Structure and function analysis of picornavirus internal ribosome entry site elements
thesisposted on 15.12.2014, 10:32 by Angela Tracy. Clark
Picornaviruses have a single-stranded, positive-sense RNA genome. An internal ribosome entry site (IRES) situated within the 5' untranslated region of the genome mediates cap-independent translation of the picornavirus RNA. In an attempt to further understand the mechanism of IRES-mediated translation, it was decided to investigate the role of several nucleotides within the encephalomyocarditis virus IRES which are absolutely conserved among all cardiovirus and aphthovirus IRES elements and in close proximity to the binding site of the translation initiation factor, eIF4GI. Four nucleotides within the J domain were randomised to generate a pool of mutant IRES elements containing up to 256 different sequences. Analysis of this pool, using a cell selection system to isolate functional IRES elements, revealed that the four nucleotides were essential for IRES activity. Furthermore, it was shown that mutations at these nucleotides severely affected the binding of eIF4GI and eIF4A to the IRES. A clear correlation was seen between the activity of the mutant IRES element and its ability to bind eIF4GI/eIF4A. This strongly suggests that the binding of eIF4GI to the IRES is functionally relevant in vivo. It was also shown that the IRES elements from several different hepatitis C virus genotypes could be used within the cell selection system. This is particularly useful since the analysis of HCV within cells is currently restricted. Finally, the role of residue 20 within the swine vesicular disease virus 2A protease was investigated. This residue is known to affect translation of the picornavirus RNA and virus virulence. To analyse the role of residue 20 to the function of 2A, this residue was substituted for each of the 20 amino acids. This revealed that amino acids substitutions were reasonably well tolerated with the exception of proline.