in linear form (Lai, 1995
; Taylor, 2003
). Ribozymes in both the HDV genome and antigenome then self-cleave the linear RNA into single units, which are ligated into a circular form (Lai, 1995
; Taylor, 2003
). During the RNA replication cycle, host enzymes called ADARs (adenosine deaminases that act on double-stranded RNA) edit a portion of HDV RNA to convert the amber stop codon (UAG) of SDAg to a tryptophan codon (UGG), which results in the production of LDAg (Casey & Gerin, 1995
; Sato et al., 2001
; Jayan & Casey, 2002
). Thereafter, LDAg inhibits HDV RNA replication and, together with SDAg and the HDV genome, assembles into a ribonucleoprotein complex, which is then transported to the cytoplasm to form a mature virion with HBsAgs. Although HDAgs have been identified in many subcellular structures, such as the nucleolus, nuclear speckles and the Golgi apparatus (Xia et al., 1992
; Wu et al., 1992
; Bichko & Taylor, 1996
; Bell et al., 2000
), the correlation of the above events with cellular locations remains largely unknown.
Many reports have revealed that post-translational modifications of HDAgs are important for the execution of HDAg function; for example isoprenylation of LDAg is crucial for its interaction with HBsAg for secretion (Glenn et al., 1992; Hwang & Lai, 1993
; Sheu et al., 1996
). Although both SDAg and LDAg are phosphoproteins, the SDAg can be phosphorylated at both serine and threonine, while the LDAg can be phosphorylated only at serine (Chang et al., 1988
; Mu et al., 1999
), which may account for their distinct biological roles. The serine residues at positions 2 and 177 of SDAg modulate HDV RNA replication but have no significant role in subviral particle formation (Yeh et al., 1996
; Yeh & Lee, 1998
; Mu et al., 1999
, 2001
). These results suggest that post-translational modifications of HDAgs may play a role in targeting HDAgs to a specific location.
Previously, we used a green fluorescent protein fused to LDAg (GFPLD) to demonstrate that translocation of GFPLD from the nucleolus to SC-35 speckles could be induced by treatment of the casein kinase II inhibitor dichlororibofuranosyl benzimidazole (DRB) (Shih & Lo, 2001). However, direct evidence showing that the dephosphorylated form of GFPLD favours residence at the SC-35 speckles is lacking. In this study, we constructed GFPLD derivatives containing a single mutation at Ser-2 or Ser-123, or a double mutation at both sites, and demonstrated that Ser-123, but not Ser-2, of GFPLD plays a role in its targeting to SC-35 speckles.
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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 in this study; one was a well-differentiated human hepatoma cell line, HuH-7, and the other was an epitheloid carcinoma cell line, HeLa. Both cell types were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum, penicillin (100 IU ml1), streptomycin (100 µg ml1), Fungizone (50 µg ml1) 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 then used for transfection. Cells at 60 % confluence in a 10 cm Petri dish were transfected with 10 µg of the indicated plasmids by the calcium phosphate/DNA precipitation method (Graham & van der Eb, 1973) or by adding lipofectamine (Invitrogen). For visualization of the various expression patterns of HDAg encoded by pSVL-d2g, HuH-7 cells at 24 h post-transfection were scrapped off and reseeded onto cover slips and cultured for an additional 310 days.
Fluorescence microscopy.
To visualize HDAg expression patterns, pSVLd2g-, pMTLD- or pMTSD-transfected cells were fixed with 4 % paraformaldehyde/PBS for 30 min at room temperature, then stained with anti-HDAg antibody followed by secondary goat anti-rabbit antibody conjugated to FITC. Cells expressing the various GFP fusion proteins (GFPLD, GFPLDS2A, GFPLDS123A or GFPLDS2/123A) were fixed and stained with anti-SC35 antibody followed by a secondary antibody conjugated to rhodamine. Cells co-transfected with pMTS and the various plasmids expressing GFP fusion proteins were fixed and stained with anti-HBs antibody followed by a secondary antibody conjugated to rhodamine. In parallel, the cells were stained with Hoechst 33258 to visualize the nucleus. Finally, cells were mounted on glass slides with mounting solution and examined using a fluorescence microscope (Olympus B-Max 60 or Leica DMIRBE) or a confocal microscope (Leica DMRE) using an FITC or rhodamine filter.
