![]() In contrast, N-terminal fusion to L resulted in an OFF-switch when PI was applied. The intramolecular insertion of the protease dimer in either the P or L protein of VSV constituted a functional ON-switch. Adding PI compounds either enabled or blocked virus replication, depending on the fusion design. Here 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. HIV PR activity can be regulated by several clinically approved protease inhibitors (PIs) that are widely used in HIV therapy. In HIV, it autocatalytically cleaves the polyprotein that is translated from the positive strand genome into functional proteins. The human immunodeficiency virus HIV protease (PR) is comprised of 99 amino acids, is active as a homodimer and functions as an aspartyl protease 18. The genes are separated by intergenic regions that enable the transcription of several viral mRNAs from one template RNA 17. The VSV RNA genome contains five viral genes coding for nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase or large-protein (L) 16. In addition, its use in veterinary medicine is limited by the risk of spread among livestock. Although numerous VSV variants are currently under clinical development 14, 15, concerns regarding potential neurotoxicity in immunocompromised hosts or in situations with exposure to the central nervous system have curbed a more rapid translational advance. Vesicular stomatitis virus (VSV), a rapidly replicating negative strand RNA virus, has been widely studied as a vaccine vector 11, oncolytic virus 12, and tracing tool 13. None of these switches could completely block virus replication. An externally tunable ON switch for RNA viruses has so far only been shown with photo responsive elements 10 which would be impractical for most clinical applications. In addition, OFF-switch control of measles virus RNA replication was shown via small molecule-assisted shutoff (SMASh)–tags fused C-terminally to the viral P protein 9. Of those, RNA-aptazymes fused to a viral gene were shown to regulate virus replication over a range of <100-fold 7 and viral transgene expression up to 30-fold 8. In recent years, a small number of systems have been explored to provide some level of RNA regulation. While control of therapeutic DNA viruses has long been established using for example tetracycline-controlled transcriptional activation 5, 6, effective external control of RNA viruses remains a challenge. ![]() If replication of viruses or viral vectors could be externally regulated on demand, their individual and environmental safety would increase dramatically. Viruses have shown great potential as gene therapy vectors 1, as oncolytic viruses in cancer therapy 2 and as vaccines in humans and animals 3, 4. ![]() This technology may also be applicable to other potentially therapeutic RNA viruses. Conversely, an N-terminal VSV polymerase tag with the HIV protease dimer constitutes an OFF switch, as application of protease inhibitor stops virus activity. Here, virus activity depends on co-application of protease inhibitor in a dose-dependent manner. 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. Virus activity can be en- or disabled by various HIV protease inhibitors. ![]() 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. Therapeutic application of RNA viruses as oncolytic agents or gene vectors requires a tight control of virus activity if toxicity is a concern. ![]()
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