Ready, steady, go - viral polymerase at work!

The hepatitis C virus (HCV) is a positive-strand RNA-virus that severely attacks health of 170 000 000 infected humans worldwide. Replication of the viral genome is carried out by the virally encoded RNA-dependent RNA-polymerase NS5B, representing an attractive drug target. Interestingly, the origin of viral genome replication forms a thermodynamically stable stem-loop RNA, thus a barrier of the polymerization reaction. Impairing this RNA-secondary structure is essentially required to incorporate nucleotides based on complementarity into a nascent RNA. Combining functional and structural data, we address to elucidate the molecular mechanisms governing the initiation of viral genome replication. Binding RNA to the HCV-polymerase monitored by fluorescence spectroscopy was found to proceed via several intermediates in intramolecular reactions subsequent to the initial bi-molecular interaction. Moreover, the polymerase-RNA-complex formation was determined to be entropically driven with different modes of binding diverse structured RNAs. Applying NMR spectroscopy being sensitive to protein backbone alterations, RNA was shown to induce a conformational change of the polymerase along with increasing local flexibility. Measuring one-dimensional ¹H NMR spectra sensitive to RNA base pairs, the RNA-induced enzyme conformation has no major consequence on the bound template RNA structure. However, a second NTP-induced conformational transition disrupts the bound stem loop RNA structure. The intrinsic equilibrium of the immobilized RNA is shifted to the single-stranded conformer. A subsequent formation of the product double-stranded RNA could be monitored by this newly established assay. Ultimately, the HCV-polymerase is demonstrated to be fully capable of initiating the viral genome replication involving conformational transitions of the enzyme itself as well as of the substrate template-RNA.