Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

Abstract

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 pyrazinecarboxamide 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.

Imprecise recombinant viruses evolve via a fitness-driven, iterative process of polymerase template-switching events

“…While the evidence highlights a role for in trans template-switching events, it remains unclear as to whether in cis events are also driving the evolution of viruses with imprecise genomes. Ongoing studies investigating the behaviour of polymerases during RNA synthesis suggests that both in trans template-switching, and in cis copy-back type recombination events can occur [ 39 , 40 ], and such research may offer valuable insights into our further understanding of this fundamental process.…”

A Randomized, Controlled Study on the Safety and Efficacy of Maraviroc and/or Favipiravir vs Currently Used Therapy in Severe COVID-19 Adults. “COMVIVIR” Trial.

“…One of these antivirals is Favipiravir (FPV), that directly inhibits viral replication and transcription by selective inhibition of the viral enzyme RNA-dependent RNA polymerase (RdRP) (37,38). FPV is a ribonucleotide analogue (fluorinated base analogue with a pyrazine carboxamide) (39). Studies of the nucleotide addition to the elongation complex of the viral RdRP showed that FPV is capable to make RdRP pause during the reading process, which leads to backtracking (an attempt to recover reading errors), that when repetitive, causes the enzyme to interpret them as a prematurely terminated product, thus stopping the elongation and therefore interrupting the viral replication process (40).…”

“…We hypothesized that an effective treatment for COVID-19 severe patients should combine a modulator of the immune response with a direct antiviral drug against SARS-CoV-2, in order to address both the hyperin ammatory effects of the immune dysregulation and the viral load, thus yielding best results. Favipiravir (FPV), directly inhibits viral replication and transcription by selective inhibition of the viral enzyme RNA-dependent RNA polymerase (RdRP) (41)(42)(43)(44).…”

A Randomized, Controlled Study on the Safety and Efficacy of Maraviroc and/or Favipiravir vs Currently Used Therapy in Severe COVID-19 Adults. “COMVIVIR” Trial.

“…One of these antivirals is Favipiravir (FPV), that directly inhibits viral replication and transcription by selective inhibition of the viral enzyme RNA-dependent RNA polymerase (RdRP)(37,38). FPV is a ribonucleotide analogue (fluorinated base analogue with a pyrazine carboxamide)(39). Studies of the nucleotide addition to the elongation complex of the viral RdRP showed that FPV is capable to make RdRP pause during the reading process, which leads to backtracking (an attempt to recover reading errors), that when repetitive, causes the enzyme to interpret them as a prematurely terminated product, thus stopping the elongation and therefore interrupting the viral replication process(40).FPV has been used successfully against A H1N1 influenza(41,42).…”