Key result
Increasing enzyme concentration and GTP stimulate de novo initiation by the HCV RNA-dependent RNA polymerase, supporting a model where initiation is stimulated by oligomeric contacts between subunits.
The study demonstrates that the de novo initiation-competent conformation of the HCV RdRp is stimulated by oligomeric contacts between individual subunits.
May guide HCV antiviral design targeting polymerase contacts; leaves open in vivo validation before therapeutic pursuit.
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Delta1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.
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Chinnaswamy et al. (2010) studied Hepatitis C Virus. Enzyme concentration and GTP was evaluated on De novo initiation of RNA synthesis. Increasing enzyme concentration and GTP stimulate de novo initiation by the HCV RNA-dependent RNA polymerase, supporting a model where initiation is stimulated by oligomeric contacts between subunits.
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