Abstract
Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, deltaAg-S and deltaAg-L, are derived from a single open reading frame by RNA editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (deltaAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (deltaAg-L), which represses replication, co-localize with delta RNA throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthesis and 3' end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA-protein complexes accumulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins, poly(A+) RNA, and RNA splicing protein factors. This implies a specific compartmentalization of ribonucleoproteins in the nucleus.
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- Bass B. L., Nishikura K., Keller W., Seeburg P. H., Emeson R. B., O'Connell M. A., Samuel C. E., Herbert A. A standardized nomenclature for adenosine deaminases that act on RNA. RNA. 1997 Sep;3(9):947–949. [PMC free article] [PubMed] [Google Scholar]
- Besse S., Vigneron M., Pichard E., Puvion-Dutilleul F. Synthesis and maturation of viral transcripts in herpes simplex virus type 1 infected HeLa cells: the role of interchromatin granules. Gene Expr. 1995;4(3):143–161. [PMC free article] [PubMed] [Google Scholar]
- Bichko V. V., Taylor J. M. Redistribution of the delta antigens in cells replicating the genome of hepatitis delta virus. J Virol. 1996 Nov;70(11):8064–8070. doi: 10.1128/jvi.70.11.8064-8070.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bohmann K., Ferreira J., Santama N., Weis K., Lamond A. I. Molecular analysis of the coiled body. J Cell Sci Suppl. 1995;19:107–113. doi: 10.1242/jcs.1995.supplement_19.16. [DOI] [PubMed] [Google Scholar]
- Bridge E., Riedel K. U., Johansson B. M., Pettersson U. Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain. J Cell Biol. 1996 Oct;135(2):303–314. doi: 10.1083/jcb.135.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carmo-Fonseca M., Cunha C., Custódio N., Carvalho C., Jordan P., Ferreira J., Parreira L. The topography of chromosomes and genes in the nucleus. Exp Cell Res. 1996 Dec 15;229(2):247–252. doi: 10.1006/excr.1996.0367. [DOI] [PubMed] [Google Scholar]
- Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carvalho C., Telhada M., do Carmo-Fonseca M., Parreira L. In situ visualisation of immunoglobulin genes in normal and malignant lymphoid cells. Clin Mol Pathol. 1995 Jun;48(3):M158–M164. doi: 10.1136/mp.48.3.m158. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casey J. L., Gerin J. L. Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA. J Virol. 1995 Dec;69(12):7593–7600. doi: 10.1128/jvi.69.12.7593-7600.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang F. L., Chen P. J., Tu S. J., Wang C. J., Chen D. S. The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8490–8494. doi: 10.1073/pnas.88.19.8490. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chao M., Hsieh S. Y., Taylor J. Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol. 1990 Oct;64(10):5066–5069. doi: 10.1128/jvi.64.10.5066-5069.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng D., Yang A., Thomas H., Monjardino J. Characterization of stable hepatitis delta expressing hepatoma cell lines: effect of HDAg on cell growth. Prog Clin Biol Res. 1993;382:149–153. [PubMed] [Google Scholar]
- Cullen J. M., David C., Wang J. G., Becherer P., Lemon S. M. Subcellular distribution of large and small hepatitis delta antigen in hepatocytes of hepatitis delta virus superinfected woodchucks. Hepatology. 1995 Oct;22(4 Pt 1):1090–1100. doi: 10.1016/0270-9139(95)90614-2. [DOI] [PubMed] [Google Scholar]
