Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contribute to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. The aims of this study were to confirm the previously identified 252-nt fragment directly upstream of vFLIP as the location of the vFLIP IRES in cellulo and to determine the structure and mechanism of action of the vFLIP IRES. Here we show that a 252-nt, within a coding region, directs translation in HEK293 cells. We have also established its RNA structure using chemical and enzymatic probing of RNA structure in solution and mutational analysis studies revealed that the domain If of the vFLIP IRES is crucial for its activity. Also, we demonstrate that IRES activity requires the presence of eIF4A and the eIF4E-eIF4G interaction. These interactions may define a new paradigm for IRES-mediated translation. Finally, we attempted to identify cellular proteins that may interact with the vFLIP IRES using several types of protein affinity chromatography, but we could detect a protein interacting with vFLIP IRES but yet to be confirmed.