1887

Abstract

Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth , and WT lethality . To the best of our knowledge, this represents the first time a recombinant virus of with integration of a reporter into a viral ORF has been compared with the WT virus . Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.

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2016-04-01
2021-10-16
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References

  1. Conzelmann K. K. 1998; Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes. Annu Rev Genet 32:123–162 [View Article][PubMed]
    [Google Scholar]
  2. Donnelly M. L., Luke G., Mehrotra A., Li X., Hughes L. E., Gani D., Ryan M. D. 2001; Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 82:1013–1025 [View Article][PubMed]
    [Google Scholar]
  3. Falzarano D., Groseth A., Hoenen T. 2014; Development and application of reporter-expressing mononegaviruses: current challenges and perspectives. Antiviral Res 103:78–87 [View Article][PubMed]
    [Google Scholar]
  4. Holst J., Vignali K. M., Burton A. R., Vignali D. A. 2006; Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat Methods 3:191–197 [View Article][PubMed]
    [Google Scholar]
  5. Kim J. H., Lee S. R., Li L. H., Park H. J., Park J. H., Lee K. Y., Kim M. K., Shin B. A., Choi S. Y. 2011; High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6:e18556 [View Article][PubMed]
    [Google Scholar]
  6. Lo M. K., Peeples M. E., Bellini W. J., Nichol S. T., Rota P. A., Spiropoulou C. F. 2012; Distinct and overlapping roles of Nipah virus P gene products in modulating the human endothelial cell antiviral response. PLoS One 7:e47790 [View Article][PubMed]
    [Google Scholar]
  7. Lo M. K., Nichol S. T., Spiropoulou C. F. 2014; Evaluation of luciferase and GFP-expressing Nipah viruses for rapid quantitative antiviral screening. Antiviral Res 106:53–60 [View Article][PubMed]
    [Google Scholar]
  8. Marsh G. A., Virtue E. R., Smith I., Todd S., Arkinstall R., Frazer L., Monaghan P., Smith G. A., Broder C. C., other authors. 2013; Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets. Virol J 10:95 [View Article][PubMed]
    [Google Scholar]
  9. Minskaia E., Ryan M. D. 2013; Protein coexpression using FMDV 2A: effect of linker residues. BioMed Res Int 2013:291730 [View Article][PubMed]
    [Google Scholar]
  10. Mizuguchi H., Xu Z., Ishii-Watabe A., Uchida E., Hayakawa T. 2000; IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 1:376–382 [View Article][PubMed]
    [Google Scholar]
  11. Szymczak A. L., Workman C. J., Wang Y., Vignali K. M., Dilioglou S., Vanin E. F., Vignali D. A. 2004; Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594 [View Article][PubMed]
    [Google Scholar]
  12. Touzelet O., Loukili N., Pelet T., Fairley D., Curran J., Power U. F. 2009; De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene. Virus Res 140:40–48 [View Article][PubMed]
    [Google Scholar]
  13. Wang Y. E., Park A., Lake M., Pentecost M., Torres B., Yun T. E., Wolf M. C., Holbrook M. R., Freiberg A. N., Lee B. 2010; Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding. PLoS Pathog 6:e1001186 [View Article][PubMed]
    [Google Scholar]
  14. Yoneda M., Guillaume V., Ikeda F., Sakuma Y., Sato H., Wild T. F., Kai C. 2006; Establishment of a Nipah virus rescue system. Proc Natl Acad Sci U S A 103:16508–16513 [View Article][PubMed]
    [Google Scholar]
  15. Yun T., Park A., Hill T. E., Pernet O., Beaty S. M., Juelich T. L., Smith J. K., Zhang L., Wang Y. E., other authors. 2015; Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real-time monitoring of viral spread in small animal models of henipavirus infection. J Virol 89:1242–1253 [View Article][PubMed]
    [Google Scholar]
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