LMP1-Induced Sumoylation Influences the Maintenance of Epstein-Barr Virus Latency through KAP1

Abstract

As a herpesvirus, Epstein-Barr virus (EBV) establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme Ubc9 via its C-terminal activating region-3 (CTAR3) and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or leakier in EBV-transformed lymphoblastoid cell lines. The cells are also more sensitive to viral reactivation induced by irradiation, which results in the increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV OriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency.

Importance: Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by a ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to antiviral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to antiviral therapies.

FIG 1

Deletion of CTAR3 did not affect viral replication but increased background levels of lytic replication. EBV WT- and EBV dCTAR3-expressing 293 cells were induced by transfection with ZTA expression plasmids or noninduced by transfection with the vector control, and cells and supernatants were collected at 48 h posttransfection. (A) Virus was harvested, and relative viral loads were quantitated by real-time PCR. The fold change in the amount of viral DNA (relative to that for noninduced EBV WT- and EBV dCTAR3-expressing 293 cells) was determined. (B) Raji cells were exposed to the remaining supernatants. The percentages of GFP-positive cells were determined by flow cytometry after 48 h. (C) For noninduced cells, the fold change in the amount of viral DNA (relative to that for noninduced EBV WT-expressing 293 cells) was determined. (D) Raji cells were exposed to the remaining supernatants. The percentages of GFP-positive cells were determined as described above. Results are shown as means ± standard deviations from experiments performed in triplicate.

FIG 2

Deletion of CTAR3 increases cell susceptibility to viral reactivation by irradiation. (A) EBV WT- and EBV dCTAR3-expressing 293 cells were grown and exposed to various doses of irradiation, and supernatants were collected at 24 h postirradiation. The supernatants were added to Raji cells, and the percentage of GFP-positive cells was determined after 48 h. (B and C) EBV WT- and EBV dCTAR3-expressing 293 cells (293 EBV cells) (B) and paired EBV WT- and EBV dCTAR3-transformed LCLs (C) were exposed to 5 Gy of irradiation, and the fold changes in viral loads (relative to the viral load for noninduced cells) were determined. All results are shown as means ± standard deviations from experiments performed in triplicate.

FIG 3

Inhibition of LMP1-induced sumoylation increases lytic viral replication following irradiation. (A) EBV-expressing 293 cells were transfected with Ubc9 C93S, Ubc9 s9RNA, or control expression vectors. At 24 h posttransfection, cells were treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid). Whole-cell lysates were collected at 48 h posttransfection. Western blot analyses were performed for SUMO-1, Ubc9, and GAPDH (loading control). (B) EBV WT-expressing 293 cells were treated with a vehicle control and transfected with scrambled siRNA and an empty expression vector, treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid), transfected with Ubc9 C93S, or transfected with Ubc9 siRNA. (C and D) EBV WT-transformed LCLs were treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid) or the vehicle control and induced (C) or noninduced (D). Fold changes in viral loads were determined 24 h after irradiation. All results are shown as means ± standard deviations from experiments performed in triplicate.

FIG 4

Inhibition of sumoylation increases cell susceptibility to cytotoxic antiviral drugs following irradiation. (A) EBV WT-transformed LCLs, EBV dCTAR3-transformed LCLs, and SUMO inhibitor (25 μM anacardic acid and 25 μM ginkgolic acid)-treated EBV WT-transformed LCLs were irradiated (5 Gy; induced) or noninduced. (B) EBV WT-expressing 293 cells, EBV dCTAR3-expressing 293 cells, and EBV WT-expressing 293 cells treated with water and transfected with scrambled siRNA and an empty expression vector, treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid), transfected with a Ubc9 C93S expression plasmid, or transfected with Ubc9 siRNA were induced or noninduced. At 24 h postirradiation, cells were treated with ganciclovir (10 mg/ml) or vehicle control (water). After 1 week, trypan blue exclusion assays were performed and fold changes in cell death were calculated. Results are shown as means ± standard deviations from experiments performed in triplicate.

