Development of a T7 RNA polymerase expressing cell line using lentivirus vectors for the recovery of recombinant Newcastle disease virus

Yeong MY 1 , Cheow PS 2 , Abdullah S 3 , Song AA 4 , Lei-Rossmann J 5 , Tan TK 6 Show all authors , Yusoff K 7 , Chia SL 8

Affiliations 

  • 1 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Electronic address: gs48337@student.upm.edu.sg
  • 2 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Electronic address: gs51522@student.upm.edu.my
  • 3 UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Electronic address: syahril@upm.edu.my
  • 4 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Electronic address: adelene@upm.edu.my
  • 5 Anticancer Viruses and Cancer Vaccines Research Group, Department of Oncology, University of Oxford, OX3 7DQ, Oxford, United Kingdom. Electronic address: janet.lei@oncology.ox.ac.uk
  • 6 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS, Oxford, United Kingdom. Electronic address: tiong.tan@rdm.ox.ac.uk
  • 7 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia; UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia; Malaysia Genome Institute, Ministry of Science, Technology and Innovation, Jalan Bangi, 43000 Kajang, Selangor Darul Ehsan, Malaysia. Electronic address: kyusoff@upm.edu.my
  • 8 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia; UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia. Electronic address: suetlin@upm.edu.my
J Virol Methods, 2021 05;291:114099.
PMID: 33592218 DOI: 10.1016/j.jviromet.2021.114099

Abstract

The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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