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. 1994 Sep;68(9):5579–5587. doi: 10.1128/jvi.68.9.5579-5587.1994

Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis.

J R Pollack 1, D Ganem 1
PMCID: PMC236958  PMID: 7520092

Abstract

Hepatitis B viruses encode a polymerase (P) protein with key roles in both reverse transcription and genomic RNA encapsidation. Genetic analysis of cis-acting signals required for viral replication implicates an RNA stem-loop structure in both RNA packaging and the initiation of reverse transcription, a process in which P protein also serves as the primer. We now show that duck hepatitis B virus (DHBV) polymerase binds specifically and with high affinity to this RNA stem-loop structure. Mutational analysis indicates that all mutations in the RNA target that inhibit the P protein-RNA interaction inhibit both in vivo RNA packaging and in vitro DNA priming to comparable extents. However, certain mutations in the loop region of the RNA have minimal impact on P protein-RNA binding but are nonetheless severely defective for packaging and DNA synthesis. Thus, P protein-RNA complex formation is necessary but not sufficient to initiate these activities. In addition, examination of RNA binding by truncated P proteins indicates that the C terminus of the polymerase, although required for RNA encapsidation in vivo, is dispensable for RNA binding and DNA priming.

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Selected References

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

  1. Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 1993 Sep;12(9):3587–3598. doi: 10.1002/j.1460-2075.1993.tb06032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartenschlager R., Junker-Niepmann M., Schaller H. The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol. 1990 Nov;64(11):5324–5332. doi: 10.1128/jvi.64.11.5324-5332.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartenschlager R., Schaller H. Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. EMBO J. 1992 Sep;11(9):3413–3420. doi: 10.1002/j.1460-2075.1992.tb05420.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnbaum F., Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol. 1990 Jul;64(7):3319–3330. doi: 10.1128/jvi.64.7.3319-3330.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Büscher M., Reiser W., Will H., Schaller H. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription. Cell. 1985 Mar;40(3):717–724. doi: 10.1016/0092-8674(85)90220-x. [DOI] [PubMed] [Google Scholar]
  6. Calvert J., Summers J. Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation. J Virol. 1994 Apr;68(4):2084–2090. doi: 10.1128/jvi.68.4.2084-2090.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang L. J., Hirsch R. C., Ganem D., Varmus H. E. Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase. J Virol. 1990 Nov;64(11):5553–5558. doi: 10.1128/jvi.64.11.5553-5558.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen Y., Robinson W. S., Marion P. L. Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging. J Virol. 1992 Feb;66(2):1282–1287. doi: 10.1128/jvi.66.2.1282-1287.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chiang P. W., Jeng K. S., Hu C. P., Chang C. M. Characterization of a cis element required for packaging and replication of the human hepatitis B virus. Virology. 1992 Feb;186(2):701–711. doi: 10.1016/0042-6822(92)90037-p. [DOI] [PubMed] [Google Scholar]
  10. Cullen B. R., Malim M. H. The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators. Trends Biochem Sci. 1991 Sep;16(9):346–350. doi: 10.1016/0968-0004(91)90141-h. [DOI] [PubMed] [Google Scholar]
  11. Das A. Control of transcription termination by RNA-binding proteins. Annu Rev Biochem. 1993;62:893–930. doi: 10.1146/annurev.bi.62.070193.004333. [DOI] [PubMed] [Google Scholar]
  12. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  13. Enders G. H., Ganem D., Varmus H. E. 5'-terminal sequences influence the segregation of ground squirrel hepatitis virus RNAs into polyribosomes and viral core particles. J Virol. 1987 Jan;61(1):35–41. doi: 10.1128/jvi.61.1.35-41.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frankel A. D., Mattaj I. W., Rio D. C. RNA-protein interactions. Cell. 1991 Dec 20;67(6):1041–1046. doi: 10.1016/0092-8674(91)90282-4. [DOI] [PubMed] [Google Scholar]
