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Transgenic expression of PB2 in 293T cells increases avian influenza virus packaging in reverse genetic systems
  • +9
  • ying Xie,
  • Ying Cui,
  • Xinghai Zhang,
  • Yuanguo Li,
  • Weiyang Sun,
  • Na Feng,
  • Tiecheng Wang,
  • Yongkun Zhao,
  • Songtao Yang,
  • Yuwei Gao,
  • Chuan Qin,
  • Xianzhu Xia
ying Xie
Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center
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Ying Cui
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Xinghai Zhang
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Yuanguo Li
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Weiyang Sun
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Na Feng
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Tiecheng Wang
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Yongkun Zhao
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Songtao Yang
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Yuwei Gao
Military Veterinary Research Institute of Academy of Military Medical Sciences
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Chuan Qin
Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center
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Xianzhu Xia
Chinese Academy of Medical Sciences (CAMS) and Comparative Medicine Center

Corresponding Author:[email protected]

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Abstract

Plasmid-based reverse genetics has transformed influenza virus research by enabling the production of recombinant influenza viruses from cloned cDNA copies of viral genome segments. Reverse genetic production of influenza viruses requires cellular expression of influenza proteins polymerase basic 1, polymerase basic 2, polymerase acidic and nucleoprotein which collectively allow for transcription of viral mRNA and synthesis of new negative-sense genomic RNA, thus enabling synthesis of all components needed to assemble infectious virus from transfected cell lines. Given the importance of these proteins in the generation of influenza viruses via reverse genetics, we sought to explore how transgenic expression of mammalian-adapted PB1, PB2, PA, or NP in the 293T packaging cell line may impact the recovery of recombinant influenza viruses. We constructed four transgenic 293T cell lines expressing PB1, PB2, PA or NP derived from the mouse-adapted 2009 pandemic influenza A virus, UI182. Transgenic expression of UI182 PB2 in 293T cells enhanced recovery of replication-competent avian influenza viruses generated by reverse genetics relative to levels achieved in unmodified 293T cells. Virus recovered from PB2-expressing 293T cells replicated with kinetics that were indistinguishable from viruses recovered from unmodified 293T cells. Provision of UI182 PB2 protein via transgenic expression in 293T cells resulted in enhanced viral polymerase activity as measured by a minigenome assay, which may account for the improved efficiency of viral packaging relative to unmodified 293T cells. Transgenic expression of mammalian-adapted PB2 in 293T cells may serve as an important tool for enhancing influenza virus recovery in reverse genetic systems.