- Dourakis S., Karayiannis P., Goldin R., Taylor M., Monjardino J., Thomas H. C. An in situ hybridization, molecular biological and immunohistochemical study of hepatitis delta virus in woodchucks. Hepatology. 1991 Sep;14(3):534–539. [PubMed] [Google Scholar]
- Fakan S. Perichromatin fibrils are in situ forms of nascent transcripts. Trends Cell Biol. 1994 Mar;4(3):86–90. doi: 10.1016/0962-8924(94)90180-5. [DOI] [PubMed] [Google Scholar]
- Fu T. B., Taylor J. The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates. J Virol. 1993 Dec;67(12):6965–6972. doi: 10.1128/jvi.67.12.6965-6972.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fu X. D., Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. doi: 10.1038/343437a0. [DOI] [PubMed] [Google Scholar]
- Gama-Carvalho M., Krauss R. D., Chiang L., Valcárcel J., Green M. R., Carmo-Fonseca M. Targeting of U2AF65 to sites of active splicing in the nucleus. J Cell Biol. 1997 Jun 2;137(5):975–987. doi: 10.1083/jcb.137.5.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glenn J. S., White J. M. trans-dominant inhibition of human hepatitis delta virus genome replication. J Virol. 1991 May;65(5):2357–2361. doi: 10.1128/jvi.65.5.2357-2361.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Govindarajan S., Hwang S., Lai M. M. Comparison of the presence of two forms of delta antigen in liver tissues of acute versus chronic delta hepatitis. Prog Clin Biol Res. 1993;382:139–143. [PubMed] [Google Scholar]
- Gowans E. J., Baroudy B. M., Negro F., Ponzetto A., Purcell R. H., Gerin J. L. Evidence for replication of hepatitis delta virus RNA in hepatocyte nuclei after in vivo infection. Virology. 1988 Nov;167(1):274–278. doi: 10.1016/0042-6822(88)90078-5. [DOI] [PubMed] [Google Scholar]
- Hsieh S. Y., Chao M., Coates L., Taylor J. Hepatitis delta virus genome replication: a polyadenylated mRNA for delta antigen. J Virol. 1990 Jul;64(7):3192–3198. doi: 10.1128/jvi.64.7.3192-3198.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hsieh S. Y., Taylor J. Regulation of polyadenylation of hepatitis delta virus antigenomic RNA. J Virol. 1991 Dec;65(12):6438–6446. doi: 10.1128/jvi.65.12.6438-6446.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jenny A., Hauri H. P., Keller W. Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit. Mol Cell Biol. 1994 Dec;14(12):8183–8190. doi: 10.1128/mcb.14.12.8183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiménez-García L. F., Spector D. L. In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. Cell. 1993 Apr 9;73(1):47–59. doi: 10.1016/0092-8674(93)90159-n. [DOI] [PubMed] [Google Scholar]
- Krause S., Fakan S., Weis K., Wahle E. Immunodetection of poly(A) binding protein II in the cell nucleus. Exp Cell Res. 1994 Sep;214(1):75–82. doi: 10.1006/excr.1994.1235. [DOI] [PubMed] [Google Scholar]
- Kuo M. Y., Chao M., Taylor J. Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol. 1989 May;63(5):1945–1950. doi: 10.1128/jvi.63.5.1945-1950.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuo M. Y., Goldberg J., Coates L., Mason W., Gerin J., Taylor J. Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications. J Virol. 1988 Jun;62(6):1855–1861. doi: 10.1128/jvi.62.6.1855-1861.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lazinski D. W., Taylor J. M. Regulation of the hepatitis delta virus ribozymes: to cleave or not to cleave? RNA. 1995 May;1(3):225–233. [PMC free article] [PubMed] [Google Scholar]