Western blotting.
To detect the secretion efficiency of various GFP fusion proteins with HBsAg, HuH-7 cells were co-transfected with pMTS and the various plasmids expressing the GFP fusion proteins GFPLD, GFPLDM, GFPLDS2A and GFPLDS123A. Growth media from 3 and 6 days post-transfection were collected and the secreted empty viral particles (EVPs) were concentrated by ultracentrifugation. Protein samples from secreted EVPs and the total lysates of cells cultured for 6 days post-transfected were separated by SDS-PAGE and electrotransferred onto PVDF membranes. The membrane containing total cell lysate was incubated with anti-HDAg antibody and anti-actin antibody (as a reference for the amount of protein loaded) in the presence of 5 % non-fat milk, and the membrane containing EVPs was probed with anti-HDAg antibody and anti-HBsAg (as a reference for the amount of protein loaded). After incubation with the secondary antibody conjugated to horseradish peroxidase, the blots were developed by enhanced chemiluminescence using a commercial kit (Amersham). The intensity of protein bands was quantified by the program ImageQuant TL software (Amersham BioSciences). The secretion ratio of GFPLD fusion proteins to HBsAg was determined.
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RESULTS AND DISCUSSION |
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Results of immunofluorescence microscopy revealed that HDAg appeared in many distribution patterns but was uniform in paired cells that had been cultured for 35 days (Fig. 1A) and in clusters of cells that had been cultured for 510 days (Fig. 1B
). HDAg appearing in the nucleolus and in various sizes of nuclear speckles (Fig. 1A, C and D
) was commonly seen in both the short- and long-term cultured cells, similar to previous reports (Xia et al., 1992
; Chang et al., 1992
; Wu et al., 1992
). In contrast, the presence of HDAg in the perinuclear region containing the Golgi apparatus was not seen in short-term cultured cells but was frequently observed in cells cultured for longer periods (Fig. 1B
, indicated by arrows; Bichko & Taylor, 1996
). This uniform staining pattern of HDAg did not occur in adjacent or clustered positively stained cells that had been transfected with plasmids expressing only LDAg or SDAg and cultured for more than 10 days (data not shown).
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Ser-123 mutated proteins of GFPLD are preferentially located in the SC-35 speckles
To confirm that CKII is regulating GFPLD phosphorylation and that dephosphorylation of GFPLD is responsible for its targeting to SC-35 speckles, we constructed three GFPLD-derived mutants, designated GFPLDS2A, GFPLDS123A and GFPLDS2/123A, and analysed their localization in transfected cells. Serine residues at positions 2 and 123 of LDAg are known target sites of CKII; thus, if the three mutants were located in the SC-35 speckles in the absence of DRB, then dephosphorylation of GFPLD must be required for its localization to the SC-35 speckles. On the other hand, if the three mutated proteins were distributed in the same way as the wild-type GFPLD, then the effect of DRB must not be on GFPLD dephosphorylation but by another mechanism that allows targeting to the SC-35 speckles.
Results obtained from fluorescence microscopy of cells expressing the four different GFP fusion proteins are shown in Fig. 2. When the distribution of GFPLD, GFPLDS2A, GFPLDS123A and GFPLDS2/123A was examined in cells 24, 48 and 72 h post-transfection, it was shown that the longer the post-transfection time of the cells, the higher the percentage of cells expressing GFPLDS123A and GFPLDS2/123A with a green fluorescent signal in the speckles (Fig. 2
GI and JL). In contrast, a higher percentage of cells expressing GFPLD and GFPLDS2A had a signal in the nucleolus, although some cells had a signal only in the speckles (Fig. 2
AC and DF). To quantify the percentage of cells showing these different distributions of GFP fusion proteins, we classified the patterns into three types: type I, with protein distribution mainly in the nucleolus; type II, with protein distributed in both the nucleolus and the speckles; and type III, with protein found only in the speckles (also see Fig. 2
legend). Three independent experiments were performed to transfect the four different plasmids into HeLa cells. Between 200 and 700 cells with green fluorescent signals were randomly selected, their patterns classified and the results summarized.
