| Summary: | KSHV infection is associated with both endothelial and B cell tumours. In KSHV the genes expressed in latency have been implicated in cell transformation. vFLIP is one of a small number of viral proteins expressed in latently infected tumour cells. In KSHV-infected primary effusion lymphoma (PEL) cells, vFLIP binds to, and persistently activates the IkB kinase complex, leading to constitutive activation of the canonical NF-kB pathway. We have previously shown in our lab that vFLIP directly interacts with the IKKy subunit of the IKK complex (Field, et al., 2003) to activate IKK. In this report, we demonstrate that vFLIP also activates the alternative NF-kB pathway, which involves processing of the p100 protein precursor and generation of the p52 subunit. Stable vFLIP expression in Jurkat cells stimulates expression of endogenous p100 and nuclear accumulation of p52 and RelB. Metabolic radiolabelling of transiently transferred 293T cells indicates that vFLIP promotes proteolysis of p100 and active generation of p52. Moreover, we show that vFLIP associates with p100 when over-expressed in Jurkat cells, or when endogenously expressed in PEL cells, and a region in the C-terminus of p100, which includes the p100 DD, is identified as the vFLIP binding region. Finally, inhibition of p100 and p52 production mediated by siRNA knockdown leads to the induction of apoptosis in PEL cells, inferring that vFLIP activation of the alternative NF-kB pathway contributes to PEL survival. These data demonstrate that vFLIP activates both canonical and alternative NF-kB pathways, a property shared with the Tax oncoprotein of HTLV-1 and LMP1 of EBV. In addition, we have examined the effect of vFLIP on primary human dermal microvascular endothelial cell (MVEC) survival, as vFLIP is expressed in the KSHV-infected cells within KS lesions. Stable vFLIP expression in MVECs induces the activation of the classical NF-kB pathway and the nuclear translocation of RelA/p65. vFLIP-mediated NF-kB activation prevents detachment-induced apoptosis (anoikis) of MVECs, but does not inhibit growth factor removal-induced apoptosis, by inducing the secretion of an additional paracrine survival factor(s). These data strongly support an important role for vFLIP in NF-kB activation, which may be crucial for cell transformation by KSHV, for the survival of infected cells, and for metastasis.
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