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B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1.
J Virol. 1997 Dec; 71(12):9134-42.JV

Abstract

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive.

Authors+Show Affiliations

Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

9371570

Citation

Yoo, L I., et al. "B-cell Lines Immortalized With an Epstein-Barr Virus Mutant Lacking the Cp EBNA2 Enhancer Are Biased Toward Utilization of the oriP-proximal EBNA Gene Promoter Wp1." Journal of Virology, vol. 71, no. 12, 1997, pp. 9134-42.
Yoo LI, Mooney M, Puglielli MT, et al. B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1. J Virol. 1997;71(12):9134-42.
Yoo, L. I., Mooney, M., Puglielli, M. T., & Speck, S. H. (1997). B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1. Journal of Virology, 71(12), 9134-42.
Yoo LI, et al. B-cell Lines Immortalized With an Epstein-Barr Virus Mutant Lacking the Cp EBNA2 Enhancer Are Biased Toward Utilization of the oriP-proximal EBNA Gene Promoter Wp1. J Virol. 1997;71(12):9134-42. PubMed PMID: 9371570.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1. AU - Yoo,L I, AU - Mooney,M, AU - Puglielli,M T, AU - Speck,S H, PY - 1997/11/26/pubmed PY - 1997/11/26/medline PY - 1997/11/26/entrez SP - 9134 EP - 42 JF - Journal of virology JO - J. Virol. VL - 71 IS - 12 N2 - During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive. SN - 0022-538X UR - https://www.unboundmedicine.com/medline/citation/9371570/B_cell_lines_immortalized_with_an_Epstein_Barr_virus_mutant_lacking_the_Cp_EBNA2_enhancer_are_biased_toward_utilization_of_the_oriP_proximal_EBNA_gene_promoter_Wp1_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=9371570 DB - PRIME DP - Unbound Medicine ER -