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. 1994 May;68(5):2879–2888. doi: 10.1128/jvi.68.5.2879-2888.1994

Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging.

D W Lazinski 1, J M Taylor 1
PMCID: PMC236776  PMID: 8151758

Abstract

The hepatitis delta virus (HDV) genome is a circular, single-stranded, rod-shaped, 1.7-kb RNA that replicates via a rolling-circle mechanism. Viral ribozymes function to cleave replication intermediates which are then ligated to generate the circular product. HDV expresses two forms of a single protein, the small and large delta antigens (delta Ag-S and delta Ag-L), which associate with viral RNA in a ribonucleoprotein (RNP) structure. While delta Ag-S is required for RNA replication, delta Ag-L inhibits this process but promotes the assembly of the RNP into mature virions. In this study, we have expressed full-length and deleted HDV RNA inside cells to determine the minimal RNA sequences required for self-cleavage, ligation, RNP packaging, and virion assembly and to assess the role of either delta antigen in each of these processes. We report the following findings. (i) The cleavage and ligation reactions did not require either delta antigen and were not inhibited in their presence. (ii) delta Ag-L, in the absence of delta Ag-S, formed an RNP with HDV RNA which could be assembled into secreted virus-like particles. (iii) Full-length HDV RNAs were stabilized in the presence of either delta antigen and accumulated to much higher levels than in their absence. (iv) As few as 348 nucleotides of HDV RNA were competent for circle formation, RNP assembly, and incorporation into virus-like particles. (v) An HDV RNA incapable of folding into the rod-like structure was not packaged by delta Ag-L.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berger J., Hauber J., Hauber R., Geiger R., Cullen B. R. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene. 1988 Jun 15;66(1):1–10. doi: 10.1016/0378-1119(88)90219-3. [DOI] [PubMed] [Google Scholar]
  2. Branch A. D., Robertson H. D., Greer C., Gegenheimer P., Peebles C., Abelson J. Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ. Science. 1982 Sep 17;217(4565):1147–1149. doi: 10.1126/science.217.4565.1147. [DOI] [PubMed] [Google Scholar]
  3. Casey J. L., Bergmann K. F., Brown T. L., Gerin J. L. Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7149–7153. doi: 10.1073/pnas.89.15.7149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chang M. F., Baker S. C., Soe L. H., Kamahora T., Keck J. G., Makino S., Govindarajan S., Lai M. M. Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity. J Virol. 1988 Jul;62(7):2403–2410. doi: 10.1128/jvi.62.7.2403-2410.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chao M., Hsieh S. Y., Taylor J. The antigen of hepatitis delta virus: examination of in vitro RNA-binding specificity. J Virol. 1991 Aug;65(8):4057–4062. doi: 10.1128/jvi.65.8.4057-4062.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen P. J., Kalpana G., Goldberg J., Mason W., Werner B., Gerin J., Taylor J. Structure and replication of the genome of the hepatitis delta virus. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8774–8778. doi: 10.1073/pnas.83.22.8774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Kuo M. Y., Sharmeen L., Dinter-Gottlieb G., Taylor J. Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol. 1988 Dec;62(12):4439–4444. doi: 10.1128/jvi.62.12.4439-4444.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lazinski D. W., Taylor J. M. Recent developments in hepatitis delta virus research. Adv Virus Res. 1994;43:187–231. doi: 10.1016/s0065-3527(08)60049-4. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lin J. H., Chang M. F., Baker S. C., Govindarajan S., Lai M. M. Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA. J Virol. 1990 Sep;64(9):4051–4058. doi: 10.1128/jvi.64.9.4051-4058.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luo G. X., Chao M., Hsieh S. Y., Sureau C., Nishikura K., Taylor J. A specific base transition occurs on replicating hepatitis delta virus RNA. J Virol. 1990 Mar;64(3):1021–1027. doi: 10.1128/jvi.64.3.1021-1027.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Perrotta A. T., Been M. D. The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant. Nucleic Acids Res. 1990 Dec 11;18(23):6821–6827. doi: 10.1093/nar/18.23.6821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ponzetto A., Cote P. J., Popper H., Hoyer B. H., London W. T., Ford E. C., Bonino F., Purcell R. H., Gerin J. L. Transmission of the hepatitis B virus-associated delta agent to the eastern woodchuck. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2208–2212. doi: 10.1073/pnas.81.7.2208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Puttaraju M., Perrotta A. T., Been M. D. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. 1993 Sep 11;21(18):4253–4258. doi: 10.1093/nar/21.18.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rackwitz H. R., Rohde W., Sänger H. L. DNA-dependent RNA polymerase II of plant origin transcribes viroid RNA into full-length copies. Nature. 1981 May 28;291(5813):297–301. doi: 10.1038/291297a0. [DOI] [PubMed] [Google Scholar]
  25. Rizzetto M., Hoyer B., Canese M. G., Shih J. W., Purcell R. H., Gerin J. L. delta Agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6124–6128. doi: 10.1073/pnas.77.10.6124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ryu W. S., Netter H. J., Bayer M., Taylor J. Ribonucleoprotein complexes of hepatitis delta virus. J Virol. 1993 Jun;67(6):3281–3287. doi: 10.1128/jvi.67.6.3281-3287.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanger H. L., Klotz G., Riesner D., Gross H. J., Kleinschmidt A. K. Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3852–3856. doi: 10.1073/pnas.73.11.3852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sharmeen L., Kuo M. Y., Dinter-Gottlieb G., Taylor J. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J Virol. 1988 Aug;62(8):2674–2679. doi: 10.1128/jvi.62.8.2674-2679.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sharmeen L., Kuo M. Y., Taylor J. Self-ligating RNA sequences on the antigenome of human hepatitis delta virus. J Virol. 1989 Mar;63(3):1428–1430. doi: 10.1128/jvi.63.3.1428-1430.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Taylor J. M. Hepatitis delta virus: cis and trans functions required for replication. Cell. 1990 May 4;61(3):371–373. doi: 10.1016/0092-8674(90)90516-h. [DOI] [PubMed] [Google Scholar]
  32. Wang K. S., Choo Q. L., Weiner A. J., Ou J. H., Najarian R. C., Thayer R. M., Mullenbach G. T., Denniston K. J., Gerin J. L., Houghton M. Structure, sequence and expression of the hepatitis delta (delta) viral genome. Nature. 1986 Oct 9;323(6088):508–514. doi: 10.1038/323508a0. [DOI] [PubMed] [Google Scholar]
  33. Wu H. N., Lin Y. J., Lin F. P., Makino S., Chang M. F., Lai M. M. Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1831–1835. doi: 10.1073/pnas.86.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zheng H., Fu T. B., Lazinski D., Taylor J. Editing on the genomic RNA of human hepatitis delta virus. J Virol. 1992 Aug;66(8):4693–4697. doi: 10.1128/jvi.66.8.4693-4697.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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