Advertisement

Molecular Biology Reports

, Volume 42, Issue 12, pp 1603–1614 | Cite as

A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism

  • Akasit Visootsat
  • Sunchai Payungporn
  • Nattanan P. T-Thienprasert
Manuscript

Abstract

Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151–1410, PRE 1520–1620 and PRE 1650–1684. PRE 1151–1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151–1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151–1410 contains several regulatory motifs. This study reported the roles of PRE 1151–1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151–1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

Keywords

Hepatitis B virus HBV PRE Nuclear export Splicing Regulatory element 

Notes

Acknowledgments

We would like to express our deep appreciation to Dr. Christopher M. Brown for plasmids (pBasic (-IN) and pSpliceLuc). AV was funded by a Graduate School Kasetsart University Grant. NPT is funded by a Faculty of Science Grant, Kasetsart University (APSP 4/2556 and ScRF-S6/2558). This study was also funded by the Kasetsart University Research and Development Institute Grant (Mor-Vor 7.56).

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Niederau C (2014) Chronic hepatitis B in 2014: great therapeutic progress, large diagnostic deficit. World J Gastroenterol 20(33):11595–11617. doi: 10.3748/wjg.v20.i33.11595 PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Seeger C, Mason WS (2000) Hepatitis B virus biology. Microbiol Mol Biol Rev 64(1):51–68PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Panjaworayan N, Roessner SK, Firth AE, Brown CM (2007) HBVRegDB: annotation, comparison, detection and visualization of regulatory elements in hepatitis B virus sequences. Virol J 4:136. doi: 10.1186/1743-422X-4-136 PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Donello JE, Beeche AA, Smith GJ 3rd, Lucero GR, Hope TJ (1996) The hepatitis B virus posttranscriptional regulatory element is composed of two subelements. J Virol 70(7):4345–4351PubMedCentralPubMedGoogle Scholar
  5. 5.
    Huang J, Liang TJ (1993) A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products. Mol Cell Biol 13(12):7476–7486PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Huang ZM, Yen TS (1994) Hepatitis B virus RNA element that facilitates accumulation of surface gene transcripts in the cytoplasm. J Virol 68(5):3193–3199PubMedCentralPubMedGoogle Scholar
  7. 7.
    Ehlers I, Horke S, Reumann K, Rang A, Grosse F, Will H, Heise T (2004) Functional characterization of the interaction between human La and hepatitis B virus RNA. J Biol Chem 279(42):43437–43447. doi: 10.1074/jbc.M402227200 CrossRefPubMedGoogle Scholar
  8. 8.
    Heise T, Sommer G, Reumann K, Meyer I, Will H, Schaal H (2006) The hepatitis B virus PRE contains a splicing regulatory element. Nucleic Acids Res 34(1):353–363. doi: 10.1093/nar/gkj440 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Heise T, Guidotti LG, Chisari FV (1999) La autoantigen specifically recognizes a predicted stem-loop in hepatitis B virus RNA. J Virol 73(7):5767–5776PubMedCentralPubMedGoogle Scholar
  10. 10.
    Horke S, Reumann K, Rang A, Heise T (2002) Molecular characterization of the human La protein.hepatitis B virus RNA.B interaction in vitro. J Biol Chem 277(38):34949–34958. doi: 10.1074/jbc.M201911200 CrossRefPubMedGoogle Scholar
  11. 11.
    Huang ZM, Zang WQ, Yen TS (1996) Cellular proteins that bind to the hepatitis B virus posttranscriptional regulatory element. Virology 217(2):573–581. doi: 10.1006/viro.1996.0152 CrossRefPubMedGoogle Scholar
  12. 12.
    Zang WQ, Fieno AM, Grant RA, Yen TS (1998) Identification of glyceraldehyde-3-phosphate dehydrogenase as a cellular protein that binds to the hepatitis B virus posttranscriptional regulatory element. Virology 248(1):46–52. doi: 10.1006/viro.1998.9255 CrossRefPubMedGoogle Scholar
  13. 13.
    Zang WQ, Li B, Huang PY, Lai MM, Yen TS (2001) Role of polypyrimidine tract binding protein in the function of the hepatitis B virus posttranscriptional regulatory element. J Virol 75(22):10779–10786. doi: 10.1128/JVI.75.22.10779-10786.2001 PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Tang H, Huang Y, Chen J, Yu C, Huang AL (2008) Cellular protein TIA-1 regulates the expression of HBV surface antigen by binding the HBV posttranscriptional regulatory element. Intervirology 51(3):203–209. doi: 10.1159/000151632 CrossRefPubMedGoogle Scholar
  15. 15.
    Smith GJ 3rd, Donello JE, Luck R, Steger G, Hope TJ (1998) The hepatitis B virus post-transcriptional regulatory element contains two conserved RNA stem-loops which are required for function. Nucleic Acids Res 26(21):4818–4827PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Huang C, Xie MH, Liu W, Yang B, Yang F, Huang J, Wu Q, Fu XD, Zhang Y (2011) A structured RNA in hepatitis B virus post-transcriptional regulatory element represses alternative splicing in a sequence-independent and position-dependent manner. FEBS J. doi: 10.1111/j.1742-4658.2011.08077.x Google Scholar
  17. 17.
    Panjaworayan N, Payungporn S, Poovorawan Y, Brown CM (2010) Identification of an effective siRNA target site and functional regulatory elements, within the hepatitis B virus posttranscriptional regulatory element. Virol J 7:216. doi: 10.1186/1743-422X-7-216 PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Huang HY, Chien CH, Jen KH, Huang HD (2006) RegRNA: an integrated web server for identifying regulatory RNA motifs and elements. Nucleic Acids Res 34(Web Server issue):W429–W434. doi: 10.1093/nar/gkl333 PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31(13):3406–3415PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Firth AE, Brown CM (2006) Detecting overlapping coding sequences in virus genomes. BMC Bioinform 7:75. doi: 10.1186/1471-2105-7-75 CrossRefGoogle Scholar