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As shown in Fig. 4, when cells expressing GFPLDS2A, GFPLDS123A and GFPLDS2/123A were treated with DRB, all fusion proteins were located in SC-35 speckles similar to GFPLD, as shown previously (Shih & Lo, 2001
). These results supported the idea that other nuclear factors may be required, in addition to Ser-123 in its non-phosphorylated form, for targeting to SC-35 speckles. This supposition is further supported by the fact that GFP fusion proteins need to associate with other factors to form a complex with a density above 1·62 g ml1 such as appears in the nucleolus (Huang et al., 2001
; Shih et al., 2004
). Modification of GFPLD might change its conformation and this could result in its association with nuclear factors that have not yet been identified. Nevertheless, the current results showed that only the Ser-123 mutant played a role in modulating the distribution of the GFP fusion protein in the nucleolus or SC-35 speckles, although CKII catalyses phosphorylation of both Ser-2 and Ser-123. Since Ser-123 in a non-phosphorylated state favours GFPLD localization to the SC-35 speckles, the question then arises as to which phosphatase is responsible for dephosphorylation of Ser-123. This is a topic that needs further exploration.
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The translocation pathway of GFPLD and its co-localization with HBsAg
Although this study did not include a dynamic study of GFPLD movement from the nucleus to the cytoplasm, it should be possible to identify the sequential process of GFPLD distribution based on the double-positive cells at seen at 24, 48 and 72 h post-transfection. Representative images were selected, as shown in Fig. 5. In the cells 24 h after transfection, most GFPLD remained inside the nucleus, appearing either in a type II or type III pattern, while HBsAg was distributed in the ER (Fig. 5A
). This image was selected to show that two adjacent cells had different distribution patterns of GFPLD, in contrast to the uniform distribution of HDAg in two adjacent cells, as shown in Fig. 1(A)
and (B). It might be that cells expressing only LDAg or SDAg have no mechanism to modify HDAg at the same time in the daughter cells as described above. In some of the cells 24 and 48 h post-transfection, green speckles of type III were found to align on the edge of the nuclear membrane (Fig. 5B
). At this stage, co-localization between GFPLD and HBsAg was occasionally observed near the nuclear membrane and cytoplasm (Fig. 5B
). Meanwhile GFPLD alone was detected in the region of the Golgi apparatus (Fig. 5B and C
). In the cells 72 h post-transfection, co-localization of GFPLD and HBsAg became more evident in the cytoplasm and the amount of GFPLD gradually diminished inside the nucleus (Fig. 5C and D
).
The results shown in Table 2 and Fig. 5
clearly demonstrated that HBsAg can facilitate GFPLD transportation from the nucleus to cytoplasm, although the underlying mechanism remains unknown. Apparently, HBsAg is not a crucial factor for LDAg export out of the nucleus, since in the absence of HBsAg, wild-type HDAg could appear in the Golgi when transfected cells had been cultured for more than 5 days (Fig. 1B
). Since LDAg alone, without SDAg and the HDV RNA genome, can be secreted with HBsAg (Sheu et al., 1996
), it is suggested that LDAg requires modification to be exported out of the nucleus. The modification of LDAg includes isoprenylation at Cys-211 and phosphorylation or dephosphorylation at Ser-123. Enzymes for LDAg modification, such as CKII, may fluctuate during the cell cycle. The presence of HBsAg may transduce signals to the nucleus and alter enzyme activity to modify LDAg. This hypothesis is supported by the demonstration of cross-talk between HBsAg and nuclear factors (Xu et al., 1997
). The reason that the majority of GFPLDM could not be transported out of the nucleus is unknown. It is possible that isoprenylation changes LDAg so that it exposes its nucleus export signal (NES) (Lee et al., 2001
). Without exposing its NES, GFPLDM can only shuttle between the nucleolus and speckles (Table 2
). Once the LDAg is exported out of the nucleus, a new conformation favours its localization to the Golgi apparatus, similar to an LDAg mutant with a deletion of the nucleus localization signal (NLS) as reported by Bichko & Taylor (1996)
.
