1887

Abstract

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Funding
This study was supported by the:
  • Takeda Science Foundation
    • Principle Award Recipient: SatoshiKomoto
  • Mochida Memorial Foundation for Medical and Pharmaceutical Research
    • Principle Award Recipient: SatoshiKomoto
  • Japan Society for the Promotion of Science (Award 18K07150)
    • Principle Award Recipient: SatoshiKomoto
  • Japan Agency for Medical Research and Development (Award 19fk0108034h1103 and 20fk0108121h0601)
    • Principle Award Recipient: SatoshiKomoto
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/content/journal/jgv/10.1099/jgv.0.001587
2021-04-12
2021-05-15
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References

  1. Tate JE, Burton AH, Boschi-Pinto C, Parashar UD. World Health Organization–Coordinated Global Rotavirus Surveillance Network Global, regional, and national estimates of rotavirus mortality in children. Clin Infect Dis 2016; 62:S96–S105 [CrossRef][PubMed]
    [Google Scholar]
  2. Troeger C, Khalil IA, Rao PC, Cao S, Blacker BF et al. Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years. JAMA Pediatr 2018; 172:956–965 [CrossRef][PubMed]
    [Google Scholar]
  3. Estes MK, Greenberg HB et al. Rotaviruses, p 1347-1401. In Knipe DM, Howley PM. (editors) Fields Virology, 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2013
    [Google Scholar]
  4. Kanai Y, Komoto S, Kawagishi T, Nouda R, Nagasawa N et al. Entirely plasmid-based reverse genetics system for rotaviruses. Proc Natl Acad Sci U S A 2017; 114:2349–2354 [CrossRef][PubMed]
    [Google Scholar]
  5. Komoto S, Fukuda S, Ide T, Ito N, Sugiyama M et al. Generation of recombinant rotaviruses expressing fluorescent proteins by using an optimized reverse genetics system. J Virol 2018; 92:e00588–18 [CrossRef][PubMed]
    [Google Scholar]
  6. Komoto S, Fukuda S, Kugita M, Hatazawa R, Koyama C et al. Generation of infectious recombinant human rotaviruses from just 11 cloned cDNAs encoding the rotavirus genome. J Virol 2019; 93:e02207–02218 [CrossRef][PubMed]
    [Google Scholar]
  7. Sánchez-Tacuba L, Feng N, Meade NJ, Mellits KH, Jaïs PH et al. An optimized reverse genetics system suitable for efficient recovery of simian, human, and murine-like rotaviruses. J Virol 2020; 94:e01294–20 [CrossRef][PubMed]
    [Google Scholar]
  8. Philip AA, Perry JL, Eaton HE, Shmulevitz M, Hyser JM et al. Generation of recombinant rotavirus expressing NSP3-UnaG fusion protein by a simplified reverse genetics system. J Virol 2019; 93:e01616–01619 [CrossRef][PubMed]
    [Google Scholar]
  9. Taniguchi K, Kojima K, Urasawa S. Nondefective rotavirus mutants with an NSP1 gene which has a deletion of 500 nucleotides, including a cysteine-rich zinc finger motif-encoding region (nucleotides 156 to 248), or which has a nonsense codon at nucleotides 153-155. J Virol 1996; 70:4125–4130 [CrossRef][PubMed]
    [Google Scholar]
  10. Hiramoto T, Tahara M, Liao J, Soda Y, Miura Y et al. Non-Transmissible mV vector with segmented RNA genome establishes different types of iPSCs from hematopoietic cells. Mol Ther 2020; 28:129–141 [CrossRef][PubMed]
    [Google Scholar]
  11. Liu Z, Chen O, Wall BJ, Zheng M, Zhou Y et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 2017; 7:2193 [CrossRef][PubMed]
    [Google Scholar]
  12. Mochiduki Y, Okita K. Methods for iPS cell generation for basic research and clinical applications. Biotechnol J 2012; 7:789–797 [CrossRef][PubMed]
    [Google Scholar]
  13. Szymczak AL, Vignali DA. Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin Biol Ther 2005; 5:627–638 [CrossRef][PubMed]
    [Google Scholar]
  14. Breen M, Nogales A, Baker SF, Martínez-Sobrido L. Replication-Competent influenza A viruses expressing reporter genes. Viruses 2016; 8:E179 [CrossRef][PubMed]
    [Google Scholar]
  15. Sakai Y, Kiyotani K, Fukumura M, Asakawa M, Kato A et al. Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication. FEBS Lett 1999; 456:221–226 [CrossRef][PubMed]
    [Google Scholar]
  16. Skiadopoulos MH, Surman SR, Durbin AP, Collins PL, Murphy BR. Long nucleotide insertions between the HN and L protein coding regions of human parainfluenza virus type 3 yield viruses with temperature-sensitive and attenuation phenotypes. Virology 2000; 272:225–234 [CrossRef][PubMed]
