1887

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Volume 102, Issue 4

Strategy for generation of replication–competent recombinant rotaviruses expressing multiple foreign genes Free

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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): multivalent viral vector , NSP1 segment , rearrangement , reverse genetics , rotavirus and self-cleaving 2A sequence
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
© 2021 The Authors
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2021-04-12
2024-04-25
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Strategy for generation of replication–competent recombinant rotaviruses expressing multiple foreign genes
J. Gen. Virol. 102, 001587 (2021); https://doi.org/10.1099/jgv.0.001587
/content/journal/jgv/10.1099/jgv.0.001587
/content/journal/jgv/10.1099/jgv.0.001587
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