- Lazinski D. W., Taylor J. M. Relating structure to function in the hepatitis delta virus antigen. J Virol. 1993 May;67(5):2672–2680. doi: 10.1128/jvi.67.5.2672-2680.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacNaughton T. B., Gowans E. J., McNamara S. P., Burrell C. J. Hepatitis delta antigen is necessary for access of hepatitis delta virus RNA to the cell transcriptional machinery but is not part of the transcriptional complex. Virology. 1991 Sep;184(1):387–390. doi: 10.1016/0042-6822(91)90855-6. [DOI] [PubMed] [Google Scholar]
- Macnaughton T. B., Gowans E. J., Jilbert A. R., Burrell C. J. Hepatitis delta virus RNA, protein synthesis and associated cytotoxicity in a stably transfected cell line. Virology. 1990 Aug;177(2):692–698. doi: 10.1016/0042-6822(90)90535-y. [DOI] [PubMed] [Google Scholar]
- Macnaughton T. B., Gowans E. J., Reinboth B., Jilbert A. R., Burrell C. J. Stable expression of hepatitis delta virus antigen in a eukaryotic cell line. J Gen Virol. 1990 Jun;71(Pt 6):1339–1345. doi: 10.1099/0022-1317-71-6-1339. [DOI] [PubMed] [Google Scholar]
- Misteli T., Cáceres J. F., Spector D. L. The dynamics of a pre-mRNA splicing factor in living cells. Nature. 1997 May 29;387(6632):523–527. doi: 10.1038/387523a0. [DOI] [PubMed] [Google Scholar]
- Moen P. T., Jr, Smith K. P., Lawrence J. B. Compartmentalization of specific pre-mRNA metabolism: an emerging view. Hum Mol Genet. 1995;4(Spec No):1779–1789. doi: 10.1093/hmg/4.suppl_1.1779. [DOI] [PubMed] [Google Scholar]
- Monneron A., Bernhard W. Fine structural organization of the interphase nucleus in some mammalian cells. J Ultrastruct Res. 1969 May;27(3):266–288. doi: 10.1016/s0022-5320(69)80017-1. [DOI] [PubMed] [Google Scholar]
- Negro F., Pacchioni D., Bussolati G., Bonino F. Hepatitis delta virus heterogeneity: a study by immunofluorescence. J Hepatol. 1991;13 (Suppl 4):S125–S129. doi: 10.1016/0168-8278(91)90043-b. [DOI] [PubMed] [Google Scholar]
- Nguyen V. T., Giannoni F., Dubois M. F., Seo S. J., Vigneron M., Kédinger C., Bensaude O. In vivo degradation of RNA polymerase II largest subunit triggered by alpha-amanitin. Nucleic Acids Res. 1996 Aug 1;24(15):2924–2929. doi: 10.1093/nar/24.15.2924. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Connell M. A., Gerber A., Keller W. Purification of human double-stranded RNA-specific editase 1 (hRED1) involved in editing of brain glutamate receptor B pre-mRNA. J Biol Chem. 1997 Jan 3;272(1):473–478. doi: 10.1074/jbc.272.1.473. [DOI] [PubMed] [Google Scholar]
- Polson A. G., Bass B. L., Casey J. L. RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature. 1996 Apr 4;380(6573):454–456. doi: 10.1038/380454a0. [DOI] [PubMed] [Google Scholar]
- Pombo A., Ferreira J., Bridge E., Carmo-Fonseca M. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells. EMBO J. 1994 Nov 1;13(21):5075–5085. doi: 10.1002/j.1460-2075.1994.tb06837.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Puvion-Dutilleul F., Bachellerie J. P., Visa N., Puvion E. Rearrangements of intranuclear structures involved in RNA processing in response to adenovirus infection. J Cell Sci. 1994 Jun;107(Pt 6):1457–1468. doi: 10.1242/jcs.107.6.1457. [DOI] [PubMed] [Google Scholar]
