| RNA viruses are causative agents of numerous human and animal diseases, such as hepatitis (hepatitis C virus), common cold (rhinoviruses), influenza, foot-and-mouth disease, hemorrhagic fever (Ebola and Dengue viruses), encephalitis and meningitis (West Nile, coxsackievirus, echoviruses), and myocarditis (coxsackievirus). Although some progress in vaccine and antiviral drug development has been made in recent years, there is still a great demand in creating more powerful and versatile antiviral compounds. The viral RNA-dependent RNA polymerase (RdRp) is a key enzyme in viral genome replication and represents a unique target for antiviral drug development. Lethal mutagenesis of the RNA viruses has been recently explored as a promising antiviral strategy. Therefore, understanding structural determinants of the polymerase fidelity will strongly facilitate rational drug design.;In this thesis, studies on structure-function relationships of poliovirus (PV) polymerase, 3Dpol, and the mechanistic basis for RdRp fidelity are described, including biochemical and biological evaluation of three 3Dpol derivatives: Glu-297, Tyr-30 and Arg-273. Previous studies of PV RdRp have shown that Asn-297 permits the enzyme to distinguish ribose from 2'-deoxyribose. Mutation of this highly conserved amino acid residue to Glu, which is present in all phage RdRps, resulted in a 3Dpol derivative with decreased efficiency of deoxyribonucleotide incorporation. At the same time, the fidelity of the ribonucleotide incorporation displayed by Glu-297 3Dpol was substantially reduced, providing a mechanistic explanation for the elevated mutation frequency observed for RNA phages. In addition, evaluation of the protein-primed initiation reaction catalyzed by 3Dpol led us to the conclusion that even though the same polymerase active site is employed in both reactions, substantial structural differences exist between initiation and elongation complexes. Moreover, Glu-297 3Dpol was able to excise deoxyribonucleotides, suggesting that RNA phage RdRps might have utilized this ability to proofread. We introduced Phe-30 to Tyr mutation in the PV 3Dpol in order to disrupt a strong interaction between the fingers and thumb subdomains of the polymerase, observed crystallographically. Contrary to our expectations, Tyr-30 mutation had no effect on the polymerase function. Unexpectedly, this mutation within 3D domain altered an unknown 3CD function after genome translation and replication. This discovery shed some light on a potential role of 3CD in virus maturation and/or viral RNA packaging; the mechanisms of both of these processes are still unknown. The last, but not least, 3Dpol derivative described in this thesis is the Arg-273 3Dpol. Although residue 273 is located about 20 A from the active site of the polymerase, mutation changing wildtype amino acid residue His-273 to Arg resulted in both a PV and a polymerase with a mutator phenotype. Biochemical evaluation of the Arg-273 3Dpol showed that the conformational change preceding the chemistry step is accountable for the relaxed fidelity observed for this polymerase; X-ray analysis of the Arg-273 3Dpol did not reveal any substantial changes in the crystal structure when compared to the WT 3Dpol, suggesting that Arg-273 may alter conformational flexibility of the enzyme. Arg-273 PV, in combination with the WT and high fidelity Ser-64 PV represents a unique experimental system for exploring relationships between virus fidelity, fitness, and pathogenicity.Together, these studies provide insight into yet unexplored properties of the 3Dpol and provide basis for further studies on the polymerase fidelity, virus maturation and virus-host interactions and towards designing novel strategies to treat RNA viruses. |