Based on the current study, we suggest a simple scheme regarding LDAg translocation and its interaction with HBsAg. The sequential pathway of LDAg could be depicted as follows. (i) LDAg is synthesized in the cytoplasm and transported into the nucleus by interaction of its NLS with importin and then accumulates in the nucleolus. (ii) Ser-123 of LDAg is dephosphorylated and interacts with unknown nuclear factors for targeting to the SC-35 speckles. (iii) Prenylation occurs at Cys-211 of LDAg and phosphorylation at Ser-123, which changes the LDAg conformation and allows its export out of the nucleus. (iv) Using its prenylated tail, LDAg inserts into the Golgi membrane and LDAg-containing vesicles bud off from Golgi and are retro-transported to the ER, where LDAg and HBsAg form a subviral particle or mature virion.
Previously, we postulated that LDAg could follow two alternative pathways (Sheu et al., 1996). Here, we have demonstrated that only one pathway occurs, in which LDAg must enter the nucleus, and isoprenylation then allows it out of the nucleus. Thus LDAg is first located in the Golgi apparatus and then moves to the ER for subviral or viral particle formation. This differs from the central paradigm of the secretion pathway and may reflect the fact that HDV requires a tighter control for its secretion than other conventional secretion pathways (Nickel, 2003
). To date, there is no evidence showing how HBV and HDV form a mature virion in the same cell. Generally, it is believed that HBV follows the conventional secretion pathway forming virions in the ER and then moving through the Golgi apparatus and secretory vesicles (Huovila et al., 1992
). HDV differs because it is present in the Golgi apparatus first and then moves to the ER, as indicated in this work by Figs 1(B) and 5(B)
and (C). Previous results showing that non-glycosylation of HBsAg retards HDV secretion but not HBV also support this hypothesis that the maturation pathway of HDV and HBV is different (Wang et al., 1996
; Sureau et al., 2003
). Nevertheless, more work is needed to elucidate both the HDV and the HBV maturation pathways.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Bichko, V. V. & Taylor, J. M. (1996). Redistribution of the delta antigens in cells replicating the genome of hepatitis delta virus. J Virol 70, 80648070.[Abstract]
Casey, J. L. & Gerin, J. L. (1995). Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA. J Virol 69, 75937600.[Abstract]
Chang, J. & Taylor, J. (2002). In vivo RNA-directed transcription, with template switching, by a mammalian RNA polymerase. EMBO J 21, 157164.
Chang, M.-F., Baker, S. C., Soe, L. H., Kamahora, T., Keck, J. G., Makino, S., Govindarajan, S. & Lai, M. M. C. (1988). Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity. J Virol 62, 24032410.[Medline]
Chang, F.-L., Chen, P.-J., Tu, S.-J., Wang, C.-J. & Chen, D.-S. (1991). The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus. Proc Natl Acad Sci U S A 88, 84908494.[Abstract]
Chang, M.-F., Chang, S. C., Chang, C.-I., Wu, K. & Kang, H.-Y. (1992). Nuclear localization signals, but not putative leucine zipper motif, are essential for nuclear transport of hepatitis delta antigen. J Virol 66, 60196027.[Abstract]
Chao, M., Hsieh, S.-Y. & Taylor, J. (1990). Role of two forms of hepatitis delta antigen: evidence for a mechanism of self-limiting genome replication. J Virol 64, 50665069.[Medline]
Chen, P.-J., Kalpana, G., Goldberg, J., Mason, W., Werner, B., Gerin, J. & Taylor, J. (1986). Structure and replication of the genome of the hepatitis delta virus. Proc Natl Acad Sci U S A 83, 87748778.[Abstract]
Chen, P.-J., Wu, H.-L., Wang, C.-J., Chia, J.-H. & Chen, D.-S. (1997). Molecular biology of hepatitis D virus: research and potential for application. J Gastroenterol Hepatol 12, 188192.
