Abstract
Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. Here we present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. Virus activity can be en- or disabled by various HIV protease inhibitors. Incorporating the HIV protease dimer in the genome of vesicular stomatitis virus (VSV) into the open reading frame of either the P- or L-protein resulted in an ON switch. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may also be applicable to other potentially therapeutic RNA viruses.
Highlights
Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern
Cells transfected with this plasmid and co-infected with a P-protein-deficient vesicular stomatitis virus (VSV) variant encoding DsRed[23] showed red fluorescence in the presence, but not absence of the protease inhibitors (PIs) amprenavir (APV) (Supplementary Fig. 1b)
Western blotting against human immunodeficiency virus (HIV) protease confirmed the proteolytic cleavage in the absence of PI with a band marking the HIV protease dimer at ~22 kDa19, compared to a band at 54 kDa corresponding to the previously reported to tolerate intramolecular insertions22 (Pprot) fusion protein in the presence of PI (Fig. 1c).To address genetic stability, we performed in vitro serial virus passages in optimal (10 μM) and suboptimal (1 μM) amprenavir conditions
Summary
Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. We present a regulator switch for RNA viruses using a conditional protease approach, in which the function of at least one viral protein essential for transcription and replication is linked to autocatalytical, exogenous human immunodeficiency virus (HIV) protease activity. An N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. This technology may be applicable to other potentially therapeutic RNA viruses. We present a control mechanism for RNA viruses by fusing the autocatalytically active HIV protease dimer and its corresponding cleavage sites into or adjacent to essential viral proteins of VSV. The conditional proteolytic switch system allowed the control of RNA-virus replication from complete inhibition to nearly wild-type replication levels
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