Abstract
Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells’ responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells’ responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.
Highlights
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus which infects over 90% of the adult population worldwide
To identify novel organic molecules that can effectively reactivate EBV lytic cycle, we screened a library of 50,240 small organic molecules from the DIVERSet screening library (ChemBridge Corp.) with AGS-BX1 cells on the High-Throughput Screening (HTS) platform established at the Department of Microbiology at the University of Hong Kong, using green fluorescent protein (GFP) as readout
The 40 hits in each cell line were first tested with Western blotting for their ability to trigger the expression of multiple EBV lytic proteins, including the IE proteins Zta and Rta, and the early protein EA-D (BMRF1) (Fig 1B and S1A Fig). 22 molecules from each cell line that induced strong expression of these proteins were selected for further comparison (Fig 1C and S1B Fig)
Summary
Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus which infects over 90% of the adult population worldwide. EBV adopts a biphasic life cycle as other herpesviruses and persists in latencies in infected cells after initial infection, expressing only a limited number of viral proteins and transcripts. Reactivation of the latent virus into lytic cycle induces the expression of a temporally regulated cascade of approximately 80 lytic proteins. The reactivation of lytic cycle in latently-infected cells can be induced by a variety of agents, e.g. anti-immunoglobulin [3, 4], tumour growth factor β (TGF-β) [5, 6], and different groups of chemicals [7]. Histone deacetylase (HDAC) inhibitors [8,9,10,11] and phorbol esters [12,13,14] are the major classes of chemical lytic inducers reported far
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