Abstract
Aim:
To substantiate the in vitro translational studies of a cross-kingdom, internal ribosome entry site (IRES), the 5′ untranslated region of the Rhopalosiphum padi virus (RhPV), can function in mammalian cells and act as a shuttle IRES between insect cells and mammalian cells.
Methods:
Cytomegalovirus (CMV) promoter-based bicistronic mammalian cell expression vectors, either in plasmids or baculovirus vectors, were generated. Plasmid transient transfection and baculovirus transduction assays were performed to test whether the RhPV IRES can mediate translation activity in versatile mammalian cell lines.
Results:
Both plasmids and recombinant baculoviruses containing the CMV promoter and the RhPV IRES can mediate bicistronic gene expression in mammalian cells. However, in the CMV promoter containing recombinant baculovirus-infected insect Sf21 cells, only the second cistron gene expression was observed. Northern blot analysis and a promoterless assay demonstrated that the RhPV IRES exhibited cryptic promoter activity in baculovirus-infected insect cells.
Conclusion:
RhPV IRES can mediate gene expression in both insect cells and mammalian cells, and this characteristic of the RhPV IRES will facilitate the development of a bicistronic baculovirus gene therapy vectors.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Pelletier J, and Sonenberg N . Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 1988; 334: 320–5.
Jang SK, Pestova TV, Hellen CU, Witherell GW, Wimmer E . Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site. Enzyme 1990; 44: 292–309.
Hellen CU, Sarnow P . Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 2001; 15: 1593–612.
Holcik M, Lefebvre C, Yeh C, Chow T, Korneluk RG . A new internal-ribosome-entry-site motif potentiates XIAP-mediated cytoprotection. Nat Cell Biol 1999; 1: 190–2.
Martinez-Salas E, Ramos R, Lafuente E, Lopez de Quinto S . Functional interactions in internal translation initiation directed by viral and cellular IRES elements, J Gen Virol 2001; 82: 973–84.
Sachs AB . Cell cycle-dependent translation initiation: IRES elements prevail. Cell 2000; 101: 243–5.
Pinkstaff JK, Chappell SA, Mauro VP, Edelman GM, Krushel LA . Internal initiation of translation of five dendritically localized neuronal mRNAs. Proc Natl Acad Sci USA 2001; 98: 2770–5.
Martinez-Salas E . Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol 1999; 10: 458–64.
van Oers, MM . Vaccines for viral and parasitic diseases produced with baculovirus vectors. Adv Virus Res 2006; 68: 193–253.
Mayo MA . A summary of taxonomic changes recently approved by ICTV. Arch Virol 2002; 147: 1655–63.
Jan E . Divergent IRES elements in invertebrates. Virus Res. 2006; 119: 16–28.
Domier LL, McCoppin NK . In vivo activity of Rhopalosiphum padi virus internal ribosome entry sites. J Gen Virol 2003; 84: 415–9.
Nakashima N, Sasaki J, Toriyama S . Determining the nucleotide sequence and capsid-coding region of himetobi P virus: a member of a novel group of RNA viruses that infect insects. Arch Virol 1999; 144: 2051–8.
Domier LL, McCoppin NK, D'Arcy CJ . Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2. Virology 2000; 268: 264–71.
Wilson JE, Powell MJ, Hoover SE, Sarnow P . Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. Mol Cell Biol 2000; 20: 4990–9.
Woolaway KE, Lazaridis K, Belsham GJ, Carter MJ, Roberts LO . The 5′untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems. J Virol 2001; 75: 10 244–9.
Terenin IM, Dmitriev SE, Andreev DE, Royall E, Belsham GJ, Roberts LO, Shatsky IN . A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry. Mol Cell Biol 2005; 25: 7879–88.
Thiem SM, Miller LK . Differential gene expression mediated by late, very late and hybrid baculovirus promoters. Gene 1990; 91: 87–94.
Sambrook J, Fritsch EF, Maniatis T . Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press: 2001.
Chen YJ, Chen WS, Wu TY . Development of a bi-cistronic baculovirus expression vector by the Rhopalosiphum padi virus 5′ internal ribosome entry site. Biochem Biophys Res Commun 2005; 335: 616–23.
Lee JC, Wu TY, Huang CF, Shih SR, Hsu TA . High-efficiency protein expression mediated by enterovirus 71 internal ribosome entry site. Biotechnol Bioeng 2005; 90: 656–72.
