Functional analysis of Kaposi's sarcoma–associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation

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 contributes 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. Here we show that a 252-nt fragment directly upstream of vFLIP, within a coding region, directs translation. We have established its RNA structure and demonstrate that IRES activity requires the presence of eIF4A and an intact eIF4G. Furthermore, and unusually for an IRES, eIF4E is part of the complex assembled onto the vFLIP IRES to direct translation. These molecular interactions define a new paradigm for IRES-mediated translation.

Eukaryotic initiation factor 4A (eIF4A) during viral infections

“…Evidence of the participation of eIF4A in the translation of other viral IRES mRNAs has established that, in some occasions, an association between eIF4A and other factors of the eIF4F complex is necessary, such as in the IRES of Kaposi's sarcoma-associated herpesvirus [43] and calicivirus mRNAs (Fig. 2c) [44].…”

“…2c) [44]. Although the importance of eIF4A binding to other translation factors is not fully understood, it is clear that such interactions increase translation efficiency [43,44].…”

Transcriptional and Post-Transcriptional Regulation of Viral Gene Expression in the Gamma-Herpesvirus Kaposi’s Sarcoma-Associated Herpesvirus

“…There is one reported internal ribosome entry sites (IRES), which drives translation of vFLIP from the v-cyclin-vFLIP transcript [26–28]. Surprisingly, IRES-mediated translation of vFLIP still requires all tested initiation factors including the cap-binding protein eIF4E, which is unique among IRESs [102]. Another protein that is expressed from a bicistronic transcript is ORF36, which is the distal ORF in the ORF35-ORF36 transcript [103].…”

Modulation of the Translational Landscape During Herpesvirus Infection

“…The vFLIP protein, which is involved in inhibiting FAS-induced apoptosis through the activation of NF-κB (100, 101), is translated as a downstream gene from a polycistronic mRNA by an IRES element located within the coding region of the upstream gene (102–104). The vFLIP IRES was shown to recruit the complete eIF4F complex, and that translation required eIF4A, as well as a functional eIF4E cap-binding protein and its ability to interact with full-length eIF4G (105). Based on data from a combination of RNA binding assays and translation inhibitors, the authors proposed that eIF4F is recruited to the vFLIP IRES via the direct interaction between the IRES, the 40S ribosome subunit, and eIF3, and that this interaction tethers ribosomes to the mRNA (105).…”

“…The vFLIP IRES was shown to recruit the complete eIF4F complex, and that translation required eIF4A, as well as a functional eIF4E cap-binding protein and its ability to interact with full-length eIF4G (105). Based on data from a combination of RNA binding assays and translation inhibitors, the authors proposed that eIF4F is recruited to the vFLIP IRES via the direct interaction between the IRES, the 40S ribosome subunit, and eIF3, and that this interaction tethers ribosomes to the mRNA (105). These observations expand the known functional requirements for IRES elements and support additional activities for the cap-binding protein in translational stimulation (106).…”