Investigation of IRES Insertion into the Genome of Recombinant MVA as a Translation Enhancer in the Context of Transcript Decapping.

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  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Genome, Viral* ; Protein Biosynthesis*; Internal Ribosome Entry Sites/*genetics ; RNA Caps/*metabolism ; Recombination, Genetic/*genetics ; Vaccinia virus/*genetics; Antigens, Viral/immunology ; Luciferases/metabolism ; Mutagenesis, Insertional/genetics ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Transgenes ; Vaccinia virus/immunology
    • Abstract:
      Recombinant modified vaccinia virus Ankara (MVA) has been used to deliver vaccine candidate antigens against infectious diseases and cancer. MVA is a potent viral vector for inducing high magnitudes of antigen-specific CD8+ T cells; however the cellular immune responses to a recombinant antigen in MVA could be further enhanced by increasing transgene expression. Previous reports showed the importance of utilizing an early poxviral promoter for increasing transgene expression and therefore enhancing cellular immune responses. However, the vaccinia D10 decapping enzyme is reported to target and decap vaccinia virus early transcripts - a mechanism that could limit the usefulness of early promoters in MVA viral vectors if this enzyme shows the same activity in this closely related virus. Therefore, we attempted to increase transgene expression in recombinant MVA by inserting the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) upstream of a transgene sequence that is controlled by the B8R early promoter, and assessed D10 enzyme decapping activity in MVA. The aim of the IRES element was to initiate translation of the transgene transcript (after the removal of the cap structure by the D10 decapping protein) in a cap-independent manner. Here, we report that overexpression of the D10 decapping protein, in trans, in MVA reduced growth and transgene expression; however, the IRES element was not able to compensate for the negative effect of the D10 decapping protein. Recombinant MVA with EMCV IRES induced levels of both gene expression and transcription that were similar to the control recombinant MVA, encoding the same transgene but without the IRES element. Both viruses were tested in BALB/c mice and induced similar magnitudes of epitope-specific CD8+ T cells. This work indicates that the MVA version of the D10 decapping enzyme, overexpressed using a plasmid, is functional, but its negative effect on transgene expression by recombinant MVA cannot be overcome by the use of the EMCV IRES inserted upstream of the transgene initiation codon.
    • References:
      Immunol Lett. 1990 Aug;25(1-3):27-31. (PMID: 1704348)
      Methods Mol Biol. 2012;890:37-57. (PMID: 22688760)
      J Gen Virol. 2013 Dec;94(Pt 12):2771-6. (PMID: 24077296)
      J Gen Virol. 2005 May;86(Pt 5):1279-90. (PMID: 15831938)
      PLoS One. 2011;6(5):e20067. (PMID: 21603645)
      J Virol. 2006 Jan;80(2):553-61. (PMID: 16378957)
      PLoS One. 2013 Jun 27;8(6):e66894. (PMID: 23826170)
      DNA Cell Biol. 2009 Mar;28(3):103-8. (PMID: 19182996)
      Nucleic Acids Res. 2005;33(4):e36. (PMID: 15731329)
      Vaccine. 2011 Apr 12;29(17):3276-83. (PMID: 21352940)
      J Virol. 2008 Apr;82(8):3822-33. (PMID: 18256155)
      Methods. 2001 Dec;25(4):402-8. (PMID: 11846609)
      J Immunol. 2008 Jun 1;180(11):7193-202. (PMID: 18490718)
      Curr Opin Struct Biol. 2009 Jun;19(3):267-76. (PMID: 19362464)
      Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2139-44. (PMID: 17283339)
      Protein Eng Des Sel. 2006 Sep;19(9):391-400. (PMID: 16857694)
      Nucleic Acids Res. 1995 Sep 25;23(18):3656-63. (PMID: 7478993)
      PLoS One. 2012;7(6):e40167. (PMID: 22761956)
      J Virol. 2006 Jul;80(13):6318-23. (PMID: 16775319)
      Virology. 1998 May 10;244(2):365-96. (PMID: 9601507)
      Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126-30. (PMID: 2548200)
      Biochim Biophys Acta. 2009 Sep-Oct;1789(9-10):542-57. (PMID: 19632368)
      Virology. 2006 Jul 5;350(2):276-88. (PMID: 16595141)
      Vaccine. 2013 Sep 6;31(39):4241-6. (PMID: 23523410)
      Biotechniques. 2006 Sep;41(3):283-4, 286, 288 passim. (PMID: 16989088)
      J Virol. 1999 Jan;73(1):791-6. (PMID: 9847390)
      Nucleic Acids Res. 2010 Nov;38(21):7599-610. (PMID: 20639534)
      Vaccine. 2010 Feb 10;28(6):1547-57. (PMID: 19969118)
      PLoS One. 2008;3(2):e1638. (PMID: 18286194)
    • Grant Information:
      G1000527 United Kingdom Medical Research Council
    • Accession Number:
      0 (Antigens, Viral)
      0 (Internal Ribosome Entry Sites)
      0 (RNA Caps)
      0 (RNA, Messenger)
      EC 1.13.12.- (Luciferases)
    • Publication Date:
      Date Created: 20150527 Date Completed: 20160418 Latest Revision: 20181113
    • Publication Date:
      20221213
    • Accession Number:
      PMC4444188
    • Accession Number:
      10.1371/journal.pone.0127978
    • Accession Number:
      26011541