Efficient and robust Paramyxoviridae reverse genetics systems

Shannon M. Beaty, Arnold Park, Sohui T. Won, Patrick Hong, Michael Lyons, Frederic Vigant, Alexander Freiberg, Benjamin R. ten Oever, W. Paul Duprex, Benhur Lee

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses.

Original languageEnglish (US)
Article numbere00376-16
JournalmSphere
Volume2
Issue number2
DOIs
StatePublished - Mar 1 2017

Keywords

  • Paramyxovirus
  • Reverse genetic analysis
  • Ribozymes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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    Beaty, S. M., Park, A., Won, S. T., Hong, P., Lyons, M., Vigant, F., Freiberg, A., ten Oever, B. R., Paul Duprex, W., & Lee, B. (2017). Efficient and robust Paramyxoviridae reverse genetics systems. mSphere, 2(2), [e00376-16]. https://doi.org/10.1128/mSphere.00376-16