Functions of KSHV-encoded FLIP
Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) was discovered in 1994. KSHV is associated with at least three types of human cancer; KS, an endothelial tumour, and two lymphoproliferative disorders, primary effusion lymphoma (PEL) and a variant of multicentric Castleman's disease (MCD). In KSHV, the genes expressed in latency have been implicated in cell transformation. One of a cluster of three latency-associated genes, that regulate proliferation and apoptosis, encodes a viral FLICE inhibitory protein (vFLIP) in open reading frame 71 (ORF71). Two roles have been proposed for vFLIP; when expressed in heterologous cells it both blocks Fas-mediated apoptosis and activates the NF-KB pathway by interaction with IKB kinase (IKK). Given the two contrasting roles assigned to vFLIP, the aim of this study was to determine the function of vFLIP in KSHV-infected cells. vFLIP was therefore immunoprecipitated from PEL cells and four associated proteins were identified by mass spectrometry: IKK components and the chaperone, Hsp90. Using gel filtration, a single population of vFLIP in the cytoplasm of PEL cells co-eluted and co-precipitated with an activated IKK complex. An inhibitor of Hsp90, geldanamycin, inhibited vFLIP-induced IKK activity and killed PEL cells, inferring that vFLIP activation of NF-KB contributes to PEL survival. In a yeast-two-hybrid screen, our collaborators identified IKK as an interacting partner of vFLIP. Fragments of IKK were expressed in mammalian cells and bacteria, and the central portion of IKK? (amino acids 150-272) was identified as the vFLIP binding region. Finally, it is suggested that vFLIP activates the alternative pathway of NF-KB activation, leading to processing of p100 and generation of p52. This process is phosphorylation dependent and results in nuclear translocation of RelB and p52. A possible mechanism of action is suggested by the physical interaction between p100/p52 and vFLIP. These data strongly support an important role for vFLIP in NF-KB activation. This may be crucial for cell transformation by KSHV, for survival of infected cells and for the maintenance of latency.