A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis

• Type of Material: Article
• Authors: Anne L. Hotard, Emory University; Fyza Y. Shaikh, Vanderbilt University School of Medicine; Sujin Lee, Emory University; Dan Yan, Emory University; Michael N. Teng, University of South Florida College of Medicine; Richard Karl Plemper, Emory University; James E. Crowe, Vanderbilt University School of Medicine; Martin L Moore, Emory University
• Language: English
• Date: 2012-12-05
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 Published by Elsevier Inc
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.virol.2012.09.022
• Title of Journal or Parent Work: Virology
• ISSN: 0042-6822
• Volume: 434
• Issue: 1
• Start Page: 129
• End Page: 136
• Grant/Funding Information: This work was also supported by the Vanderbilt Medical Scientist Training Program NIGMS/NIH T32 GM007347 and by a grant from the March of Dimes to JEC, as well as NIAID 5R01AI081977 to MNT, and NIH R01 AI071002 to RKP.; This work was supported by the following grants awarded to MLM: NIH 1R01AI087798, NIH 1U19AI095227, and a Children’s Healthcare of Atlanta (CHOA) Center for Immunology and Vaccines Pilot Grant.
• Keywords: reverse genetics; bacterial artificial chromosome; respiratory syncytial virus; recombination-mediated mutagenesis; RNA virus
• Research Categories: Biology, Virology; Biology, Genetics

Abstract

We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered two RSV reporter viruses, one expressing the red fluorescent protein monomeric Katushka 2 (A2-K-line19F) and one expressing Renilla luciferase (A2-RL-line19F). As proof of principle, we efficiently generated a RSV gene deletion mutant (A2-line19FΔNS1/NS2) and a point mutant (A2-K-line19F-I557V) by recombination-mediated BAC mutagenesis. Together with sequence-optimized helper expression plasmids, BAC-RSV is a stable, versatile, and efficient reverse genetics platform for generation of a recombinant Pneumovirus.