RNA replication by poliovirus RNA-dependent RNA polymerase: effects of residue 5 on elongation complex stability and processivity

Abstract

The poliovirus (PV) RNA-dependent RNA polymerase (RdRP) is a small, single-subunit, enzyme that is responsible for the replication of the viral genome. PV genome replication is much more involved than just repetitively incorporating a single nucleotide into an oligonucleotide primer. The polymerase must first go through multiple steps of initiation before processive replication can happen. During primer-dependent initiation, the polymerase must bind the primer/template RNA substrate and undergo a conformational change to form a stable RNA-polymerase complex. After the stable RNA-polymerase complex is formed the polymerase undergoes a second conformational change associated with the addition of the first nucleotide to the primer. It is only after these few steps of initiation that the polymerase can begin processive elongation. The work presented in this dissertation addresses a structure-function relationship related to initiation and processivity of the PV RdRP. Specifically, my work shows that the PV RdRP residue 5 is involved in forming and maintaining the stable, elongation-competent complex. Also, the complex stability can be modulated by downstream RNA interactions or by the number of nucleotides that are incorporated to form the stable complex. Lastly, my data demonstrate evidence of an RNA rearrangement during elongation complex formation and show that the maintenance of a stable elongation complex is required for processive RNA replication, which is required for virus replication. To end, these data elucidate a more complete understanding of the structure-function relationships of the viral RNA polymerase and could eventually facilitate in the design of specific polymerase inhibitors.