*3.1. Clozapine Blocked the Induction of EBV Lytic Genes*

We investigated the response of EBV to the atypical antipsychotic drug clozapine. The experiments were performed in the HH514-16 EBV-positive Burkitt lymphoma cell line, derived from Jijoye and P3HR1 cells [8]. The degree of viral reactivation was measured by expression of the viral BZLF1 gene, an immediate early gene that encodes a transcription transactivator [9]. The expression of BZLF1 initiates the reactivation of the Epstein–Barr virus lytic cycle. Treatment of cells with the known inducing agent NaB for 24 h caused an approximately 200-fold increase in BZLF1 expression compared to untreated cells (Figure 2A). Clozapine alone did not induce BZLF1 expression or decrease basal levels of expression. When clozapine (50 μM) was added to cells with NaB, BZLF1 induction was significantly decreased (Figure 2A). Induction of another EBV immediate early gene, BRLF1, was also significantly inhibited by clozapine (Figure 2B). To determine if the reduction in these viral immediate early genes by clozapine affects the expression of a downstream lytic gene, levels of the BMLF1 gene that encodes an mRNA export factor were measured [10,11]. Expression of BMLF1 was induced in cells treated with butyrate, as expected. The addition of clozapine plus butyrate decreased BMLF1 expression to levels comparable to untreated cells (Figure 2C).

**Figure 2.** Clozapine inhibited the induction of Epstein–Barr virus (EBV) lytic gene expression. Expression of each gene, (**A**) BZLF1, (**B**) BRLF1, (**C**) BMLF1, was measured by RT-qPCR in untreated HH514-16 cells compared to treatment for 24 h with NaB (3 mM), clozapine (50 μM), or the combination of NaB and clozapine. Values are the average fold induction compared to the untreated control of four or more biological replicates. Error bars show the standard deviation. Differences with a *p*-value < 0.001 are denoted with \*\*\*, *p*-value < 0.01 with \*\*, and not significantly different (*p*-value > 0.05) with n.s.

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