Structure of the Picornavirus Replication Platform: A Potential Drug Target for Inhibiting Virus Replication

Picornaviruses are small, positive-stranded RNA viruses, divided into twelve different genera. Members of the Picornaviridae family cause a wide range of human and animal diseases including the common cold, poliomyelitis, foot and mouth disease, and dilated cardiomyopathy. The picornavirus genome is replicated via a highly conserved mechanism involving a presumed cloverleaf structure located at the 5’ noncoding region of the virus genome. The 5’ cloverleaf consists of three stem loops (B, C, and D) and one stem (A), which interact with a variety of virus and host cell proteins during replication. In this dissertation, human rhinovirus serotype 14 (HRV-14) SLB and the 5’cloverleaf (5’CL) solution structures were determined using nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS). HRV-14 SLB adopts a predominantly A-form helical structure. The stem contains five Watson−Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations in the helical parameters, but does not appear to introduce flexibility. The helix major groove appears to be more accessible than in typical standard A-form RNA. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the poly(C) binding protein, is the most disordered region of the structure. The solution structure of HRV-14 5ʹCL was determined in the absence and presence of magnesium. In the absence of magnesium, the structure adopts an open, somewhat extended conformation. In the presence of magnesium, the structure compacts, bringing SLB and SLD into close contact, a geometry that creates an extensive accessible major groove surface, and permits interaction between the proteins that target each stem loop. A deeper understanding of these structures will offer invaluable information regarding the picornavirus replication mechanism. The results from these studies have the potential to elucidate unique drug targets with broad spectrum efficacy against a range of picornaviruses