The analysis of a conserved RNA structure in the 3D polymerase encoding region of Human Parechovirus 1

Picornaviruses are important causes of human illness and it is necessary to understand more about how these viruses function. Human parechoviruses (HPeV) are common pathogens and studies have shown that 95% of people become infected with HPeV at a very early age, usually with symptoms such as mild diarrhoea and fever. However, one virus type HPeV3, is implicated in much more serious cases of neonatal disease and so it is important to understand HPeVs to increase the opportunity to develop drugs or vaccines against the infection. The HPeV1 genome encodes a single polyprotein that is cleaved into structural and non-structural proteins. Analysis of one region of the genome (encoding the polymerase, 3Dpol) shows that some codons are perfectly conserved, suggesting functions in addition to protein coding. This region seems to fold into an RNA secondary structure made up of three stem-loops and a tertiary structure “kissing” interaction. The structure was validated by comparing all the available HPeV sequences and found to be highly conserved. To investigate if the structure has a role in RNA stability, an EGFP fluorescent assay was used. Sequences containing the structure was added to the 3’ UTR of the EGFP gene. A mutant with 21 mutations which completely destroys the RNA structure was also used. A FACS-based method was used to measure expression levels of EGFP. The results showed that there was a significant reduction in fluorescence from the mutant construct. The effect of the structure was also investigated in infected cells and in cells exposed to different stresses which could mimic virus infection. The results suggest that the structure can positively affect RNA stability/translation. Further investigation on other possible roles such as RNA replication and translation should be performed to improve the understanding of the biology of the structure in HPeVs and a Renilla Luciferase reporter gene system was assembled to facilitate the studies in the future.