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. 2015 May;89(9):5002-11.
doi: 10.1128/JVI.00121-15. Epub 2015 Feb 25.

Cellular STAT3 functions via PCBP2 to restrain Epstein-Barr Virus lytic activation in B lymphocytes

Siva Koganti  1 , Carissa Clark  1 , Jizu Zhi  2 , Xiaofan Li  1 , Emily I Chen  3 , Sharmistha Chakrabortty  4 , Erik R Hill  1 , Sumita Bhaduri-McIntosh  5
Affiliations

Cellular STAT3 functions via PCBP2 to restrain Epstein-Barr Virus lytic activation in B lymphocytes

Siva Koganti et al. J Virol. 2015 May.

Abstract

A major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV(+)) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV(+) cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers.

Importance: Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV(+) cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies.

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Figures

FIG 1
FIG 1
Comparison of gene expression levels in sorted lytic and refractory cells by using microarray and proteomic approaches. (A) Venn diagram showing the overlap between the numbers of genes with at least 2-fold increased levels of gene products in lytic cells (top half) and those in refractory cells (bottom half). Numbers outside circles indicate the total number of genes with increased levels of mRNA (by microarray analysis [left]) or protein (by proteomic analysis [right]). Numbers inside small circles indicate the number of genes whose products were observed in lytic or refractory cells only.
FIG 2
FIG 2
Candidate genes contributing to the RNA processing GO term show increased transcript levels in refractory cells. Untreated but mock-sorted HH514-16 BL cells, NaB-treated sorted refractory cells, and NaB-treated sorted lytic cells were subjected to qRT-PCR using primers targeting four candidate genes that are transcriptional targets of STAT3 and contribute to the “RNA processing” GO biological process pathway. Results represent means of relative amounts of RNA normalized to the level of 18S rRNA ± standard errors of the means from three technical replicates from each of three independent sorting experiments (*, P < 0.05).
FIG 3
FIG 3
Cells expressing high levels of PCBP2 protein are refractory to spontaneous and induced EBV lytic activation. (A to C) HH514-16 cells were untreated (A) or treated with NaB (B and C). Cells were harvested 48 h later and evaluated for lytic cells (using reference EBV-seropositive serum [top] or reference EBV-seronegative serum [bottom] in panels A and B) and PCBP2 expression by flow cytometry. Numbers in panels A and B represent the percentages of cells in quadrants; numbers in rectangular gates in panel C represent the percentages of PCBP2hi (top), PCBP2int (middle), and PCBP2lo/− (bottom) cells undergoing lytic activation. Staining with a matched isotype control antibody for PCBP2 is shown at the bottom of panels A and B. (D) HH514-16 cells were transfected with control scrambled siRNA or siRNA targeting PCBP2, harvested 24 h later, and subjected to immunoblotting with anti-PCBP2 and anti-β-actin antibodies. Numbers indicate the relative amounts of PCBP2 protein after normalization to β-actin levels. These experiments were performed at least twice. Data for representative experiments are shown.
FIG 4
FIG 4
Impairment of STAT3 results in suppression of PCBP2 transcript levels. (A) HH514-16 cells were transfected with scrambled siRNA (open bars) or siRNA targeting STAT3 (black bars) and harvested 24 h later for determination of relative amounts of PCBP2 and STAT3 mRNAs by qRT-PCR after normalization to 18S rRNA levels using the ΔΔCT method. (B) Seven-week-old LCLs derived from 3 healthy subjects and 3 AD-HIES patients were examined for relative levels of PCBP2 transcripts by qRT-PCR as described above for panel A. (C) Healthy subject-derived and AD-HIES patient-derived LCLs were transfected with scrambled siRNA (open bars) or siRNA targeting STAT3 (black bars) and harvested 24 h later for determination of the relative amounts of PCBP2 and STAT3 mRNAs by qRT-PCR as described above for panel A. Error bars are standard errors of the means of data from 3 technical replicates from each of 2 transfection experiments in panels A and C (*, P < 0.05). (D) Healthy subject-derived LCLs were transfected with scrambled siRNA (SiScram) (top) or STAT3 siRNA (bottom), harvested 24 h later, and examined for STAT3 expression by immunofluorescence. Representative nuclei (∼15% of cells transfected with siRNA targeting STAT3, consistent with our typical transfection efficiency) are shown. Experiments were performed twice.
FIG 5
FIG 5
Suppression of PCBP2 results in EBV lytic activation. (A) HH514-16 cells were transfected with scrambled siRNA (open bars) or siRNA targeting PCBP2 (black bars) and harvested 24 h later for determination of relative amounts of transcripts from 3 EBV lytic genes, BZLF1, BMRF1, and BFRF3, as well as PCBP2 by qRT-PCR after normalization to 18S rRNA levels by using the ΔΔCT method. Error bars indicate standard errors of the means of data from 3 technical replicates from each of 2 transfection experiments (*, P < 0.05). (B) HH514-16 cells were transfected with either PCBP2 siRNA and FITC+ scrambled siRNA (PCBP2) at a 3:1 ratio (the latter to mark transfected cells) or FITC-negative and FITC+ scrambled siRNA at a 3:1 ratio (Sc). After 24 h, the FITC+ (i.e., transfected) population was examined for lytic cells by flow cytometry using reference EBV-seropositive and -seronegative human sera. Numbers indicate the percentages of transfected cells that were spontaneously lytic. Data are representative of two experiments.
FIG 6
FIG 6
Overexpression of PCBP2 results in repression of the EBV lytic cycle. (A to C) HH514-16 cells were transfected with an empty vector (EV) or the PCBP2 plasmid, exposed to NaB after 12 h, and harvested after another 24 h for determination of the relative amounts of transcripts from 3 EBV lytic genes, BZLF1, BMRF1, and BFRF3, as well as PCBP2 by qRT-PCR after normalization to 18S rRNA levels by using the ΔΔCT method (A) and examined for PCBP2 expression (after comparison with cells stained with the isotype control antibody) (B) or for lytic cells by using reference EBV-seropositive and -seronegative human sera after gating on “live cells” based on forward- and side-scatter distribution by flow cytometry (C). Error bars in panel A indicate standard errors of the means of data from 3 technical replicates from each of 2 transfection experiments (*, P < 0.05). The number in panel B indicates the percentage of live, PCBP2-transfected cells expressing high levels of PCBP2. Numbers in panel C indicate the percentages of live cells that were lytic. (D) HH514-16 cells were transfected at the same time as in panels A and B with FITC+ scrambled siRNA (or were mock transfected as a control), harvested 36 h later, and examined for the percentage of FITC-positive cells (numbers in dot plots) by flow cytometry. Data are representative of two experiments.
FIG 7
FIG 7
STAT3 functions via PCBP2 to restrict EBV lytic cycle activation. HH514-16 cells were transfected with the empty vector and scrambled siRNA (control), the STAT3 plasmid and scrambled siRNA, or the STAT3 plasmid and siRNA targeting PCBP2; exposed to NaB after 12 h; and harvested after another 24 h for determination of the relative amounts of transcripts from the EBV immediate early lytic genes BZLF1 (open bars) and BRLF1 (black bars) (A) as well as STAT3 (open bars), PCBP2 (black bars), and PCBP1 (gray bars) (B) by qRT-PCR after normalization to 18S rRNA levels by using the ΔΔCT method. Error bars are standard errors of the means of data from 3 technical replicates from each of 2 transfection experiments (*, P < 0.05).
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