KSHV RTA Abolishes NFκB Responsive Gene Expression during Lytic Reactivation by Targeting vFLIP for Degradation via the Proteasome

Elana S Ehrlich,Jennifer C Chmura,Nene N Kalu,Gary S Hayward,John C Smith,Shou-Jiang Gao

doi:10.1371/journal.pone.0091359

Abstract

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 NFκB pathway. Inhibitors of either vFLIP expression or the NFĸB pathway result in enhanced lytic reactivation and apoptosis. We have observed a decrease in vFLIP protein levels and of NFκB activation in the presence of the KSHV lytic switch protein RTA. Whereas vFLIP alone induced expression of the NFĸB responsive genes ICAM1 and TNFα, inclusion of RTA decreased vFLIP induced ICAM1 and TNFα 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 NFĸB responsive gene expression early during lytic reactivation.

Highlights

Kaposi’s Sarcoma Herpesvirus (KSHV), known as human herpesvirus 8 (HHV8), is the causative agent of Kaposi’s Sarcoma (KS) and is associated with primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD)
To determine whether regulator of transcription activation (RTA) has an effect on NF B signaling, 293T cells were transfected with Viral FLICE inhibitory protein (vFLIP) and/or RTA and NFkB activity was assessed using a luciferase reporter containing five NFkB response elements. vFLIP expression induced a 15-fold NFkB activation as expected, cotransfection with RTA reduced the activation three-fold (Fig. 1a)
While co-transfection with RTA consistently resulted in a dose dependent 2–3 fold decrease in vFLIP induced NFkB activation, RTA had no significant effect on NFkB activation stimulated by TNFa, suggesting that RTA was targeting an aspect of vFLIPinduced NF B activation, rather than a component of the signal transduction pathway (Fig. 1b)

Summary

Introduction

Kaposi’s Sarcoma Herpesvirus (KSHV), known as human herpesvirus 8 (HHV8), is the causative agent of Kaposi’s Sarcoma (KS) and is associated with primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). Lytic replication is initiated via expression of the viral regulator of transcription activation (RTA) protein both directly in primary infection and as a lytic switch factor during reactivation [3]. VFLIP has been shown to activate NFkB by interacting with the IkB kinase complex (IKK) [4,5,6,7] This occurs via activation of IKK followed by phosphorylation of IkB and subsequent translocation of NFkB into the nucleus [8,9]. Chemical inhibition of NFkB by Bay11–7082 in PEL cells promotes lytic reactivation while activation of NFkB inhibits lytic promoters [11,12].

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Conclusion