EqHV is the most closely related virus to HCV, and is proposed to have diverged from HCV within the last 1000 years. Studying viruses which share a close evolutionary relationship, yet differ greatly in their ability to cause disease, provides a unique opportunity to facilitate comparative analysis of both replication strategies and pathogenic mechanisms. It has previously been demonstrated that, like HCV, the EqHV 5’UTR functioned as an IRES. Here we set out to undertake a more detailed structural and functional analysis of the 5’UTR.
A mutational analysis of the structural features of the EqHV 5′UTR was undertaken to investigate their roles in its function as an IRES. A complementary study utilising 2’ hydroxyl acylation and analysis by primer extension (SHAPE), to experimentally confirm the predicted structure of the EqHV 5’UTR in order to reveal how this related to function, was also conducted. This combined approach revealed that miR122 mediated enhancement of translation was dependent on a seed site located directly upstream of stem-loop (SL) II and that SLI was not required for EqHV IRES mediated translation. SLIII was absolutely essential, as was SLIIId and the conserved GGG motif. An essential role for SLIIIb was demonstrated for translation from an EqHV sub-genomic replicon. SHAPE experiments revealed that the conserved GGG motif was essential for EqHV 5′UTR:40S ribosomal subunit interactions, and that a GTC sequence in the apical loop of SLIIIb mediated an interaction with eIF3.
This study also set out to establish an EqHV sub-genomic replicon capable of replicating in vitro in mammalian cells; however, this could not be achieved. When taken with published data it is possible to conclude that current EqHV isolates are not able to replicate in currently available cell culture systems and it is likely that new isolates, or cell lines, will be required to achieve in vitro replication.
