Structural Insights into the Enterovirus Replication Platform: A Potential Target for Inhibiting Viral Replication

Viral replication is a complex and vital part of the picornavirus life cycle. A key structure in this process is the predicted cloverleaf located at the 5’ end of the positive-sense strand (5’CL) or at the 3’ end of the negative-sense strand (3’CL). To develop effective antiviral treatments against enteroviruses, it is crucial to better understand the role of the cloverleaf structure in viral replication. This dissertation aims to deepen the understanding of the conservation, structure, and dynamics of the enterovirus replication platform through three projects. First, the conservation and divergence of 209 enterovirus 5’CL were analyzed using bioinformatic tools. By classifying the CLs into six distinct groups, regions of high conservation found in all studied 5’CLs included known protein interaction sites as well as regions with unknown functions. Therefore, this work offers a more detailed understanding of both sequence and structural conservation and divergence, which could be useful for developing a broad-spectrum antiviral. Second, the conformational structure of the 5’CLs in solution was studied using various experimental techniques, including circular dichroism (CD), size exclusion chromatography (SEC), and small-angle X-ray scattering (SAXS). It was found that the behavior of the 5’CL in solution greatly depends on factors such as serotype, ionic conditions, and the tendency to dimerize. Therefore, this work highlights the importance of combining crystal and solution methods to better understand the CL structure. Finally, the tertiary structure of the 3’CL of Coxsackievirus B3 (CVB3) is examined using experimental methods, such as NMR and SAXS, combined with computational methods. Investigating the template tertiary structure provided insights into the monomer structure of the CVB3 3’CL, as well as its tendency for dimerization. This work aims to establish a structural baseline for the 3’CL to gain insight into potential antiviral targets. Overall, the work presented in this dissertation contributes valuable insights into the structural properties and conservation of the enterovirus cloverleaf, enhancing our understanding of the potential of the 5’CL or 3’CL as an antiviral target.