  15. Hellen C. U., Witherell G. W., Schmid M., Shin S. H., Pestova T. V., Gil A., Wimmer E. A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7642–7646. doi: 10.1073/pnas.90.16.7642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature. 1990 Apr 5;344(6266):552–555. doi: 10.1038/344552a0. [DOI] [PubMed] [Google Scholar]
  17. Hirsch R. C., Loeb D. D., Pollack J. R., Ganem D. cis-acting sequences required for encapsidation of duck hepatitis B virus pregenomic RNA. J Virol. 1991 Jun;65(6):3309–3316. doi: 10.1128/jvi.65.6.3309-3316.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirsch R., Colgrove R., Ganem D. Replication of duck hepatitis B virus in two differentiated human hepatoma cell lines after transfection with cloned viral DNA. Virology. 1988 Nov;167(1):136–142. doi: 10.1016/0042-6822(88)90062-1. [DOI] [PubMed] [Google Scholar]
  19. Junker-Niepmann M., Bartenschlager R., Schaller H. A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO J. 1990 Oct;9(10):3389–3396. doi: 10.1002/j.1460-2075.1990.tb07540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katze M. G., Wambach M., Wong M. L., Garfinkel M., Meurs E., Chong K., Williams B. R., Hovanessian A. G., Barber G. N. Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system. Mol Cell Biol. 1991 Nov;11(11):5497–5505. doi: 10.1128/mcb.11.11.5497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Knaus T., Nassal M. The encapsidation signal on the hepatitis B virus RNA pregenome forms a stem-loop structure that is critical for its function. Nucleic Acids Res. 1993 Aug 25;21(17):3967–3975. doi: 10.1093/nar/21.17.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  23. Lavine J., Hirsch R., Ganem D. A system for studying the selective encapsidation of hepadnavirus RNA. J Virol. 1989 Oct;63(10):4257–4263. doi: 10.1128/jvi.63.10.4257-4263.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Linial M. L., Miller A. D. Retroviral RNA packaging: sequence requirements and implications. Curr Top Microbiol Immunol. 1990;157:125–152. doi: 10.1007/978-3-642-75218-6_5. [DOI] [PubMed] [Google Scholar]
  25. Mattaj I. W. RNA recognition: a family matter? Cell. 1993 Jun 4;73(5):837–840. doi: 10.1016/0092-8674(93)90265-r. [DOI] [PubMed] [Google Scholar]
  26. Pollack J. R., Ganem D. An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation. J Virol. 1993 Jun;67(6):3254–3263. doi: 10.1128/jvi.67.6.3254-3263.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roychoudhury S., Faruqi A. F., Shih C. Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus. J Virol. 1991 Jul;65(7):3617–3624. doi: 10.1128/jvi.65.7.3617-3624.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schwartz S. P., Aisenthal L., Elisha Z., Oberman F., Yisraeli J. K. A 69-kDa RNA-binding protein from Xenopus oocytes recognizes a common motif in two vegetally localized maternal mRNAs. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11895–11899. doi: 10.1073/pnas.89.24.11895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seeger C., Baldwin B., Tennant B. C. Expression of infectious woodchuck hepatitis virus in murine and avian fibroblasts. J Virol. 1989 Nov;63(11):4665–4669. doi: 10.1128/jvi.63.11.4665-4669.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sprengel R., Kuhn C., Will H., Schaller H. Comparative sequence analysis of duck and human hepatitis B virus genomes. J Med Virol. 1985 Apr;15(4):323–333. doi: 10.1002/jmv.1890150402. [DOI] [PubMed] [Google Scholar]
  32. Tang C. K., Draper D. E. Unusual mRNA pseudoknot structure is recognized by a protein translational repressor. Cell. 1989 May 19;57(4):531–536. doi: 10.1016/0092-8674(89)90123-2. [DOI] [PubMed] [Google Scholar]
  33. Tavis J. E., Perri S., Ganem D. Hepadnavirus reverse transcription initiates within the stem-loop of the RNA packaging signal and employs a novel strand transfer. J Virol. 1994 Jun;68(6):3536–3543. doi: 10.1128/jvi.68.6.3536-3543.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang G. H., Seeger C. Novel mechanism for reverse transcription in hepatitis B viruses. J Virol. 1993 Nov;67(11):6507–6512. doi: 10.1128/jvi.67.11.6507-6512.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang G. H., Seeger C. The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell. 1992 Nov 13;71(4):663–670. doi: 10.1016/0092-8674(92)90599-8. [DOI] [PubMed] [Google Scholar]

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