Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1

Abstract

The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation.

Importance: EBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

FIG 1

Latent EBV infection correlates with increased PAR levels. (A) PAR levels in EBV-negative B cells and type I and type III latently infected EBV-positive B cells. Stars indicate corresponding isogenic cell lines. Results show averages ± standard deviations (SD) and are representative of those from three experiments. (B) Western blot of EBV-positive cells shown in panel A probed for the EBV proteins EBNA1, EBNA2, and LMP1 or the host protein PARP1. Actin served as a loading control. Western blots are representative of those from three independent experiments.

FIG 2

LMP1 expression increases PAR levels. DG75 (A) and Rat1 (B) cells were transfected with an LMP1 expression construct or plasmid vector, and PAR levels were measured by ELISA. Results are averages ± SD and are representative of three experiments. LMP1 expression was confirmed by Western blotting, and actin served as a loading control. (C) Rat1 cells were transfected with EBNA1 or an empty vector, and PAR levels were measured by ELISA. Results show averages ± SD and are representative of three experiments. Western blot for EBNA1 confirms expression. Actin served as a loading control. (D) LCL, GM13605, and Raji cells were transduced with shLMP1 or a scrambled lentiviral construct (shCtr) for 96 h, and protein extracts were analyzed by Western blotting for LMP1 and actin (loading control). IB, immunoblot. (E) PAR levels were measured by ELISA in cells treated as described for panel D. Results are averages ± SD and are representative of three experiments.

FIG 3

LMP1 increases PAR through PARP1. (A) Western blot for PARP1 in WT MEF and MEF PARP1^−/− cells confirms PARP1 deficiency. Actin served as a loading control. MEF PARP1^−/− (B) and WT MEF (C) were transfected with an empty plasmid vector or LMP1 expression construct, and PAR levels were measured by ELISA. Results show averages ± SD and are representative of three experiments. Western blot for LMP1 confirms expression. Actin served as a loading control. (D) MEF PARP1^−/− cells stably expressing pBabe (empty vector) or HA-LMP1 via retroviral transduction were transfected with an empty vector, WT PARP1, or a catalytically inactive PARP1 construct (PARP1 E998A). LMP1 and PARP1 expression were confirmed by Western blotting, and actin served as a loading control. The asterisk indicates the PARP1 band. (E) PAR levels in MEF PARP1^−/− cells shown in panel D were measured by ELISA and are graphed as percent PARP1 activity, with PARP1 activity in MEF PARP1^−/− cells stably expressing pBabe and transfected with the empty vector set at 100%. Results are averages ± SD and are representative of those from three experiments. Statistical significance is indicated by asterisks (*, P < 0.1).

FIG 4

LMP1 decreases global H3K27me3 levels through PARP1. (A) H3K27me3 levels measured by ELISA from histone extracts from the isogenic Kem I and Kem III cell lines. Bars represent the mean level (±SD) of H3K27me3 normalized to total histones. Results are representative of three experiments. WT MEF (B) and MEF PARP1^−/− (C) cells were transfected with a plasmid vector or an LMP1 expression construct, and H3K27me3 levels in isolated histone extracts were measured by ELISA. Bars represent the mean level of H3K27me3 normalized to total histones ± SD. Results are representative of three experiments. (D) Western blot of histone extracts from WT MEF or MEF PARP1^−/− transfected with an empty vector or LMP1 and probed for H3K27me3. Histone H3 serves as a loading control. The image is representative of three experiments.

FIG 5

PARP inhibition offsets LMP1-induced activation of its target genes, c-Fos and EGR1. (A) Quantitative chromatin immunoprecipitation (ChIP) assay for H3K27me3 occupancy at the c-Fos (left) or EGR1 (right) transcription start site (TSS) in control cells or LCLs treated with 2.5 μM olaparib for 72 h. Results are expressed as the percentage of input chromatin material ± SD and are representative of two independent experiments. (B) c-Fos (left) and EGR1 (right) mRNA transcripts were measured by RT-qPCR in LCLs left untreated or treated with 2.5 μM olaparib for 72 h. c-Fos and EGR1 mRNA levels in untreated cells were normalized to 1, and results are expressed relative to the untreated cells. Bars represent the mean level of expression ± SD and are representative of three independent experiments.

FIG 6

LMP1 regulates the expression of c-Fos and EGR1 through PARP1. MEF PARP1^−/− (A) and WT MEF (B) cells were transfected with an empty plasmid vector or an LMP1 expression construct. Bars represent the mean level of c-Fos or EGR1 mRNA expression normalized to the empty vector ± standard errors of the means (SEM) as measured by RT-qPCR. Experiments were performed in triplicate, and results are representative of three independent experiments. (C and D) MEF PARP1^−/− cells stably expressing HA-LMP1 were transfected with an empty plasmid vector or a WT PARP1 expression construct. Bars represent the mean level of c-Fos or EGR1 mRNA expression normalized to the empty vector ± SD as measured by RT-qPCR. Experiments were performed in triplicate, and results are representative of three independent experiments.

FIG 7

PARP inhibition reduces LMP1-mediated cellular transformation. (A) Rat1 cells were retrovirally transduced with the pBABE vector or HA-LMP1. Stably transduced pBABE- or HA-LMP1-expressing cells were treated with 1.25 μM olaparib, 2.5 μM olaparib, or DMSO for 3 days. Medium was then replaced with fresh medium and focus formation was analyzed after 14 days. The image is representative of six independent experiments. (B) Colony intensity for each condition in panel A was determined by the ImageJ plugin ColonyArea (n = 6; means ± SEM). A.u, arbitrary units. Statistical significance is indicated by asterisks (***, P ≤ 0.001).

FIG 8

PARP1 knockdown reduces LMP1-mediated cellular transformation. (A) Rat1 cells stably expressing HA-LMP1 were transduced with shPARP1 or a scrambled lentiviral construct (shCtr) for 72 h, and protein extracts were analyzed by Western blotting for PARP1, PARP2, LMP1, and actin (loading control). The Western blots are representative of those from three independent experiments. (B) Focus formation analysis of Rat1 cells stably expressing LMP1 following PARP1 depletion by an shPARP1 lentiviral construct. Puromycin was added 24 h after PARP1 depletion for selection. Cells were stained 7 days postlentiviral transduction. The image is representative of three independent experiments. (C) Number of colonies for each condition shown in panel B determined by counting. Statistical significance is indicated by asterisks (**, P < 0.05). (D) Quantification of colony intensity using ImageJ software analysis. Statistical significance is indicated by asterisks (*, P < 0.1). (E) Photomicrographs of phase contrast and GFP fluorescence of Rat1 cells stably expressing LMP1 transduced with shControl or shPARP1 GFP lentiviral particles. Images were acquired with an Envos FL cell imaging system using a 20× objective; the white bar represents 400 μm.