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.Technology for the regulation of gene expression in mammalian cells and tissues is of primary importance for a wide variety of basic and applied biological research areas, including functional genomics, gene therapy, animal models for human diseases, and biopharmaceutical protein production. All these applications require that production of the protein(s) of interest be regulated in both a quantitative and temporal way. For this purpose, artificial gene expression systems have been developed that are controlled by effector molecules in a dose-dependent and reversible manner. The most frequently used regulatory circuit is the so-called Tet system, which allows stringent control of gene expression by tetracycline (Tc) 1 or its derivative doxycycline (dox) (1-3). The Tet system is based on the specific, high affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) sequence. Tc and dox induce a conformational change in TetR, which impedes the interaction with tetO. Fusion of the activation domain of the herpes simplex virus VP16 protein to TetR resulted in the transcriptional activator tTA, which induces gene expression from promoters placed downstream of tetO elements (P tet ) in eukaryotic cells. The presence of Tc or dox abolishes this gene expression. A tTA variant with four amino acid substitutions in the TetR moiety exhibits a reverse phenotype (4). This reverse tTA (rtTA) binds to P tet , and activates gene expression in the presence of dox but not in its absence. The Tet system is now widely applied to control gene expression in eukaryotes, including mammals, plants, and insects (reviewed in Ref. 1). Since the Tet system originates from a bacterial regulatory system, it seems likely that the components can be optimized for their new transcriptional function i...

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