CO-TRANSFECTION OF TWO HUMAN CELL LINES WITH pSV2-NE0 AND CLONED RESTRICTION FRAGMENTS OF EPSTEIN-BARR VIRUS B95-8 DNA.

Abstract

Introduction of Epstein-Barr virus DNA into mammalian cell lines by microinjection and transfection has been an effective method to study transient viral antigen expression and gene mapping (Boyd, et al., 1985; Glaser et al., 1983; Graessmann, et al., 1980; Stoerker et al., 1981; Takada et al., 1986b). Here I describe a method for the stable introduction of subgenomic fragments of EBV B95-8 DNA into two EBV negative human cell lines, the nasopharyngeal carcinoma line, CNE, and the fibroblast line AD/AH. CNE and AD/AH cells were co-transfected with pSV2-neoᴿ, which contains the bacterial Tn5 neo gene, and recombinant pBR322 plasmids containing individual EcoR12 restriction fragments of B95-8 DNA. Co-transfectants were selected for stable expression of Tn5 neo in G418 supplemented media. Fifty-two AD/AH-neoᴿ and 16 CNE-neoᴿ clones were isolated and screened for expression of viral antigens by indirect immunofluorescence using pre-characterized human sera and monoclonal antibodies specific for EBV antigens. Expression of the nuclear antigen EBNA-3 was detected in the CNE-neoᴿ K clone which had received the EcoR1 restriction fragment. CNE-neoᴿ clones co-transfected with the EcoR1 D, F, H, I, J, L, and M restriction fragments reacted with human sera against EBV early antigens. Two clones, CNE-neoᴿ Cl and CNE-neoᴿ C8, which received the EcoR1 C restriction fragment reacted with anti-VCA human sera. CNEneoᴿ clones were expanded and analyzed for stable maintenance of EBV DNA by Southern blot and stable expression of EBV antigens by Western blot and radio-immunoprecipitation. Two of the expanded CNE-neoᴿ cell lines were found to contain EBV DNA as demonstrated by hybridization of cDNA EBV radiolabeled probes to extracted cellular DNA (H. Ying, personal communication). Expansion of immuno-positive CNE-neoᴿ clones, however, resulted in the loss of EBV antigen expression. Further study is required to determine if loss of antigen expression is due to the lack of EBV DNA stability in neoᴿ co-transformants or due to repression of viral gene expression. The cell lines generated here provide the opportunity to further explore the possible restrictions involved with EBV gene expression.