The genomes of RNA viruses are relatively small. To overcome the small-size limitation, RNA viruses assign distinct functions to the processed viral proteins and their precursors. This is exemplified by poliovirus 3CD protein. 3C protein is a protease and RNA-binding protein. 3D protein is an RNA-dependent RNA polymerase (RdRp). 3CD exhibits unique protease and RNA-binding activities relative to 3C and is devoid of RdRp activity. The origin of these differences is unclear, since crystal structure of 3CD revealed “beads-on-a-string” structure with no significant structural differences compared to the fully processed proteins. We performed molecular dynamics (MD) simulations on 3CD to investigate its conformational dynamics. A compact conformation of 3CD was observed that was substantially different from that shown crystallographically. This new conformation explained the unique properties of 3CD relative to the individual proteins. Interestingly, simulations of mutant 3CD showed altered interface. Additionally, accelerated MD simulations uncovered a conformational ensemble of 3CD. When we elucidated the 3CD conformations in solution using small-angle X-ray scattering (SAXS) experiments a range of conformations from extended to compact was revealed, validating the MD simulations. The existence of conformational ensemble of 3CD could be viewed as a way to expand the poliovirus proteome, an observation that may extend to other viruses.

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.