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. 2020 Oct 14;94(21):e01179-20.
doi: 10.1128/JVI.01179-20. Print 2020 Oct 14.

A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents

Xiaofan Li #  1 , Ibukun A Akinyemi #  2 , Jeehyun Karen You  3 , Mohammad Ali Rezaei  4 , Chenglong Li  4 , Michael T McIntosh  2   5 , Maurizio Del Poeta  3   6   7 , Sumita Bhaduri-McIntosh  8   5
Affiliations

A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents

Xiaofan Li et al. J Virol. .

Abstract

Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers, including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that through the activities of the viral protein kinase conserved across herpesviruses and two cellular proteins, ATM and KAP1, a lytic cycle amplification loop is established, and disruption of this loop disables the EBV lytic cascade. We therefore devised a high-throughput screening assay, screened a small-molecule-compound library, and identified 17 candidates that impair the release of lytically replicated EBV. The identified compounds will (i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses, and (ii) be developed into anticancer agents, as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for antiherpesviral and anticancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small-molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even antiherpesviral potential, as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both antiherpesviral and anticancer drugs.

Keywords: In-Cell Western assay; KAP1; anti-EBV; antiviral agents; high-throughput assay; lytic cycle.

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Figures

FIG 1
FIG 1
In-Cell Western (ICW) screening assay for identifying chemical inhibitors of EBV lytic cycle. (A) Positive feedback loop comprising EBV lytic proteins ZEBRA and vPK and cellular proteins ATM and KAP1 contributes to amplification of the EBV lytic cycle. This loop involves ZEBRA-mediated transcriptional activation of vPK leading to vPK-mediated phosphorylation of ATM at S2996; ATM then phosphorylates KAP1 at S824, resulting in derepression of BZLF1 (which encodes ZEBRA). Derepression of BZLF1 and other EBV lytic genes (many of which are also transcriptional targets of ZEBRA) results in amplification of the EBV lytic cascade. (B) Workflow of ICW assay that measures p-S824 KAP1 signal normalized to total KAP1 in CLIX-FZ BL cells in which EBV is induced into the lytic phase through addition of doxycycline. (C) An example of an ICW assay showing images of wells in which cells were left untreated, treated with doxycycline, or treated with doxycycline plus an ATM inhibitor (KU55933) and then stained with an antibody to p-S824-KAP1 versus total KAP1. (D) Distribution of SSMD values for initial screen. Assay quality was assessed using strictly standardized mean difference (SSMD; β) using doxycycline-induced (D+) and uninduced (D−) CLIX-FZ BL cells.
FIG 2
FIG 2
Validation of 30 candidate compounds by measuring their effects on KAP1 phosphorylation. CLIX-FZ BL cells were treated with doxycycline, doxycycline plus 10 μM (each) for 30 candidate compounds, or left untreated. Twenty-four hours later, cells were harvested for ICW as imaged (A) and analyzed for ratios between p-S824 KAP1 and total KAP1 (B). Error bars represent SEM of biological triplicates. Red line, mean of p-S824 KAP1:total KAP1 in doxycycline-treated cells. *, P < 0.05; **, P < 0.01; ns, not significant.
FIG 3
FIG 3
Evaluating toxicity of 21 candidates on CLIX-FZ BL cells. CLIX-FZ BL cells were treated with doxycycline or doxycycline along with 21 individual compounds at different concentrations (10 nM, 1 μM, or 100 μM). At 24 h posttreatment, cell cultures were mixed with WST-1 assay substrate and incubated for 2 h for measurement. Error bars represent SEM of biological triplicates.
FIG 4
FIG 4
Testing the effects of 21 candidates on release of encapsidated EBV from cells. CLIX-FZ BL cells were treated with doxycycline, doxycycline along with 1-μM or 10-μM concentrations of 21 individual compounds, or left untreated. Released virus particles were collected from culture supernatants and quantified via qPCR at 48 h (A) or 72 h (B) after treatment. An equal amount of supernatant from each sample was used as PCR template. Error bars represent SEM of 3 technical replicates from 2 experiments. Red line, mean encapsidated extracellular virions in doxycycline-treated cells. *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
Examination of the effects of 21 candidates on EBV replication. CLIX-FZ BL cells were induced with doxycycline, doxycycline plus 21 individual compounds at two different concentrations (1 μM or 10 μM), or left untreated. Cell-associated total DNA was extracted at 18 h (A) or 24 h (B) after treatment and quantitated by qPCR after normalizing to 18S genomic fragment. Error bars represent SEM of 3 technical replicates from 2 experiments. Red line, mean cell-associated EBV DNA in doxycycline-treated cells. *, P < 0.05; **, P < 0.01.
FIG 6
FIG 6
Investigating the effects of 21 candidates on expression of the EBV latent-to-lytic switch protein ZEBRA. CLIX-FZ BL cells were treated with doxycycline, doxycycline plus 10 μM concentrations of 21 individual compounds, or left untreated. After 24 h, cells were harvested for immunoblotting with anti-ZEBRA antibody. Numbers reflect ratios between endogenous ZEBRA and doxycycline-inducible FLAG-ZEBRA. Numbers in green indicate inhibition > 25%. Experiments were performed at least twice.
FIG 7
FIG 7
Examination of the effects of 9 candidates on expression and phosphorylation of EBV early lytic gene product EA-D. CLIX-FZ BL cells were induced with doxycycline, doxycycline plus 10 μM concentrations of 9 individual compounds, or left untreated. Cells were harvested at 36 h posttreatment for immunoblotting with indicated antibodies. The top row of numbers indicates the ratio between phosphorylated EA-D (higher-molecular-mass band) and unphosphorylated EA-D (lower-molecular-mass band). The bottom row of numbers represents relative amounts of EA-D protein after normalization to FLAG-ZEBRA. Numbers in green indicate inhibition >25%. Experiments were performed at least twice.
FIG 8
FIG 8
IC50s and toxicity of compounds 21H10-8 and 21H10-10. (A) Chemical structures of compounds 21H10-8 and 21H10-10. (B and C) CLIX-FZ BL cells were treated with doxycycline or doxycycline plus indicated doses of 21H10-8 (B) or 21H10-10 (C) and harvested 72 h later for qPCR quantification of released virus particles in culture supernatants that were concentrated by centrifugation as described in Materials and Methods. (D and E) A549 cells were exposed to 21H10-8 (D) or 21H10-10 (E) at doses corresponding to those in panels B and C. After 24 h, cell cultures were mixed with WST-1 assay substrate and read after another 2 h. Error bars represent SEM of triplicate technical repeats derived from two biological repeats.
FIG 9
FIG 9
Selected candidate compounds inhibit phosphorylation of KAP1 in an independent readout. CLIX-FZ BL cells were induced with doxycycline or doxycycline plus individual compounds at indicated concentrations or left untreated. Cells were harvested at 18 h posttreatment for immunoblotting with indicated antibodies. Compounds 21H10-8 and 21H10-10 were tested using concentrations approximating their IC50 values (2 and 5 μM, respectively) and 10 μM, while 47A4-9 and 47A4-10 were tested at 1-μM and 10-μM concentrations. The experiment was performed twice.
FIG 10
FIG 10
Compounds 21H10-8 and 21H10-10 inhibit release of encapsidated EBV from a lymphoblastoid cell line. Cells were exposed to the lytic trigger sodium butyrate and compound 21H10-8 or 21H10-10 at indicated concentrations, or left untreated. Culture supernatants were harvested 72 h later, concentrated by centrifugation as described in Materials and Methods, and assayed for released virus particles by qPCR. Error bars represent SEM of 3 technical replicates. *, P < 0.05; **, P < 0.01.
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References