Fu, T.-B. & Taylor, J. (1993). The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates. J Virol 67, 69656972.[Abstract]
Glenn, J. S., Watson, J. A., Havel, C. M. & White, J. M. (1992). Identification of a prenylation site in delta virus large antigen. Science 256, 13311333.[Medline]
Graham, E. L. & van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456467.[Medline]
Hu, H.-M., Shih, K.-N. & Lo, S. J. (1996). Disulfide bond formation of the in vitro-translated large antigen of hepatitis D virus. J Virol Methods 60, 3946.[CrossRef][Medline]
Huang, W.-H., Yung, B. Y. M., Syu, W. J. & Lee, Y.-H. W. (2001). The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication. J Biol Chem 276, 2516625175.
Huovila, A.-P. J., Eder, A. M. & Fuller, S. D. (1992). Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol 118, 13051320.[Abstract]
Hwang, S. B. & Lai, M. M. C. (1993). Isoprenylation mediates direct proteinprotein interactions between hepatitis large delta antigen and hepatitis B surface antigen. J Virol 67, 76597662.[Abstract]
Jayan, G. C. & Casey, J. L. (2002). Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2. J Virol 76, 38193827.
Kos, A., Dijkema, R., Arnberg, A. C., van de Meide, P. H. & Schelekens, H. (1986). The hepatitis delta () virus possesses a circular RNA. Nature 323, 558560.[Medline]
Kuo, M. Y.-P., Chao, M. & Taylor, J. (1989). Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol 63, 19451950.[Medline]
Lai, M. M. C. (1995). The molecular biology of hepatitis delta virus. Annu Rev Biochem 64, 259286.[CrossRef][Medline]
Lee, C.-H., Chang, S. C., Wu, C. H. & Chang, M.-F. (2001). A novel chromosome region maintenance 1-independent nuclear export signal of the large form hepatitis delta antigen that is required for the viral assembly. J Biol Chem 276, 81428148.
MacNaughton, T. B., Shi, S. T., Modahl, L. E. & Lai, M. M. C. (2002). Rolling circle replication of hepatitis delta virus RNA carried out by two different cellular RNA polymerases. J Virol 76, 39203927.
Meisner, H. & Czech, M. P. (1991). Phosphorylation of transcriptional factors and cell-cycle-dependent proteins by casein kinase II. Curr Opin Cell Biol 3, 474483.[Medline]
Modahl, L. E., MacNaughton, T. B., Zhu, N., Johnson, D. L. & Lai, M. M. C. (2000). RNA-dependent replication and transcription of hepatitis delta virus RNA involve a distinct cellular RNA polymerase. Mol Cell Biol 73, 60306039.[CrossRef]
Mu, J.-J., Wu, H.-L., Chiang, B.-L., Chang, R.-P., Chen, D.-S. & Chen, P.-J. (1999). Characterization of phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens. J Virol 73, 1054010545.
Mu, J.-J., Chen, D.-S. & Chen, P.-J. (2001). The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication. J Virol 75, 90879095.
Nickel, W. (2003). The mystery of non-classical protein secretion: a current review on cargo proteins and potential export routes. Eur J Biochem 270, 21092119.
