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, 74 (20), 9755-61

Latent Membrane Protein 1 of Epstein-Barr Virus Inhibits as Well as Stimulates Gene Expression

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Latent Membrane Protein 1 of Epstein-Barr Virus Inhibits as Well as Stimulates Gene Expression

M L Sandberg et al. J Virol.

Abstract

The latent membrane protein 1 (LMP-1) of Epstein-Barr virus (EBV) functionally resembles a constitutively active, CD40-like receptor and contributes to the maintenance of proliferation of EBV-infected primary human B lymphocytes. LMP-1 is targeted to the plasma membrane, where it binds TRAF, TRADD, and JAK molecules to activate NF-kappaB-, AP-1-, and STAT-dependent pathways as does CD40. Yet LMP-1 appears to lack a ligand to regulate its signaling. We have found that LMP-1, when expressed at physiologic levels, inhibits gene expression detectably. Higher levels of LMP-1 expression eventually inhibit both the steady-state level of RNA produced from a BamHI C promoter reporter and general cellular protein synthesis. These findings indicate that LMP-1 can limit its signaling and that this control is manifest at two levels. The domain of LMP-1 that binds TRAF, TRADD, and JAK/STAT molecules is not required for this regulation. A derivative of LMP-1 that contains only its amino-terminal and membrane-spanning domains is sufficient to inhibit reporter activity when the reporter genes are expressed from the BamHI C and LMP-1 promoters. This same derivative of LMP-1 in parallel assays is sufficient to inhibit wild-type LMP-1's stimulation of NF-kappaB-dependent gene expression. We suggest that LMP-1 encodes stimulatory and inhibitory activities; the latter could limit signaling in the apparent absence of ligand-dependent down-regulation.

