A fundamental problem in studying the latent-to-lytic switch of Epstein-Barr virus (EBV) and the viral lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction. Strategies to target EBV-positive tumors by inducing the viral lytic cycle with chemical agents are hindered by inefficient responses to stimuli. In vitro, even in the most susceptible cell lines, more than 50% of cells latently infected with EBV are refractory to induction of the lytic cycle. The mechanisms underlying the refractory state are not understood. We separated lytic from refractory Burkitt lymphoma-derived HH514-16 cells after treatment with an HDAC inhibitor, sodium butyrate. Both refractory-and lytic-cell populations responded to the inducing stimulus by hyperacetylation of histone H3. However, analysis of host cell gene expression showed that specific cellular transcripts Stat3, Fos, and interleukin-8 (IL-8) were preferentially upregulated in the refractory-cell population, while IL-6 was upregulated in the lytic population. STAT3 protein levels were also substantially increased in refractory cells relative to untreated or lytic cells. This increase in de novo expression resulted primarily in unphosphorylated STAT3. Examination of single cells revealed that high levels of STAT3 were strongly associated with the refractory state. The refractory state is manifest in a unique subpopulation of cells that exhibits different cellular responses than do lytic cells exposed to the same stimulus. Identifying characteristics of cells refractory to lytic induction relative to cells that undergo lytic activation will be an important step in developing a better understanding of the regulation of the EBV latent to lytic switch.
Epstein-Barr virus (EBV) is a gammaherpesvirus that per-sists as a lifelong infection by remaining in the latent phase of its life cycle within B lymphocytes (17). EBV is associated with human cancers such as Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and EBV-associated lymphoproliferative disease in immunocompromised individuals (11). Efforts to eliminate EBV-positive tumor cells by nucleoside analogue antiviral agents following induction of the viral lytic cycle have shown promising results (15,16,18,38,44,52). These efforts have been preceded by extensive studies on the switch from latency to the EBV lytic cycle in lymphoid cell lines. A fundamental problem in studying the latent to lytic switch and the lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction (45). In cell culture, EBV can be induced into the lytic cycle by a variety of chemical stimuli, including agents currently being used or investigated as chemotherapeutic drugs, such as the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) and the DNA methyltransferase inhibitor azacytidine (Aza) (3, 57). However, following treatment of cells latently infected with EBV, only a fraction of cells enter the lytic cycle; the remainder of the population is refractory to lyti...