Characterizing a Unique IRES in Kaposi's Sarcoma-Associated Herpesvirus

The synthesis of proteins during eukaryotic translation involves four major phases, where initiation is often the rate-limiting step and target for translational control with a large number of factors involved. Some viruses have evolved a mechanism that allows them to bypass the canonical initiation, with an internal ribosome entry site (IRES). IRESs can functionally replace the function of the 5’ mRNA cap structure and many of the proteins that are involved in recruiting the ribosome. Found in Kaposi’s sarcoma-associated herpesvirus, the vFLIP IRES is a unique IRES in that it is found in a DNA virus, within the coding region of a protein, and requires most of the host factors involved in translation initiation. To further characterize this IRES, an in vitro transcription approach was developed in order to generate milligram amounts of RNA required for crystallography. X-ray diffraction and structure determination of IRES crystals would lead to important insight into the viral IRES’s mechanism of action. Although amounts sufficient for crystallography were not obtained during this work, optimization of the transcription approach showed that the RNA synthesis reactions would be amenable for scaling up. Secondly, a bicistronic reporter was developed suitable for functional investigations of the viral IRES in an in vitro translation assay as well as screening and testing of compounds for selective inhibitor of viral translation initiation