Rizzetto, M., Canese, M. G., Gerin, J. L., London, W. T., Sly, D. L. & Purcell, R. H. (1980). Transmission of the hepatitis B virus-associated delta antigen to chimpanzees. J Infect Dis 141, 590602.[Medline]
Ryu, W. S., Bayer, M. & Taylor, J. (1992). Assembly of hepatitis delta virus particles. J Virol 66, 23102315.[Abstract]
Sato, S., Wong, S. K. & Lazinski, D. W. (2001). Hepatitis delta virus minimal substrates competent for editing by ADAR1 and ADAR2. J Virol 75, 85478555.
Sheu, S. Y. & Lo, S. J. (1994). Biogenesis of the hepatitis B viral middle (M) surface protein in a human hepatoma cell line: demonstration of an alternative secretion pathway. J Gen Virol 75, 30313039.[Abstract]
Sheu, S. Y., Chen, K.-L., Lee, Y.-H. W. & Lo, S. J. (1996). No intermolecular interaction between the large hepatitis delta antigens is required for the secretion with hepatitis B surface antigen: a model of empty HDV particle. Virology 218, 275278.[CrossRef][Medline]
Shih, K.-N. & Lo, S. J. (2001). The HDV large-delta antigen fused with GFP remains functional and provides for studying its dynamic distribution. Virology 285, 138152.[CrossRef][Medline]
Shih, K.-N., Chuang, Y.-T., Liu, H. & Lo, S. J. (2004). Hepatitis D virus RNA editing is inhibited by a GFP fusion protein containing a C-terminally deleted delta antigen. J Gen Virol 85, 947957.
Stetler, D. A. & Rose, K. M. (1982). Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis. Biochemistry 21, 37213728.[Medline]
Sureau, C., Fournier-Wirth, C. & Maurel, P. (2003). Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus. J Virol 77, 55195523.
Taylor, J. M. (2003). Replication of human hepatitis delta virus: recent developments. Trends Microbiol 11, 185190.[CrossRef][Medline]
Wang, K.-S., Choo, Q.-L., Weiner, A. J. & 7 other authors (1986). Structure, sequence and expression of the hepatitis delta () viral genome. Nature 323, 508514.[Medline]
Wang, C.-J., Sung, S.-Y., Chen, D.-S. & Chen, P.-J. (1996). N-glycosylation of hepatitis B surface antigen is involved but not essential in the assembly of hepatitis delta virus. Virology 220, 2836.[CrossRef][Medline]
Weiner, A. J., Choo, Q.-L., Wang, K.-S., Govindarajan, S., Redeker, A. G., Gerin, J. L. & Houghton, M. (1988). A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 and p27
. J Virol 62, 594599.[Medline]
Wu, J. C., Chen, C.-L., Lee, S.-D., Sheen, I.-J. & Ting, L.-P. (1992). Expression and localization of the small and large delta antigens during the replication cycle of hepatitis D virus. Hepatology 16, 11201127.[Medline]
Xia, Y.-P., Yeh, C.-T., Ou, J.-H. & Lai, M. M. C. (1992). Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex. J Virol 66, 914921.[Abstract]
Xu, Z., Jensen, G. & Yen, T. S. (1997). Activation of hepatitis B virus S promoter by the viral large surface protein via induction of stress in the endoplasmic reticulum. J Virol 71, 73877392.[Abstract]
Yamaguchi, Y., Filipovska, J., Yano, K. & 7 other authors (2001). Stimulation of RNA polymerase II elongation by hepatitis D antigen. Science 293, 124127.
Yeh, T.-S. & Lee, Y.-H. W. (1998). Assembly of hepatitis delta virus particles: package of multimeric hepatitis delta virus genomic RNA and the role of phosphorylation. Virology 249, 1220.[CrossRef][Medline]
Yeh, T.-S., Lo, S. J., Chen, P.-J. & Lee, Y.-H. W. (1996). Casein kinase II and protein kinase C modulate hepatitis delta virus RNA replication but not empty viral particle assembly. J Virol 70, 61906198.[Abstract]
Received 2 October 2003;
accepted 23 January 2004.