Artikel
Systematic Analysis of Combination Effects of HHV-8 genes on NF-κB Activation
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Veröffentlicht: | 16. April 2008 |
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Gliederung
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Introduction: Human herpesvirus-8 (HHV-8) is the etiologic agent of Kaposi¡¦s sarcoma (KS), an endothelial cell-derived tumor with pronounced inflammation. Nuclear factor-kappa B (NF-κB) is a key regulatory molecule in inflammation. Using reverse transfection cell arrays (RTCA) as an unbiased systems biology approach the effects on NF-κB-activation of all HHV-8-encoded genes individually and of pairwise combinations of all K- and latent genes were investigated. More than 15.000 transfections were performed.
Materials and methods: All 86 HHV-8 genes were cloned in expression vectors, the resulting plasmids were confirmed in sequence and the expressed proteins were controlled by western blotting and immunofluorescent stainings. NF-κB-activation of all HHV-8-encoded genes individually and of pairwise combinations of all K- and latent genes was investigated through co-transfection with a reporter plasmid expressing GFP under the control of NF-κB in the RTCA manner. Classical transfection experiments were performed with a reporter plasmid expressing firefly luciferase under the control of NF-κB. EMSA was carried out as described previously [Ref. 1].
Results: The strongest activation of NF-κB was observed in combinations of vFLIP, a known activator of NF-κB, with the latent genes vCYC or LNA-1. Combined activities of these genes were clearly synergistic, could be abrogated by co-expression of a constitutively active IκB-α molecule and were confirmed in classical transfection experiments and EMSA. In addition to the known activator vFLIP, ORF75 was detected as a previously unknown activator of NF-κB. Interestingly NF-κB-activation by ORF75 could not be inhibited by co-expression of a constitutively active IκB-α molecule, suggesting that ORF75 activates NF-κB in the alternative pathway.
Conclusion: Our results indicate that co-operative effects of vFLIP, vCYC and LNA-1, which are all encoded within a tricistronic latency-associated gene locus in the viral genome, and ORF75 are key to the robust NF-κB-activation observed in HHV-8-infected cells.