Advertisement

In Vivo Analyses of Viral RNA Translation

  • William R. Staplin
  • W. Allen Miller
Part of the Methods in Molecular Biology™ book series (MIMB, volume 451)

Abstract

Positive-strand RNA viruses often use noncanonical strategies to usurp the host translational machinery for their own benefit. These strategies have been analyzed using transient expression assays in the absence of replication, with reporter genes replacing viral genes. A sensitive and convenient reporter assay is the dual luciferase system using Renilla (Renilla reniformis) and firefly (Photinus pyralis) reporter genes. Use of recombinant viral constructs containing the reporter luciferase gene allows us to discern whether a particular RNA sequence or secondary structure elicits an effect on initiation of translation or recoding. This chapter describes a standard luciferase protocol that can be molded to fit any viral sequence, in order to detect cis-acting regulatory elements in viral RNA.

Keywords

Cap-independent translation Electroporation Oat protoplast Plant cell suspension culture Recoding Ribosomal frameshift 

References

  1. 1.
    1. Kneller, E.R, Rakotondrafara, A.M., Miller, W.A., Cap-independent translation of plant vial RNAs. Virus Res, 2005. 119: 63–75.PubMedCrossRefGoogle Scholar
  2. 2.
    Matsuda, D., Dreher, T., In Vivo Translation Studies of Plant Viral RNAs Using Reporter Genes. Current Protocols in Microbiology. 2005. 16K.2.1–16K.2.11.Google Scholar
  3. 3.
    Gallie, D.R., The cap and poly (A) tail function synergistically to regulate mRNA translational efficiency. Cold Spring Harbor Laboratory, 1991: p. 2108–2116.Google Scholar
  4. 4.
    4. Gallie, D.R., Kobayashi, M., The role of the 3′ Untranslated region of nonpolyadenylated plant mRNAs in regulating translational efficiency. Gene, 1994. 142: 159–165.PubMedCrossRefGoogle Scholar
  5. 5.
    5. Brasier, A.R.a.F, J.J., Nonisotopic assays for reporter gene activity. In: Current Protocols in Molecular Biology, R. Brent. F.M. Ausubel, R.E. Kingston, D.D. Moore, J.G Seidman, J.A. Smith, K. Struhl (eds.). 1995, Hoboken, N.J.: John Wiley & Sons. 9.7.12–9.7–21.Google Scholar
  6. 6.
    6. Matsuda, D., Bauer, L., Tinnesand, K., Dreher, T.W., Expression of the two nested overlapping reading frames of TYMV RNA is enhanced by a 5′ cap and by 5′ and 3′ viral sequences. J Virol, 2004. 78: 9325–9335.PubMedCrossRefGoogle Scholar
  7. 7.
    7. Yamaji, Y., Kobayashi, T., Hamada, K., Sakurai, K, Yoshii, A, Suzuki, M., Namba, S., Hibi, T. et al., In vivo interaction between Tobacco mosaic virus RNA-dependent RNA polymerase and host translation elongation factor 1A. Virology, 2006. 347, 100–108.PubMedCrossRefGoogle Scholar
  8. 8.
    8. Brierley, I., Dos Ramos, F.J., Programmed ribosomal frameshifting in HIV-1 and the SARS-CoV. Virus Res, 2006. 119(1): 29–42.PubMedCrossRefGoogle Scholar
  9. 9.
    9. Baril, M., Brakier-Gingras, L., Translation of the F protein of hepatitis C virus is initiated at a non-AUG codon in a + 1 reading frame relative to the polyprotein. Nucleic Acids Res, 2005. 33(5): 1474–1486.PubMedCrossRefGoogle Scholar
  10. 10.
    10. Plant, E.R, Perez-Alvarado, G.C., Jacobs, J.L., Mukhopadhyay, B., Hennig, M., Dinman, J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal. PLoS Biol, 2005. 3(6): el72.CrossRefGoogle Scholar
  11. 11.
    11. Dreher, T.W., Miller, W.A., Translational control in positive strand RNA plant viruses. Virology, 2006. 344(1): 185–197.PubMedCrossRefGoogle Scholar
  12. 12.
    12. McInerney, P., T. Mizutani, T. Shiba, Inorganic polyphosphate interacts with ribosomes and promotes translation fidelity in vitro and in vivo. Mol Microbiol, 2006. 60(2): 438–447.PubMedCrossRefGoogle Scholar
  13. 13.
    Rakotondrafara, A.M., Jackson, J.R., Pettit Kneller, E., Miller, WA. Preparation and electroporation of oat protoplasts from cell suspension culture. Curr. Protocols Micro., 2007. 16D.3.1–16D.3.12.Google Scholar
  14. 14.
    14. Kozak, M., New ways of initiating translation in eukaryotes? Mol Cell Biol, 2001. 21: 1899–1907.PubMedCrossRefGoogle Scholar
  15. 15.
    15. Thoma, C, et al., Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly (A) tail is independent of intact eIF4G and PABP. Mol Cell, 2004. 15(6): 925–935.PubMedCrossRefGoogle Scholar
  16. 16.
    16. Sanchez, M., et al., Iron Regulation and the Cell Cycle: Identification of an iron-responsive element in the 3′ -untranslated region of human cell division cycle 14A mRNA by a refined micro array-based screening strategy J Biol Chem, 2006. 281(32): 22865–22874.PubMedCrossRefGoogle Scholar
  17. 17.
    17. Meulewaeter, F., Van Montagu, M., Cornelissen, M., Features of the autonomous function of the translational enhancer domain of satellite tobacco necrosis virus. RNA, 1998. 4(11): 1347–1356.PubMedCrossRefGoogle Scholar
  18. 18.
    18. Qin, X., Sarnow, P., Preferential translation of internal ribosome entry site-containing mRNAs during the mitotic cycle in mammalian cells. J Biol Chem, 2004. 279: 13721–13728.PubMedCrossRefGoogle Scholar
  19. 19.
    19. Barry, J.K., Miller, W.A., A-l ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA. Proc Natl Acad Sci U S A, 2002. 99(17): 11133–11138.PubMedCrossRefGoogle Scholar
  20. 20.
    20. Rakotondrafara, A.M., et al., Oscillating kissing stem-loop interactions mediate 5′ scanning-dependent translation by a viral 3′-cap-independent translation element. RNA, 2006. 12: 1893–1906.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • William R. Staplin
    • 1
  • W. Allen Miller
    • 1
  1. 1.Molecular Cellular and Developmental Biology, Department of Plant PathologyIowa State UniversityAmesUSA

Personalised recommendations