    [Google Scholar]
  17. Takeda M, Nakatsu Y, Ohno S, Seki F, Tahara M et al. Generation of measles virus with a segmented RNA genome. J Virol 2006; 80:4242–4248 [CrossRef][PubMed]
    [Google Scholar]
  18. Barro M, Patton JT. Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci U S A 2005; 102:4114–4119 [CrossRef][PubMed]
    [Google Scholar]
  19. Graff JW, Mitzel DN, Weisend CM, Flenniken ML, Hardy ME. Interferon regulatory factor 3 is a cellular partner of rotavirus NSP1. J Virol 2002; 76:9545–9550 [CrossRef][PubMed]
    [Google Scholar]
  20. Kim JH, Lee SR, Li L-H, Park H-J, Park HJ et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 2011; 6:18556 [CrossRef][PubMed]
    [Google Scholar]
  21. Sharma P, Yan F, Doronina VA, Escuin-Ordinas H, Ryan MD et al. 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucleic Acids Res 2012; 40:3143–3151 [CrossRef][PubMed]
    [Google Scholar]
  22. Ito N, Takayama-Ito M, Yamada K, Hosokawa J, Sugiyama M et al. Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system. Microbiol Immunol 2003; 47:613–617 [CrossRef][PubMed]
    [Google Scholar]
  23. Taniguchi K, Nishikawa K, Kobayashi N, Urasawa T, Wu H et al. Differences in plaque size and VP4 sequence found in SA11 virus clones having simian authentic VP4. Virology 1994; 198:325–330 [CrossRef][PubMed]
    [Google Scholar]
  24. Komoto S, Sasaki J, Taniguchi K. Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus. Proc Natl Acad Sci U S A 2006; 103:4646–4651 [CrossRef][PubMed]
    [Google Scholar]
  25. Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA. Embo J 1994; 13:4195–4203[PubMed]
    [Google Scholar]
  26. Fukuda S, Hatazawa R, Kawamura Y, Yoshikawa T, Murata T et al. Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics. J Gen Virol 2020; 101:806–815 [CrossRef][PubMed]
    [Google Scholar]
  27. Taniguchi K, Morita Y, Urasawa T, Urasawa S. Cross-Reactive neutralization epitopes on VP3 of human rotavirus: analysis with monoclonal antibodies and antigenic variants. J Virol 1987; 61:1726–1730 [CrossRef][PubMed]
    [Google Scholar]
  28. Urasawa S, Urasawa T, Taniguchi K. Three human rotavirus serotypes demonstrated by plaque neutralization of isolated strains. Infect Immun 1982; 38:781–784 [CrossRef][PubMed]
    [Google Scholar]
  29. Kumamoto K, Iguchi T, Ishida R, Uemura T, Sato M et al. Developmental downregulation of LIS1 expression limits axonal extension and allows axon pruning. Biol Open 2017; 6:1041–1055 [CrossRef][PubMed]
    [Google Scholar]
  30. Komoto S, Kanai Y, Fukuda S, Kugita M, Kawagishi T et al. Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell culture. J Virol 2017; 91:e00695–17 [CrossRef][PubMed]
    [Google Scholar]
  31. Philip AA, Doucette KK, Rasal TA, Patton JT. Rotaviruses as neonatal vaccine expression vectors against other enteric pathogens. Proceedings 2020; 50:53 [CrossRef]
    [Google Scholar]
  32. Philip AA, Patton JT. Expression of separate heterologous proteins from the rotavirus NSP3 genome segment using a translational 2A stop-restart element. J Virol 2020; 94:e00959–20 [CrossRef][PubMed]
    [Google Scholar]
  33. Hundley F, McIntyre M, Clark B, Beards G, Wood D et al. Heterogeneity of genome rearrangements in rotaviruses isolated from a chronically infected immunodeficient child. J Virol 1987; 61:3365–3372 [CrossRef][PubMed]
    [Google Scholar]
  34. McIntyre M, Rosenbaum V, Rappold W, Desselberger M, Wood D et al. Biophysical characterization of rotavirus particles containing rearranged genomes. J Gen Virol 1987; 68:2961–2966 [CrossRef][PubMed]
    [Google Scholar]
  35. Desselberger U. What are the limits of the packaging capacity for genomic RNA in the cores of rotaviruses and of other members of the Reoviridae?. Virus Res 2020; 276:197822 [CrossRef][PubMed]
    [Google Scholar]
  36. Komoto S, Fukuda S, Hatazawa R, Murata T, Taniguchi K. Generation of recombinant rotaviruses from just 11 cDNAs encoding a viral genome. Virus Res 2020; 286:198075 [CrossRef][PubMed]
    [Google Scholar]
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