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

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 NF?B 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 NF?B activation in the presence of the KSHV lytic switch protein RTA. Whereas vFLIP alone induced expression of the NF?B responsive genes ICAM1 and TNF?, inclusion of RTA decreased vFLIP induced ICAM1 and TNF? 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.

SUBMITTER: Ehrlich ES

PROVIDER: S-EPMC3948842 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Ehrlich Elana S ES Chmura Jennifer C JC Smith John C JC Kalu Nene N NN Hayward Gary S GS

PloS one 20140310 3

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 de ...[more]

PMID: 24614587

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Project description:The oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.

Dataset Information

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

Publications

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

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