Wu TY, Wu CY, Chen YJ, Chen CY, Wang CH . The 5′untranslated region of Perina nuda virus (PnV) possesses a strong internal translation activity in baculovirus-infected insect cells. FEBS Lett 2007; 581: 3120–6.
Groppelli E, Belsham GJ, Roberts LO . Identification of minimal sequences of the Rhopalosiphum padi virus 5′untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems. J Gen Virol 2007; 88: 1583–8.
Teng CY and Wu TY . Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system. Biotechnol Lett 2007; 29: 1019–24.
Hu YC . Baculovirus vectors for gene therapy. Advances Virus Res 2006; 68: 287–320.
Condreay JP, Kost TA . Baculovirus expression vectors for insect and mammalian cells. Curr Drug Targets 2007; 8: 1126–31.
Wu TY, Liono L, Chen SL, Chen CY, Chao YC . Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system. J Biotechnol 2000; 80: 75–83.
Liu YK, Chu CC, Wu TY . The baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells. Acta Phamacol Sin 2006; 27: 321–7.
O'Reilly DR, Miller LK, and Luckow VA . Baculovirus Expression Vector: a laboratory manual. NY: WH Freeman and Co; 1992.
Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, et al. Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc Natl Acad Sci USA 2002; 99: 5301–6.
Finkelstein Y, Faktor O, Elroy-Stein O, Levi BZ . The use of bi-cistronic transfer vectors for the baculovirus expression system. J Biotechnol 1999; 75: 33–44.
Pijman GP, Roode EC, Fan X, Roberts LO, Belsham GJ, Valk JM, et al. Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic tanscript. J Biotechol 2006; 123: 13–21.
Royall E, Woolaway KE, Schacherl J, Kubick S, Belsham GJ, Roberts LO . The Rhopalosiphum padi virus 5′ internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system. J Gen Virol 2004; 85: 1565–9.
Pal N, Boyapalle S, Beckett R, Miller WA, Bonning BC . A baculovirus-expressed dicistrovirus that is infectious to aphids. J Virol 2007; 81: 9339–45.
van Strien EA, Zuidema D, Goldbach RW, Valk, JM . Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus. J Gen Virol 1992; 73: 2813–21.
Liu Z, Dong Z, Han B, Yang Y, Liu Y, Zhang, JT . Regulation of expression by promoters versus internal ribosome entry site in the 5′-untranslated sequence of the human cyclin-dependent kinase inhibitor p27kip1. Nucleic Acids Res 2005; 33: 3763–71.
Wang Z, Weaver M, Magnuson NS . Cryptic promoter activity in the DNA sequence corresponding to the pim-1 5′-UTR. Nucleic Acids Res 2005; 33: 2248–58.
Dumas E, Staedel C, Colombat M, Reigadas S, Chabas S, Astier-Gin T, et al. A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5′UTR. Nucleic Acids Res 2003; 31: 1275–81.
Ongus JR, Roode EC, Pleij CWA, Valk JM, van Oers MM . The 5′ non-translated region of Varroa destructor virus (genus iflavirus): structure prediction and IRES activity in Lymantria dispar cells. J Gen Virol 2006; 87: 415–9.
Gao H, Wang Y, Li N, Peng WP, Sun Y, Tong GZ, Qiu HJ . Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells. J Biotechnol 2007; 131: 138–43.
Author information
Authors and Affiliations
Corresponding author
Additional information
This work was supported by grants (No NSC-96-2317-B-033-001 and NSC-96-2627-M-033-001, 002) to Tzong-yuan Wu from the National Science Council of Taiwan, China.
Rights and permissions
About this article
Cite this article
Wu, Yj., Teng, Cy., Chen, Yj. et al. Internal ribosome entry site of Rhopalosiphum padi virus is functional in mammalian cells and has cryptic promoter activity in baculovirus-infected Sf21 cells . Acta Pharmacol Sin 29, 965–974 (2008). https://doi.org/10.1111/j.1745-7254.2008.00820.x
Received:
Accepted:
Issue date:
DOI: https://doi.org/10.1111/j.1745-7254.2008.00820.x
Keywords
This article is cited by
-
Baculovirus as an efficient vector for gene delivery into mosquitoes
Scientific Reports (2018)