Figures

FIG. 1
FIG. 1
Diagrams of the B95-8 strain of EBV, oriP-Bam Cp-luciferase, and oriP-LMP-1p-luciferase reporters. Shown are the elements expressed and used by EBV in latent infection of resting B cells in cell culture. Not shown are the more than 80 genes used during the lytic phase of infection. Letters inside the circle indicate the fragments of the B95-8 strain of EBV resulting from digestion of the genome with BamHI. The promoters used in latent infection are indicated by arrowheads, with the primary RNA transcripts generated from these promoters indicated by dashed lines and the open boxes representing exons of these transcripts. The origin of DNA replication used in the latent life cycle (oriP), the origin of DNA replication used in the lytic life cycle (ori Lyt), and the site of DNA circularization after infection, the terminal repeats (TR), are indicated by black boxes. Above the genome are diagrams of the oriP-Bam Cp-luciferase and oriP-LMP-1p-luciferase reporters used. oriP consists of 20 EBNA-1 binding sites found in the family of repeats (FR) and four binding sites for EBNA-1 in the dyad symmetry element (DS). The luciferase open reading frame was inserted at the locations indicated. The base pair numbers indicate locations of the corresponding DNAs found in the B95-8 strain of EBV (1).
FIG. 2
FIG. 2
LMP-1's effect on expression from oriP-Bam Cp-luciferase and oriP-LMP-1p-luciferase. (A) Increasing amounts of SVLMP-1 (0, 1, 3, 9, and 27 μg) were transfected as indicated into BJAB cells along with 5 μg of oriP-EBNA-1 and 5 μg of oriP-Bam Cp-luciferase. In all transfections, the same total amount of DNA was used with an empty vector added as a filler. The average number of LMP-1 molecules per transfected cell was calculated as described below. Relative activity of oriP-Bam Cp-luciferase was determined 48 h posttransfection by setting the luciferase detected in the presence oriP-EBNA-1 and absence of SVLMP-1 to 100%. The average RLU detected at 100% of oriP-Bam Cp-luciferase activity is 1.5 × 106. The data shown are averages of three independent transfections. Error bars indicate 1 standard deviation from the mean; where no error bar is indicated, the standard error was smaller than the size of the symbol. Wild-type LMP-1 as it resides in the plasma membrane is shown as an inset. (B) oriP-LMP-1p-luciferase (10 μg) was cotransfected into BJAB cells with 0, 1, 3, or 9 μg of SVLMP-1. The cells were assayed for luciferase activity 48 h later. The average number of LMP-1 molecules per transfected cell was calculated as described below. The data represent the averages of four independent experiments, with 100% oriP-LMP-1p-luciferase activity corresponding to the 5.5 × 103 RLU detected in the absence of SVLMP-1. Error bars indicate 1 standard deviation from the mean. (C) Measuring LMP-1 expression levels in the EBV-immortalized B-cell line 721 and transfected BJAB cells. Luciferase data are taken directly from the experiments represented in panel A. The average number of LMP-1 molecules per cell was calculated by the following method. GST–LMP-1(181-386) was isolated from E. coli DH5α as described previously (35) and found to be 50% pure. GST–LMP-1(181-386) extracts of transfected cells were assayed for LMP-1 by quantitative Western blotting (35) with LMP-1 signals corrected for the efficiency of transfection, which was approximately 50%. All data represent the averages of three independent experiments.
FIG. 3
FIG. 3
Measuring LMP-1's effect on oriP-Bam Cp-CAT in BJAB cells by CAT assay and S1 nuclease mapping. (A) Extracts of BJAB cells cotransfected with the indicated DNAs were assayed for CAT activity as described previously (37); 5 μg of oriP-Bam Cp-CAT, 5 μg of oriP-EBNA-1, and 9 μg of SVLMP-1 were cotransfected where indicated. The percentage of chloramphenicol acetylated in each extract is indicated at the bottom. (B) S1 nuclease mapping of GAPDH and CAT RNAs in the cell extracts shown in panel A was performed as described previously (14). The expected sizes of signals of undigested probes and digested probes are indicated at the right. Lane 1 contains markers; their sizes are indicated in nucleotides at the left. Above each lane is indicated the probe used in the S1 reaction and the DNAs transfected in each set of BJAB cells. Lane 3 is the GAPDH probe digested in the absence of any cellular RNA. The fold induction of CAT activity was determined by averaging the CAT activity observed in four independent experiments, with the signal of oriP-Bam Cp-CAT set to 1. The difference between the CAT activity observed for each point was determined by the Wilcoxon rank sum test and has a P value of 0.01. The CAT RNA levels were determined by PhosphorImager quantitation of signals in the S1 nuclease mapping gels shown in panel B. The fold induction of CAT RNA is the average of four independent S1 nuclease mapping experiments after normalizing each for its detected GAPDH signals. The statistical significance of the levels of CAT RNA in each point was determined by the Wilcoxon rank sum test and has a P value of 0.01. (C) Metabolic labeling of cells was performed by incubating 107 BJAB cells with 100 μCi of [35S]Met-Cys for 60 min at 37°C 48 h after transfection. The cells were washed once with RPMI 1640 containing 10% fetal bovine serum and then lysed in 1 ml of 0.2 N NaOH for 5 min at 25°C; 10 ml of 10% trichloroacetic acid containing unlabeled methionine (30 μg/ml) was added, and the samples were bound to glass fiber filters. The filters were washed twice with 10 ml of 10% trichloroacetic acid–unlabeled methionine (30 μg/ml) and once with 10 ml of 100% ethanol. Filters were measured for bound radioactivity in a liquid scintillation counter. Normalized 35S incorporation was determined by setting the radioactivity detected in labeled BJAB cells transfected with the oriP-Bam Cp-CAT reporter alone to 1. 35S incorporation data are averages of three independent experiments; ± indicates 1 standard deviation from the mean.
FIG. 4
FIG. 4
6MHALMP-1EE inhibits gene expression. Shown in the inset is 6MHALMP-1EE and its predicted secondary structure in the plasma membrane. (A) The oriP-Bam Cp-luciferase reporter was not or was cotransfected with 100 ng, 300 ng, 1 μg, 3 μg, or 9 μg of an expression vector for 6MHALMP-1EE into BJAB (squares) and GG68 cells (circles). After 48 h, the cells were assayed for luciferase activity; 100% reporter activity corresponds to the 1.3 × 106 RLU in BJAB cells and 3 × 104 RLU in GG68 cells detected in the absence of 6MHALMP-1EE. oriP-EBNA-1 was not included in the experiments performed in GG68 cells because these cells express EBNA-1 constitutively. The data represent the averages of three independent experiments. Error bars indicate 1 standard deviation from the mean. (B) BJAB cells were cotransfected with a constant amount of a NF-κB-responsive luciferase reporter, the indicated amounts of 6MHALMP-1EE, and either 1 μg of SVLMP-1 (circles) or 30 ng of an expression vector for a NF-κB p50/p65 fusion protein (squares); 100% reporter activity is calculated from the luciferase activity detected in the absence of 6MHALMP-1EE and in the presence of 1 μg of transfected SVLMP-1 (average of 2 × 105 RLU) or 30 ng of transfected p50/65 expression vector (average of 1.5 × 105 RLU). Results shown are the averages of three independent transfection experiments. Error bars indicate 1 standard deviation from the mean. (C) A clone of BJAB cells which expresses 6MHALMP-1EEGFP conditionally was tested for inhibition of protein synthesis by this truncated derivative of LMP-1. 35S-incorporation was measured 48 h after addition of the indicated amounts of tetracycline. Cells were labeled as described in the legend to Fig. 3, with 35S-incorporation in the uninduced cells set to 1. The percent 35S incorporated is the average of four independent experiments, with ± indicating 1 standard deviation from the mean. The average number of 6MHALMP-1EEGFP molecules per cell was determined by measuring the amount of 6MHALMP-1EEGFP expressed in one of the four experiments used to determine the percent 35S incorporated.

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