  21. 21.
    Hofacker IL, Fekete M, Stadler PF (2002) Secondary structure prediction for aligned RNA sequences. J Mol Biol 319(5):1059–1066. doi: 10.1016/S0022-2836(02)00308-X CrossRefPubMedGoogle Scholar
  22. 22.
    Perez I, Lin CH, McAfee JG, Patton JG (1997) Mutation of PTB binding sites causes misregulation of alternative 3′ splice site selection in vivo. RNA 3(7):764–778PubMedCentralPubMedGoogle Scholar
  23. 23.
    Yuan X, Davydova N, Conte MR, Curry S, Matthews S (2002) Chemical shift mapping of RNA interactions with the polypyrimidine tract binding protein. Nucleic Acids Res 30(2):456–462PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Zhang L, Ashiya M, Sherman TG, Grabowski PJ (1996) Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA. RNA 2(7):682–698PubMedCentralPubMedGoogle Scholar
  25. 25.
    Pudi R, Srinivasan P, Das S (2004) La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation. J Biol Chem 279(29):29879–29888. doi: 10.1074/jbc.M403417200 CrossRefPubMedGoogle Scholar
  26. 26.
    Garson JA, Grant PR, Ayliffe U, Ferns RB, Tedder RS (2005) Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control. J Virol Methods 126(1–2):207–213. doi: 10.1016/j.jviromet.2005.03.001 CrossRefPubMedGoogle Scholar
  27. 27.
    Sun D, Rosler C, Kidd-Ljunggren K, Nassal M (2010) Quantitative assessment of the antiviral potencies of 21 shRNA vectors targeting conserved, including structured, hepatitis B virus sites. J Hepatol 52(6):817–826. doi: 10.1016/j.jhep.2009.10.038 CrossRefPubMedGoogle Scholar
  28. 28.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108CrossRefPubMedGoogle Scholar
  29. 29.
    Hofacker IL, Fekete M, Flamm C, Huynen MA, Rauscher S, Stolorz PE, Stadler PF (1998) Automatic detection of conserved RNA structure elements in complete RNA virus genomes. Nucleic Acids Res 26(16):3825–3836PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Hofacker IL, Stadler PF (1999) Automatic detection of conserved base pairing patterns in RNA virus genomes. Comput Chem 23(3–4):401–414CrossRefPubMedGoogle Scholar
  31. 31.
    Chen A, Panjaworayan TTN, Brown CM (2014) Prospects for inhibiting the post-transcriptional regulation of gene expression in hepatitis B virus. World J Gastroenterol 20(25):7993–8004. doi: 10.3748/wjg.v20.i25.7993 PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Thongthae N, Payungporn S, Poovorawan Y, T-Thienprasert NP (2014) A rational study for identification of highly effective siRNAs against hepatitis B virus. Exp Mol Pathol 97(1):120–127. doi: 10.1016/j.yexmp.2014.06.006 CrossRefPubMedGoogle Scholar
  33. 33.
    Tang J, Huang ZM, Chen YY, Zhang ZH, Liu GL, Zhang J (2012) A novel inhibitor of human La protein with anti-HBV activity discovered by structure-based virtual screening and in vitro evaluation. PLoS One 7(4):e36363. doi: 10.1371/journal.pone.0036363 PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Ni Q, Chen Z, Yao HP, Yang ZG, Liu KZ, Wu LL (2004) Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells. World J Gastroenterol 10(14):2050–2054PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Sherf BA, Wood KV (1994) Firefly luciferase engineered for improved genetic reporting. Promega Notes 49:14–21Google Scholar
  36. 36.
    Fukuhara T, Kambara H, Shiokawa M, Ono C, Katoh H, Morita E, Okuzaki D, Maehara Y, Koike K, Matsuura Y (2012) Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus. J Virol 86(15):7918–7933. doi: 10.1128/JVI.00567-12 PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Basu S, Bhattacharyya SN (2014) Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells. Nucleic Acids Res 42(11):7170–7185. doi: 10.1093/nar/gku346 PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Meex SJ, Andreo U, Sparks JD, Fisher EA (2011) Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion? J Lipid Res 52(1):152–158. doi: 10.1194/jlr.D008888 PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Vecchi C, Montosi G, Pietrangelo A (2010) Huh-7: a human “hemochromatotic” cell line. Hepatology 51(2):654–659. doi: 10.1002/hep.23410 CrossRefPubMedGoogle Scholar
  40. 40.
    Schlaak JF, Hilkens CM, Costa-Pereira AP, Strobl B, Aberger F, Frischauf AM, Kerr IM (2002) Cell-type and donor-specific transcriptional responses to interferon-alpha. Use of customized gene arrays. J Biol Chem 277(51):49428–49437. doi: 10.1074/jbc.M205571200 CrossRefPubMedGoogle Scholar
  41. 41.
    Wan Y, Kertesz M, Spitale RC, Segal E, Chang HY (2011) Understanding the transcriptome through RNA structure. Nat Rev Genet 12(9):641–655. doi: 10.1038/nrg3049 CrossRefPubMedGoogle Scholar
  42. 42.
    Gluzman Y (1981) SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23(1):175–182CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Akasit Visootsat
    • 1
  • Sunchai Payungporn
    • 2
  • Nattanan P. T-Thienprasert
    • 1
  1. 1.Department of Biochemistry, Faculty of ScienceKasetsart UniversityBangkokThailand
  2. 2.Department of Biochemistry, Faculty of MedicineChulalongkorn UniversityBangkokThailand

Personalised recommendations