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. 2023 Feb 2;15(2):423.
doi: 10.3390/v15020423.

Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas

Joanna W Y Ho  1   2 , Lili Li  3 , Kai Yau Wong  1 , Gopesh Srivastava  1 , Qian Tao  3   4
Affiliations

Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas

Joanna W Y Ho et al. Viruses. .

Abstract

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Keywords: CpG methylation; EBV; LMP1; Zta; latency; peripheral T-cell lymphoma; promoter.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Autoradiograph images showing Southern blot hybridization of RT-PCR products for various EBV gene transcripts detected in seven PTCL cases. The B95-8 cell line was used as a positive control.
Figure 2
Figure 2
Detection of EBV protein expression by immunohistochemistry (IHC). (A) LMP1 protein shown by cytoplasmic staining in a pleomorphic medium-sized and large T-cell lymphoma case (case 2); (B) ZEBRA protein shown by strong nuclear staining in the same case. (C) LMP1 protein, shown by strong cytoplasmic staining in a case (case t3). Black arrowheads indicate EBV protein-positive cells.
Figure 3
Figure 3
Promoter usage for EBNA1 expression. Autoradiograph showing Southern blot hybridization of RT-PCR products from EBV gene transcripts differentially initiated from Cp, Wp, and Fp promoters in seven PTCL cases. B95-8 was the positive control.
Figure 4
Figure 4
Analysis of the CpG methylation status of EBV Cp and Wp in PTCL by methylation-specific PCR (MSP) using bisulfite genomic DNA. “m” primers were used for methylated promoters, while “u” primers were used for unmethylated promoters.
Figure 5
Figure 5
ISH for the detection of BHLF1 and EBER transcripts. (A) B95-8, an EBV+ lytic cell line, was used as a lytic positive control for BHLF1. (B) BJAB, an EBV-negative cell line, was used as a negative control. (C) A PTCL case (case t3) was shown to be EBV+ by being EBER+. The vast majority of tumor cells were EBER+ with a diffuse pattern of distribution. (D) The BHLF1 transcript was shown to be expressed in rare, isolated EBV+ cells in EBER+ case 3t.
Figure 6
Figure 6
EBV genotyping of PTCL cases, including the EBNA3C variation, the LMP1 gene 30 bp deletion, and the BamHI F/f variation. (A) Autoradiograph images showing Southern blot hybridization of PCR products. (B) Agarose gel electrophoresis of PCR products from a separate assay. EBNA3C had a 153 bp product for type-A EBV and a 246 bp product for type-B EBV. A 30 bp deletion at the LMP1 carboxyl terminus is shown by the shorter PCR product of 286 bp rather than a 316 bp band for the wild-type gene. BamHI F is shown by an uncut PCR product band of 222 bp, whereas the BamHI f gene is shown by a cut PCR product with two bands of 125 bp and 97 bp. The controls used are as follows: for the EBNA3C gene, the B95-8 cell line is type-A, and the AG876 cell line is type-B. For the LMP1 gene, B95-8 is wild-type, and Jijoye or AG876 is deletion-type. For BamHI F/f variation, B95-8 is the positive control for BamHI F and Jijoye for BamHI f.
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This work was supported by Hong Kong HMRF (#20190152; #16151042; #21200842) and RGC.

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