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. 2001 Mar 27;98(7):4119-24.
doi: 10.1073/pnas.051004198.

Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator

J Chen  1 , K Ueda, S Sakakibara, T Okuno, C Parravicini, M Corbellino, K Yamanishi
Affiliations

Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator

J Chen et al. Proc Natl Acad Sci U S A. .

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposi's sarcoma, primary effusion lymphomas, and a subset of multicentric Castleman's disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV/Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.

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Figures

Figure 1
Figure 1
Immunofluorescence assay (IFA) of BCBL-1 cells. Cells were collected untreated or treated (for 48 h) with TPA, 5-azacytidine (AZC), or AZC plus phosphonoformic acid (AZC + PFA), and checked by mAb against Lyta, ORF K9, ORF 59, and ORF K8.1 of KSHV. The negative cells were stained red (with Evans blue) and the positive cells, bright yellow.
Figure 2
Figure 2
Southern blotting results. (A) Untreated BCBL-1 DNA were hybridized with different digoxigenin-labeled probes. The restriction enzyme HpaII (H) and MspI (M) are isoschizomers that cleave the same target sequence in DNA but have a different response to its state of methylation. MspI cleaves all CCGG sequences whether or not they are methylated at the second C, but HpaII cleaves only nonmethylated CCGG tetramers. (B) Lyta promoter probed with different cells shows the same pattern in BCBL-1 and BC-3 cells, but not in Ramos cells which are KSHV negative.
Figure 3
Figure 3
Methylation status in the Lyta promoter region. (A) Sequencing image shows the bisulfite modified sequence. All of the cytosines except for methyl-cytosine were converted to thymine when treated with bisulfite. Arrows indicate the CpG sites. Numbers indicate the position relative to the Lyta transcription start site. (B) Methylation status in the Lyta promoter region of BCBL-1 cells untreated or treated with TPA for 24 h. Each included six independent clones. Solid circles indicate methylated CpG, and open circles indicate demethylated CpG. (C) Time course of TPA treatment. DNA samples from BCBL-1 cells treated with TPA at time intervals of 1, 4, 8, 12, and 24 h were bisulfite-modified, PCR-amplified, and sequenced directly without the separation of a single clone. Arrows show the CpG sites, which were mixed with C (methylated C) and T (demethylated C). T, A, G, and C are shown as red, green, black, and blue, respectively, in the Applied Biosystems sequencing image.
Figure 4
Figure 4
Methylation status in the LANA promoter region in latent (untreated) BCBL-1 cells. Solid circles indicate methylated CpG, and open circles indicate demethylated CpG. Numbers indicate the position relative to the transcription start site of LANA.
Figure 5
Figure 5
Luciferase reporter assay of the Lyta promoter compared with other promoters and in different cell lines. Open bars in A, B, and C represent untreated TPA. Solid bars in A, B, and C represent TPA treated immediately after transfection. Cells were collected 48 h after transfection; luciferase activity values were normalized to a cotransfected internal control plasmid (pCMV-β-gal) and shown as folded, as compared with a promoterless vector (pGL3), which is defined as 1. (A) Compared with other virus IE promoters (human cytomegalovirus and simian virus 40) in 293T cells. (B) Compared with other gene promoters of KSHV [K9.P (ORF K9 promoter), POL.P (DNA polymerase promoter)] in 293T cells. (C) Lyta promoter activity in different cell lines with or without TPA treatment. (D) Lyta promoter activity in 293T cells cotransfected with pcDNA3.1 vector (open bar) or pcDNA3.1-ORF 50 cDNA clone (solid bar).
Figure 6
Figure 6
Methylation cassette assay of the Lyta promoter. (A) Outline of the methylation cassette assay. (B) Confirms the effect of methylation with SssI in vitro. Mock: plasmid DNA prepared from E. coli was mock methylated as a control. Methylated: the same equivalent DNA of the Lyta promoter fragment was methylated with a methylase-SssI. Both mock and methylated DNA were digested by HapII (H) and MspI (M) under the same conditions. (C) Lyta promoter ablates its luciferase activity when methylated by SssI in vitro. Open box: Mock-methylated Lyta promoter. Its reporter activity is shown as 100%. Solid box: the relative methylated Lyta promoter–reporter activity.
Figure 7
Figure 7
Determination of elements in the Lyta promoter region. (A) Schematic diagram of the Lyta promoter and a series of nested deletion mutants. Numbers indicate the position relative to the transcription start site of Lyta (7). A hypermethylated region is shown in the promoter region between −315 to −255 base. Closed circles: methylated CpGs in latently infected KSHV. (B) Luciferase activation of the Lyta promoter and the corresponding deletion mutants. Rev.: Promoter orientation-reverse construct.
Figure 8
Figure 8
Methylation status of KSHV-lyta promoter in KSHV-related diseases. Solid circles indicate methylated CpG, and open circles indicate demethylated CpG. (A) MCD; (B) PEL; and (C) KS. (D) Peripheral blood mononuclear cells (PBMC) and spleen samples from a latent KSHV carrier.
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