Key result
The pyrazine-carboxamide ribonucleotide analogue T-1106 induces viral RNA-dependent RNA polymerase pausing and backtracking, promoting copy-back RNA synthesis and homologous RNA recombination.
The study identifies RdRp-catalyzed intra- and intermolecular template switching as a novel mechanistic target for broad-spectrum antivirals, demonstrating that ribonucleotide analogues like T-1106 can promote the formation of defective viral genomes.
T-1106-induced RdRp pausing promotes defective viral genomes; hypothesis-generating for RdRp-targeted antivirals, translation to humans remains open.
The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.
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Janissen et al. (2020) studied RNA virus infection (Poliovirus, Enterovirus A71). T-1106 (pyrazine-carboxamide ribonucleotide analogue) vs. Control (absence of T-1106) was evaluated on RdRp elongation dynamics and viral recombination. The pyrazine-carboxamide ribonucleotide analogue T-1106 induces viral RNA-dependent RNA polymerase pausing and backtracking, promoting copy-back RNA synthesis and homologous RNA recombination.
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