title: Viral evolution as a tool to improve the tetracycline-regulated gene expression system.
abstract: We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.
authors.affiliation: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors.forename: Atze T
authors.initials: AT
authors.lastname: Das
authors.forename: Xue
authors.initials: X
authors.lastname: Zhou
authors.forename: Monique
authors.initials: M
authors.lastname: Vink
authors.forename: Bep
authors.initials: B
authors.lastname: Klaver
authors.forename: Koen
authors.initials: K
authors.lastname: Verhoef
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Marzio
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D014644:Genetic Variation; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D009875:Operator Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection / Q000379:methods; D014779:Virus Replication / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline