Research Center of Emerging Viral Infections, Chang Gung University
The ever-increasing emerging viral threats contribute to the mounting demand for antivirals. By applying the magnetic tweezer technique, different mechanisms of antiviral nucleotides at single-molecule level were revealed. Besides the chain terminator and mutagen, a novel nucleotide antiviral, T-1106, is found to be capable of inducing heavy genetic recombination to cause genome rewriting, hence the viral replication is inhibited. Our findings highlight the importance of selecting appropriate nucleotide antivirals to tackling RNA viruses, especially for virus with proofreading activity such as SARS-CoV-2.
The evolutionary conserved RNA polymerases of RNA virus are ideal targets for broad-spectrum antiviral development, thus different nucleotide antivirals have been developed. However, the error-prone replication of RNA polymerase not only contributes to viral evolution, but also results in the emergence of drug-resistant virus. Meanwhile, recombination of viral genomes further accelerates these processes. Understanding the underlying mechanism at the molecular level inevitably serves as the foundation of evaluating the efficacy of the antivirals. With the support of Human Frontier Science Program (HFSP), an international collaborative research project led by Dr. Shin-Ru Shih (Chang Gung University, Taiwan), Dr. Nynke Dekker (TU Delft, Netherlands) and Dr. Craig Cameron (Pennsylvania State University, USA) has been initiated since 2015 to solve the mystery of recombination by combinatorial strategies, including next-generation sequencing, cell-based recombination assay, singe-molecule technique, structural modeling, and animal studies.
Modes of action of different nucleotide antivirals against viral replication vary. For example, incorporation of nucleotide analogue remdesivir triphosphate leads to the termination of RNA polymerase while ribavirin alters the base-pairing property to increase the error rate of the RNA polymerase. Our results uncovered another mechanism utilized by nucleotide antivirals where recombination is promoted. Using the magnetic tweezers, proceeding of the RNA polymerase can be monitored near single-base resolution to distinguish the four polymerization steps: stalling (pause), dissociation (backtracking), re-initiation and reversal. Enterovirus A71 (EV-A71) RNA polymerase is featured by extended pauses and increased reversals. The reversal that is barely observable in other viral polymerase is unique to EV-A71 polymerase possibly due to a more open structure adopted near the active center. The open conformation allows the jump of the polymerase from the template to the newly synthesized strand where elongation is re-initiated, this process is called copy-back synthesis. Incorporation of T-1106 triphosphate markedly increased the duration of the pause and more reversals were observed, while another mutation (Y276H) altered the open confirmation to prevent the polymerase dissociation, thus blocking the cell-based recombination and specifically diminishing the reversals. RNA sequencing analyses of the EV-A71 infected cells further confirmed that the T-1106 treatment increased the transcripts of the copy-back synthesis without obvious changes in the mutation rate. Thus T-1106 antagonized EV-A71 replication by inducing recombination catastrophe (error catastrophe?) to generate defective viral genomes.
The importance of our study lies beyond the discovery of antivirals capable of inhibiting viral replication. Besides the introduction of aberrant replication products, copy-back synthesis also produces the double-stranded RNAs which are able to activate the innate immunity. The distinct responses to identical nucleotide antiviral resulted from the subtle variation in the catalytic center of the conserved polymerase is another exciting finding. More aspects of the clinical application regarding the use of nucleotide antivirals can be taken into consideration when combating the RNA virus; meanwhile, structure-based design of antiviral drug candidates opens new avenues to rapidly respond to emerging viruses and design better antivirals in a virus-specific manner in the future. These findings have been published in Molecular cell, 2021, Nov 4; 81(21):4467-4480. (https://pubmed.ncbi.nlm.nih.gov/34687604/)
Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses. Richard Janissen, Andrew Woodman, Djoshkun Shengjuler, Thomas Vallet, Kuo-Ming Lee, Louis Kuijpers, Ibrahim M Moustafa, Fiona Fitzgerald, Peng-Nien Huang, Angela L Perkins, Daniel A Harki, Jamie J Arnold, Belén Solano, Shin-Ru Shih, Marco Vignuzzi, Craig E Cameron, Nynke H Dekker. Mol. Cell (2021), 81(21):4467-4480