Signal Transducer and Activator of Transcription 3 Limits Epstein-Barr Virus Lytic Activation in B Lymphocytes

Abstract

g Lytic activation of Epstein-Barr virus (EBV) is central to its life cycle and to most EBV-related diseases. However, not every EBVinfected B cell is susceptible to lytic activation. This lack of uniform susceptibility to lytic activation also directly impacts the success of viral oncolytic therapy for EBV cancers, yet determinants of susceptibility to lytic induction signals are not well understood. To determine if host factors influence susceptibility to EBV lytic activation, we developed a technique to separate lytic from refractory cells and reported that EBV lytic activation occurs preferentially in cells with lower levels of signal transducer and activator of transcription 3 (STAT3). Using this tool to detect single cells, we now extend the correlation between STAT3 and lytic versus refractory states to EBV-infected circulating B cells in patients with primary EBV infection, leading us to investigate whether STAT3 controls susceptibility to EBV lytic activation. In loss-of-function and gain-of-function studies in EBV-positive B lymphoma and lymphoblastoid cells, we found that the levels of functional STAT3 regulate susceptibility to EBV lytic activation. This prompted us to identify a pool of candidate cellular genes that might be regulated by STAT3 to limit EBV lytic activation. From this pool, we confirmed increases in transcript levels in refractory cells of a set of genes known to participate in transcription repression. Taken together, our findings place STAT3 at a critical crossroads between EBV latency and lytic activation, processes fundamental to EBV lymphomagenesis. E pstein-Barr virus (EBV) infects most humans and persists silently in B lymphocytes. Primary infection with EBV can cause infectious mononucleosis (IM). Under certain circumstances, EBV can cause B-cell lymphomas and epithelial cell cancers (1). Several studies suggest that EBV lytic activation is an important step in the pathogenesis of such EBV-related diseases (2-5). From a therapeutic standpoint, efforts to eliminate EBV-positive tumors using nucleoside analogues after induction of viral lytic activation have shown promise (6-8). However, EBV-infected tumor cells are not fully permissive to lytic induction, as only a fraction of EBV-infected B cells exposed to lytic cycle-inducing agents enters the lytic cycle; the remainder of the population is refractory to lytic induction (9, 10). These refractory cells are not susceptible to oncolytic therapy, necessitating further investigations into the physiology underlying both lytic and refractory states.A general problem with investigating EBV lytic activation is that a mixed population of refractory and lytic cells results from exposure to lytic cycle-inducing stimuli. We therefore developed a technique to separate lytic cells from refractory cells in a mixed population of EBV-infected B cells (9). Our previous studies using this technique showed that host cell determinants regulate susceptibility of EBV-infected B cells to lytic cycle-inducing stimuli (9, 11) and that higher levels o...

Cellular STAT3 Functions via PCBP2 To Restrain Epstein-Barr Virus Lytic Activation in B Lymphocytes

“…EBV lymphoblastoid cell lines (EBV-LCLs) derived from 3 healthy subjects and 3 patients with autosomal dominant hyper-IgE syndrome (AD-HIES) were described previously (9). Two AD-HIES patients had a mutation in the SH2 domain and the third patient had a mutation in the DNA-binding domain of their STAT3 gene.…”

“…Staining and microscopy were performed as described previously (9,12). Briefly, cells were stained as described above for flow cytometry by using rabbit anti-human STAT3 (Santa Cruz Biotechnology) followed by FITC-anti-rabbit IgG (Life Technologies), washed, cytospun onto glass slides, air dried, and mounted with 4=,6-diamidino-2-phenylindole (DAPI) Prolong Gold Antifade (Life Technologies).…”

“…Total cell extracts were electrophoresed in 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and reacted with antibodies (Abs) as described previously (9). Mouse monoclonal Abs were used to detect PCBP2 (1/500 dilution) (catalog number sc-101136; Santa Cruz Biotechnology) and ␤-actin (1/3,000 dilution) (catalog number AC-15; Sigma).…”

“…By probing a cellular microarray using RNA from separated cells, we then identified STAT3 (signal transducer and activator of transcription 3) as a key regulator of the refractory state. Specifically, we found that high levels of cellular STAT3 restrict the susceptibility of latently infected cells to lytic cycle activation signals (8,9). In this study, we examined the proteome of EBV ϩ sorted refractory and lytic cells to identify PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells.…”

“…However, with this approach, killing of cancer cells is restricted by the ability of cells to support the lytic phase of the viral life cycle. Studies have shown that only about half the number of latently infected cells in a population responds to lytic cycle-activating agents (8,9). Consequently, a substantial fraction of cells in a population is refractory to oncolytic killing.…”