title: Efficient and RobustParamyxoviridaeReverse Genetics Systems.
abstract: The notoriously low efficiency ofParamyxoviridaereverse 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 majorParamyxoviridaegenera (from ~1 in 106-107to ~1 in 102-103transfected 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.IMPORTANCEThe ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors.
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Beaty
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Arnold
authors.identifier: 0000-0001-8916-2230
authors.initials: A
authors.lastname: Park
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Sohui T
authors.initials: ST
authors.lastname: Won
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Hong
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Lyons
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Frederic
authors.initials: F
authors.lastname: Vigant
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Freiberg
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: tenOever
authors.affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA.
authors.forename: W Paul
authors.initials: WP
authors.lastname: Duprex
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Benhur
authors.initials: B
authors.lastname: Lee
keywords: paramyxovirus; reverse genetic analysis; ribozymes