- Rebelo L., Almeida F., Ramos C., Bohmann K., Lamond A. I., Carmo-Fonseca M. The dynamics of coiled bodies in the nucleus of adenovirus-infected cells. Mol Biol Cell. 1996 Jul;7(7):1137–1151. doi: 10.1091/mbc.7.7.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ryu W. S., Bayer M., Taylor J. Assembly of hepatitis delta virus particles. J Virol. 1992 Apr;66(4):2310–2315. doi: 10.1128/jvi.66.4.2310-2315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saldanha J., Homer E., Goldin R., Thomas H. C., Monjardino J. Cloning and expression of an immunodominant region of the hepatitis delta antigen. J Gen Virol. 1990 Feb;71(Pt 2):471–475. doi: 10.1099/0022-1317-71-2-471. [DOI] [PubMed] [Google Scholar]
- Schul W., Groenhout B., Koberna K., Takagaki Y., Jenny A., Manders E. M., Raska I., van Driel R., de Jong L. The RNA 3' cleavage factors CstF 64 kDa and CPSF 100 kDa are concentrated in nuclear domains closely associated with coiled bodies and newly synthesized RNA. EMBO J. 1996 Jun 3;15(11):2883–2892. [PMC free article] [PubMed] [Google Scholar]
- Singer R. H., Green M. R. Compartmentalization of eukaryotic gene expression: causes and effects. Cell. 1997 Oct 31;91(3):291–294. doi: 10.1016/s0092-8674(00)80411-0. [DOI] [PubMed] [Google Scholar]
- Spector D. L., Fu X. D., Maniatis T. Associations between distinct pre-mRNA splicing components and the cell nucleus. EMBO J. 1991 Nov;10(11):3467–3481. doi: 10.1002/j.1460-2075.1991.tb04911.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spector D. L. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. doi: 10.1146/annurev.cb.09.110193.001405. [DOI] [PubMed] [Google Scholar]
- Spector D. L. Nuclear organization and gene expression. Exp Cell Res. 1996 Dec 15;229(2):189–197. doi: 10.1006/excr.1996.0358. [DOI] [PubMed] [Google Scholar]
- Taylor J. M. The structure and replication of hepatitis delta virus. Annu Rev Microbiol. 1992;46:253–276. doi: 10.1146/annurev.mi.46.100192.001345. [DOI] [PubMed] [Google Scholar]
- Taylor J., Mason W., Summers J., Goldberg J., Aldrich C., Coates L., Gerin J., Gowans E. Replication of human hepatitis delta virus in primary cultures of woodchuck hepatocytes. J Virol. 1987 Sep;61(9):2891–2895. doi: 10.1128/jvi.61.9.2891-2895.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vincent M., Lauriault P., Dubois M. F., Lavoie S., Bensaude O., Chabot B. The nuclear matrix protein p255 is a highly phosphorylated form of RNA polymerase II largest subunit which associates with spliceosomes. Nucleic Acids Res. 1996 Dec 1;24(23):4649–4652. doi: 10.1093/nar/24.23.4649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weis K., Rambaud S., Lavau C., Jansen J., Carvalho T., Carmo-Fonseca M., Lamond A., Dejean A. Retinoic acid regulates aberrant nuclear localization of PML-RAR alpha in acute promyelocytic leukemia cells. Cell. 1994 Jan 28;76(2):345–356. doi: 10.1016/0092-8674(94)90341-7. [DOI] [PubMed] [Google Scholar]
- Wu J. C., Chen C. L., Lee S. D., Sheen I. J., Ting L. P. Expression and localization of the small and large delta antigens during the replication cycle of hepatitis D virus. Hepatology. 1992 Nov;16(5):1120–1127. [PubMed] [Google Scholar]
- Zeng C., Kim E., Warren S. L., Berget S. M. Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity. EMBO J. 1997 Mar 17;16(6):1401–1412. doi: 10.1093/emboj/16.6.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zirbel R. M., Mathieu U. R., Kurz A., Cremer T., Lichter P. Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries. Chromosome Res. 1993 Jul;1(2):93–106. doi: 10.1007/BF00710032. [DOI] [PubMed] [Google Scholar]
- van Driel R., Wansink D. G., van Steensel B., Grande M. A., Schul W., de Jong L. Nuclear domains and the nuclear matrix. Int Rev Cytol. 1995;162A:151–189. doi: 10.1016/s0074-7696(08)61231-0. [DOI] [PubMed] [Google Scholar]