Title: Structural and biochemical studies of the poliovirus precursor protein 3CD and the components of the viral replication complex
Description: The replication of poliovirus RNA requires the formation of a replication complex composed of both viral and cellular proteins. The viral protein 3CD is a multi-functional precursor to the poliovirus protease, 3C pro , and the RNA-dependent RNA polymerase, 3D pol . Both 3CD and 3D pol facilitate the uridylylation of the viral peptide VPg, which serves to prime RNA synthesis. However, the composition and specific interactions between the proteins and RNA of the initiation and uridylylation complexes are not well understood. To further our understanding of these viral processes, the crystal structures of 3CD and two 3D pol fidelity mutants were solved. The structures of the polymerase fidelity mutants, 3D pol G64S and 3D pol H273R, were solved to a resolution of 3.0 Å. Both mutants show subtle variations in structure that likely contribute to their unique phenotypes. The crystal structure of poliovirus 3CD was also solved to a resolution of 3.4 Å. The 3C and 3D domains of 3CD are joined by a poorly ordered polypeptide linker in an arrangement that precludes autocatalysis of 3CD. Although 3CD is not an active polymerase, the 3D pol active site remains largely intact in the 3CD structure. A hydrogen-bonding network present at the N-terminus of 3D pol is absent in 3CD, suggesting that changes in molecular flexibility may be important factors in polymerase activity and fidelity. The crystal structure of 3CD reveals significant contacts between the 3D domain of one 3CD molecule and the 3C and 3D domains of another molecule. The arrangement and size of these interfaces suggests a possible role in the stabilization of a uridylylation complex. The biological relevance of these interfaces was validated through in vitro uridylylation assays.

Fluorescence anisotropy experiments were designed to quantitatively determine binding constants between 3CD, the cellular protein PCBP2, and the 5' cloverleaf RNA, which together form a ternary complex that regulates viral replication and translation. A qualitative assessment of the raw anisotropy data suggests that 3CD recognizes specific structural elements within the stem-loop RNA rather than binding to a particular RNA sequence.