KSHV RTA abolishes NF B responsive gene expression during lytic reactivation by targeting vFLIP for degradation via the proteasome

Kaposi's sarcoma herpesvirus (KSHV) is a gamma-2 herpesvirus present in all cases of Kaposi's sarcoma, primary effusion lymphoma (PEL), and some cases of multicentric Castleman's disease. Viral FLICE inhibitory protein (vFLIP) is a latently expressed gene that has been shown to be essential for survival of latently infected PEL cells by activating the NFkB pathway. Inhibitors of either vFLIP expression or the NF B pathway result in enhanced lytic reactivation and apoptosis. We have observed a decrease in vFLIP protein levels and of NFkB activation in the presence of the KSHV lytic switch protein RTA. Whereas vFLIP alone induced expression of the NF B responsive genes ICAM1 and TNFa, inclusion of RTA decreased vFLIP induced ICAM1 and TNFa expression in both co-transfected 293T cells and in doxycycline induced TREx BCBL1 cells. RTA expression resulted in proteasome dependent destabilization of vFLIP. Neither RTA ubiquitin E3 ligase domain mutants nor a dominant-negative RAUL mutant abrogated this effect, while RTA truncation mutants did, suggesting that RTA recruits a novel cellular ubiquitin E3 ligase to target vFLIP for proteasomal degradation, allowing for inhibition of NF B responsive gene expression early during lytic reactivation.