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. 1996 Oct;70(10):6803–6809. doi: 10.1128/jvi.70.10.6803-6809.1996

A host factor that binds near the termini of hepatitis B virus pregenomic RNA.

S Perri 1, D Ganem 1
PMCID: PMC190725  PMID: 8794319

Abstract

The terminal regions of hepatitis B virus (HBV) pregenomic RNA (pgRNA) harbors sites governing many essential functions in the viral life cycle, including polyadenylation, translation, RNA encapsidation, and DNA synthesis. We have examined the binding of host proteins to a 170-nucleotide region from the 5' end of HBV pgRNA; a large portion of this region is duplicated at the 3' end of this terminally redundant RNA. By UV cross-linking labeled RNA to HepG2 cell extracts, we have identified a 65-kDa factor (p65) of nuclear origin which can specifically bind to this region. Two discrete binding sites were identified within this region; in vitro cross-competition experiments suggest that the same factor binds to both elements. One binding site (termed UBS) overlaps a portion of the highly conserved stem-loop structure (epsilon), while the other site (termed DBS) maps 35 nucleotides downstream of the hexanucleotide polyadenylation sequence. Both binding sites are highly pyrimidine rich and map to regions previously found to be important in the regulation of viral polyadenylation. However, functional analysis of mutant binding sites in vivo indicates that p65 is not involved in the polyadenylation of HBV pgRNA. Potential roles for the factor in viral replication in vivo are discussed.

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Selected References

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  1. 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]
  2. 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]
  3. Chang L. J., Ganem D., Varmus H. E. Mechanism of translation of the hepadnaviral polymerase (P) gene. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5158–5162. doi: 10.1073/pnas.87.13.5158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang L. J., Pryciak P., Ganem D., Varmus H. E. Biosynthesis of the reverse transcriptase of hepatitis B viruses involves de novo translational initiation not ribosomal frameshifting. Nature. 1989 Jan 26;337(6205):364–368. doi: 10.1038/337364a0. [DOI] [PubMed] [Google Scholar]
  5. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cherrington J., Ganem D. Regulation of polyadenylation in human immunodeficiency virus (HIV): contributions of promoter proximity and upstream sequences. EMBO J. 1992 Apr;11(4):1513–1524. doi: 10.1002/j.1460-2075.1992.tb05196.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cherrington J., Russnak R., Ganem D. Upstream sequences and cap proximity in the regulation of polyadenylation in ground squirrel hepatitis virus. J Virol. 1992 Dec;66(12):7589–7596. doi: 10.1128/jvi.66.12.7589-7596.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Constantoulakis P., Campbell M., Felber B. K., Nasioulas G., Afonina E., Pavlakis G. N. Inhibition of Rev-mediated HIV-1 expression by an RNA binding protein encoded by the interferon-inducible 9-27 gene. Science. 1993 Feb 26;259(5099):1314–1318. doi: 10.1126/science.7680491. [DOI] [PubMed] [Google Scholar]
  10. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DuBridge R. B., Tang P., Hsia H. C., Leong P. M., Miller J. H., Calos M. P. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol. 1987 Jan;7(1):379–387. doi: 10.1128/mcb.7.1.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Enders G. H., Ganem D., Varmus H. Mapping the major transcripts of ground squirrel hepatitis virus: the presumptive template for reverse transcriptase is terminally redundant. Cell. 1985 Aug;42(1):297–308. doi: 10.1016/s0092-8674(85)80125-2. [DOI] [PubMed] [Google Scholar]
  14. Gatignol A., Buckler-White A., Berkhout B., Jeang K. T. Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991 Mar 29;251(5001):1597–1600. doi: 10.1126/science.2011739. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Jang S. K., Wimmer E. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560–1572. doi: 10.1101/gad.4.9.1560. [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. 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]
  21. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loeb D. D., Hirsch R. C., Ganem D. Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements. EMBO J. 1991 Nov;10(11):3533–3540. doi: 10.1002/j.1460-2075.1991.tb04917.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mason W. S., Halpern M. S., England J. M., Seal G., Egan J., Coates L., Aldrich C., Summers J. Experimental transmission of duck hepatitis B virus. Virology. 1983 Dec;131(2):375–384. doi: 10.1016/0042-6822(83)90505-6. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Pollack J. R., Ganem D. Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis. J Virol. 1994 Sep;68(9):5579–5587. doi: 10.1128/jvi.68.9.5579-5587.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Russnak R., Ganem D. Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses. Genes Dev. 1990 May;4(5):764–776. doi: 10.1101/gad.4.5.764. [DOI] [PubMed] [Google Scholar]
  27. Sawadogo M., Roeder R. G. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4394–4398. doi: 10.1073/pnas.82.13.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schlicht H. J., Radziwill G., Schaller H. Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins. Cell. 1989 Jan 13;56(1):85–92. doi: 10.1016/0092-8674(89)90986-0. [DOI] [PubMed] [Google Scholar]
  29. Seeger C., Ganem D., Varmus H. E. Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus. J Virol. 1984 Aug;51(2):367–375. doi: 10.1128/jvi.51.2.367-375.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sells M. A., Chen M. L., Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A. 1987 Feb;84(4):1005–1009. doi: 10.1073/pnas.84.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Simonsen C. C., Levinson A. D. Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids. Mol Cell Biol. 1983 Dec;3(12):2250–2258. doi: 10.1128/mcb.3.12.2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Singh R., Valcárcel J., Green M. R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. doi: 10.1126/science.7761834. [DOI] [PubMed] [Google Scholar]
  33. Staprans S., Loeb D. D., Ganem D. Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. J Virol. 1991 Mar;65(3):1255–1262. doi: 10.1128/jvi.65.3.1255-1262.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Summers J., Mason W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell. 1982 Jun;29(2):403–415. doi: 10.1016/0092-8674(82)90157-x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Wang G. H., Zoulim F., Leber E. H., Kitson J., Seeger C. Role of RNA in enzymatic activity of the reverse transcriptase of hepatitis B viruses. J Virol. 1994 Dec;68(12):8437–8442. doi: 10.1128/jvi.68.12.8437-8442.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zamore P. D., Green M. R. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]

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