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. 2023 Oct 25;11(11):2624.
doi: 10.3390/microorganisms11112624.

Establishment of Epstein-Barr Virus (EBV) Latent Gene-Expressing T-Cell Lines with an Expression Vector Harboring EBV Nuclear Antigen 1

Hiroyuki Kanno  1 , Tomohiro Osada  1 , Ayako Tateishi  1
Affiliations

Establishment of Epstein-Barr Virus (EBV) Latent Gene-Expressing T-Cell Lines with an Expression Vector Harboring EBV Nuclear Antigen 1

Hiroyuki Kanno et al. Microorganisms. .

Abstract

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and is associated with EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders, which frequently lead to the development of life-threatening complications, such as virus-associated hemophagocytic syndrome and EBV-positive apparent leukemia/lymphoma mainly in T- and NK-cell lineages. In order to clarify the EBV genes responsible for the diseases, we introduced the plasmid coding sequences of EBV-encoded small RNAs (EBERs) and/or latent membrane protein (LMP) 1 into human T-lymphocyte virus-I-negative human T-cell lines using a gene expression vector harboring EBV nuclear antigen 1, established the G418-resistant transformants of five T-cell lines, and quantitatively examined the expression of EBERs and LMP1 using real-time reverse transcriptase-polymerase chain reaction. The expression levels of EBERs in T-cell transformants with EBER DNA paralleled those in EBV-positive human T- and NK-cell lines, SNTK cells. The expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and the expression of LMP1 mRNA in T-cell lines expressing both EBERs and LMP1 was much lower than that in the same cell line expressing LMP1 mRNA alone. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders.

Keywords: EBV-encoded small RNAs; Epstein–Barr virus; T-cell lines; chronic active EBV infection; latent membrane protein 1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of EBER1 and EBER2 in EBV-positive T- or NK-cell lines. Real-time RT-PCR specific for EBER1 or EBER2 was performed as described in Materials and Methods Section. Bars indicate the mean of absolute copy number of EBER1 or EBER2 in 50 ng of total RNA of each cell line.
Figure 2
Figure 2
Expression of EBER1 and EBER2 in the stable transformants of human T-cell lines transfected with pEBMulti-Neo-EKS6. Real-time RT-PCR specific for EBER1 or EBER2 was performed as described in Materials and Methods Section. Bars indicate the mean of absolute copy number of EBER1 or EBER2 in 50 ng of total RNA of each transformant.
Figure 3
Figure 3
Expression of LMP1 mRNA in EBV-positive T- or NK-cell lines and in the stable transformants of human T-cell lines transfected with pEBMulti-Neo-LMP1 alone or both pEBMulti-Neo-EKS6 and pEBMulti-Neo-LMP1. Real-time RT-PCR specific for LMP1 mRNA was performed as described in Materials and Methods Section. Bars indicate the relative expression level of mRNA compared to that in TK6 cells, which was standardized with the expression of mRNA of glyceraldehyde-3-phosphate dehydrogenase, represented as permillage of that in TK6 cells (LCLs).
Figure 4
Figure 4
Expression of EBER1 and EBER2 in the stable transformants of human T-cell lines transfected with pEBMulti-Neo-EKS6 alone or both pEBMulti-Neo-EKS6 and pEBMulti-Neo-LMP1. Real-time RT-PCR specific for EBER1 or EBER2 was performed as described in Materials and Methods Section. Bars indicate the mean of absolute copy number of EBER1 or EBER2 in 50 ng of total RNA of each transformant.
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