Abstract
Kaposi’s sarcoma associated herpesvirus (KSHV), is a gammaherpesvirus from the Herpesviridae family and causes a number of proliferative diseases such as Kaposi sarcoma, primary effusion lymphoma and Castleman disease. The prime effectors of the cell proliferation occurring during KSHV infections are the latent gene products LANA, vCyclin and vFLIP. The analysis of the mRNA latent gene transcripts produced by KSHV showed no monocistronic transcript encoding vFLIP suggesting its expression was regulated by a non-canonical translation mechanism. Indeed, previous studies proposed that an IRES element located within the vCyclin ORF is responsible for vFLIP expression. We have now shown that a minimum fragment of 252 nt of within the vCyclin ORF and directly upstream of vFLIP can function as IRES. The KSHV IRES 252 fragment showed IRES activity in Rabbit Reticulocyte Lysate (RRL) but not in KS-derived cell lines. Due to lack of information on the mechanism of internal initiation mediated by the KSHV IRES, we have analyzed the role of eIF4A, eIF4E and eIF4G for the IRES function in RRL. Surprisingly, the KSHV IRES require the whole eIF4F complex for it to function. In addition, several IRES trans-acting factors (ITAFs) have been shown to interact with KSHV IRES by Mass spectrometry analysis. These are Y-box protein 1 (YB-1), eukaryotic translation elongation factor 1α (EEF1A1 and EEF1A2), heterogenous nuclear ribonucleoprotein K (hnRPK), heterogenous nuclear ribonucleoprotein C1/C2 (hnRP C1/C2), and Poly(rc) binding protein 1 (PCBP1). Although the interaction between the KSHV IRES and ITAFs has yet to be confirmed by direct interaction studies, analysis on the YB-1 showed an interaction with KSHV IRES based on Western blot result. With several ITAFs interacting with the KSHV IRES and the requirement for eIF4F, we then characterized the RNA secondary structure of the KSHV IRES in solution to understand how the IRES structure could coordinate such interactions. Chemical and enzymatic probing, combined with structure folding prediction using Mfold revealed 3 domains with domain I is the most structured domain. In search of structure similarities among IRESes, X-linked inhibitor of apoptosis (XIAP) was found to be the closest, although the XIAP IRES contains only two domains. The two IRESes also share similarities for a polypyrimidine sequence located in domain II. For the first time, we have showed the requirements of the KSHV IRES for canonical, non-canonical initiation factors and preliminary prediction of secondary structure which shows that the KSHV IRES does not belong to any existing functional groups of IRESes and a novel DNA virus IRES.