    1. Jha HC, Banerjee S, Robertson ES. 2016. The role of gammaherpesviruses in cancer pathogenesis. Pathogens 5:18. doi:10.3390/pathogens5010018. - DOI - PMC - PubMed
    1. Young LS, Yap LF, Murray PG. 2016. Epstein-Barr virus: more than 50 years old and still providing surprises. Nat Rev Cancer 16:789–802. doi:10.1038/nrc.2016.92. - DOI - PubMed
    1. Hong GK, Gulley ML, Feng WH, Delecluse HJ, Holley-Guthrie E, Kenney SC. 2005. Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model. J Virol 79:13993–14003. doi:10.1128/JVI.79.22.13993-14003.2005. - DOI - PMC - PubMed
    1. Mueller N, Evans A, Harris NL, Comstock GW, Jellum E, Magnus K, Orentreich N, Polk BF, Vogelman J. 1989. Hodgkin's disease and Epstein-Barr virus. Altered antibody pattern before diagnosis. N Engl J Med 320:689–695. doi:10.1056/NEJM198903163201103. - DOI - PubMed
    1. van Esser JW, van der Holt B, Meijer E, Niesters HG, Trenschel R, Thijsen SF, van Loon AM, Frassoni F, Bacigalupo A, Schaefer UW, Osterhaus AD, Gratama JW, Löwenberg B, Verdonck LF, Cornelissen JJ. 2001. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative disease following T-cell-depleted SCT. Blood 98:972–978. doi:10.1182/blood.v98.4.972. - DOI - PubMed

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