FIG 5

LMP1 expression correlates with endogenous KAP1 sumoylation in a CTAR3-dependent manner. (A) Denaturing immunoprecipitations (IP) were performed on BL41 EBV-negative, BL41 EBV-positive, and BL41 P3HR1 mutant-infected cells with SUMO-1-specific antibodies or IgG control antibodies. Western blot (WB) analyses were used to detect KAP1 covalently modified by SUMO-1. (B) Densitometric analysis of repeat experiments was performed. Results are shown as means ± standard deviations from experiments performed in triplicate. pos, positive; neg, negative. (C) Western blot analyses were used to detect KAP1 covalently modified by SUMO-1 in paired EBV WT- and EBV dCTAR3-transformed LCLs with SUMO-1-specific antibodies or IgG control antibodies. (D) Densitometric analysis of repeat experiments was performed. Results are shown as means ± standard deviations from experiments performed in triplicate. (E) 293 cells were transfected with GFP-KAP1 and either FLAG-LMP1, FLAG-LMP1 dCTAR3, or vector control expression constructs. At 48 h posttransfection, whole-cell lysates were collected and immunoblotting assays were performed to detect LMP1 (FLAG) and KAP1 (GFP) levels. GAPDH was used as a loading control.

FIG 6

LMP1 CTAR3-induced sumoylation regulates KAP1 function. (A) Whole-cell lysates, chromatin-enriched extracts (chrom), and the corresponding supernatants (sup) collected from 293 cells transfected with LMP1 expression constructs or control expression constructs; (B) paired EBV WT- and EBV dCTAR3-transformed LCLs and EBV WT-transformed LCLs treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid); (C) 293 cells transfected with a vector control expression construct and scrambled siRNA, LMP1 dCTAR3 expression constructs, LMP1 expression constructs, LMP1 expression constructs treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid), LMP1 and Ubc9 C93S expression constructs, and LMP1 expression constructs treated with Ubc9 siRNA. (A) Western blot analyses were performed, and KAP1 and GAPDH levels were detected. (B and C) Densitometric analysis of repeat immunoblots and slot blot analyses were performed, and the KAP1 levels in chromatin fractions relative to the levels in whole-cell lysates were determined. Fold changes in KAP1 chromatin association were determined. (D and E) Paired EBV WT- and EBV dCTAR3-transformed LCLs and EBV WT-transformed LCLs treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid) (D) and EBV WT-expressing 293 cells, EBV dCTAR3-expressing 293 cells, and EBV WT-expressing 293 cells treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid), transfected with Ubc9 C93S-expression constructs, or transfected with Ubc9 siRNA (E) were grown, and chromatin immunoprecipitations were performed with KAP1-specific antibodies or control IgG antibodies. Real-time PCR analyses were performed to examine the KAP1 association with the GABPB2, EFCAB7, and ZNF gene promoters. The fold change in DNA binding (relative to that for the input controls) was determined. Results are shown as means ± standard deviations from experiments performed in triplicate.

FIG 7

KAP1 binds to the EBV OriLyt and the immediate early promoters. Paired EBV WT-transformed LCLs, EBV dCTAR3-transformed LCLs, and EBV WT-transformed LCLs treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid) (A) and EBV WT-expressing 293 cells, EBV dCTAR3-expressing 293 cells, and EBV WT-expressing 293 cells treated with the vehicle control (water) and transfected with scrambled siRNA and an empty expression vector and treated with SUMO inhibitors (25 μM anacardic acid and 25 μM ginkgolic acid), transfected with Ubc9 C93S expression constructs, or transfected with Ubc9 siRNA (B) were grown, and chromatin immunoprecipitations were performed with KAP1-specific antibodies or control IgG antibodies. Real-time PCR analyses were done for OriLyt, the ZTA promoter, the RTA promoter, the BALF5 promoter, the BNRF1 promoter, the BCRF1 promoter, and Qp. The fold change in DNA binding (relative to input controls) was determined. Results are shown as means ± standard deviations from experiments performed in triplicate.