Chemogenetic ON and OFF switches for RNA virus replication

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.

A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition

“…Based on our previous experience designing conditional viruses with the HIV-1 protease 19 , we generated an inhibitor-off VSV-based system with SARS-CoV-2 3CLpro. The inhibitor-off system is based on a polyprotein consisting of an N-terminal reporter, followed by the SARS-CoV-2 3CLpro, and the C-terminal VSV polymerase L. Cognate 3CLpro cleavage sites are located between the reporter and 3CLpro as well as between 3CLpro and L. This virus is replication-proficient due to efficient 3CLpro-catalyzed cleavage and release of functional L. Virus replication leads to an accumulation of strong reporter signal over time (e.g., GFP and luciferase in Supplementary Fig.…”

“…To facilitate SARS-CoV-2 3CLpro characterization and drug development, we sought to create a safe, biosafety level 1-based cellular assay based on our prior work using vesicular stomatitis virus (VSV) 19 . VSV is a non-segmented negative-strand RNA virus of the family of Rhabdoviridae 20 .…”

“…We recently leveraged this knowledge by generating two chimeric VSV constructs. In the inhibitor-off system, the viral polymerase L is tagged on the N-terminus with HIV-1 protease, eGFP, and an HIV-1 protease cleavage site 19 . Only after tag removal by proteolytic cleavage is L protein function restored and VSV able to replicate.…”

“…Thus, protease inhibitors prevent cleavage and inhibit virus replication. In the inhibitor-on system, the HIV-1 protease flanked by its cognate cleavage sites are inserted into the viral P protein 19 . This chimeric construct is unable to replicate due to HIV-1 protease catalyzing the cleavage of the VSV P protein into separate non-functional pieces.…”

Engineering RNA viruses with unnatural amino acid to evoke adjustable immune response in mice

“…RNA viruses account for the majority of human viral infections 1 and remain difficult to control despite the availability of several working vaccine strategies [2][3][4] . While synthetic biology holds great potential for biomedical engineering, it is limited in accessing vector design for gene therapy [5][6][7][8] , which renders it far from practical in vaccine production 9,10 . Therefore, by integrating advances in reverse genetics 11,12 with insights from system vaccinology 13,14 , viral-engineering methods could be adapted to effective and smart vaccine design in a novel way.…”