Relación estructura-función de la polimerasa del virus de la hepatitis C (NS5B)

Abstract

The hepatitis C Virus (HCV) RNA-dependent RNA-polimerase (NS5B) shows a great genetic diversity reflected in the high number of circulating genotypes and subtypes. This polymerase initiates RNA synthesis by a de novo mechanism. This process implies conformational changes that direct transition from the initiating complex to a processive elongation complex. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, while information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The Vmax/Km ratios were statistically different between genotype 1 and the other genotypes, mainly due to differences in Vmax values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the ¿F helix as a structural component probably involved in NS5B-NS5B interactions. In order to address the importance of this process, we have analyzed the mechanisms of intermolecular interactions to control conformational changes, dissecting biochemical characteristics of the initiation and the elongation process. Using protein-protein docking and Molecular Dynamics we propose an energetically plausible model for this interaction, connecting different structural elements from NS5B as ß-loop and T-helix at ¿thumb¿ subdomain, and F-helix at ¿fingers¿ subdomain. Based on this model and previous experimental data, we have designed a series of interaction mutants (Glu128Ala, Asp220Ala, His502A, Asp220Ala/His502Ala, and Arg114Glu to address the relevance of NS5B-NS5B interactions in the different steps of RNA synthesis. Both structural and biochemical data suggest that NS5B self-interaction regulates early steps of RNA synthesis, controlling the rate limiting process of transition from the distributive initiation to the proccesive elongation.