Abstract
A group of repetitive sequences, known as the Family of Repeats (FR), is a critical cis-acting sequence required for EBV latent infection. The FR sequences are heterogeneous among EBV strains, and they are sometimes subject to partial deletion when subcloned in E. coli-based cloning vectors. However, the FR stability in EBV-BAC (bacterial artificial chromosome) system has never been investigated. We found that the full length FR of the Akata strain EBV was not stably maintained in a BAC vector. By contrast, newly obtained BAC clones of the B95-8 strain of EBV stably maintained the full length FR during recombinant virus production and B-cell transformation. Investigation of primary DNA sequences of Akata–derived EBV-BAC clones indicates that the FR instability is most likely due to a putative secondary structure of the FR region. We conclude that the FR instability in EBV-BAC clones can be a pitfall in E. coli-mediated EBV genetics.
Highlights
Epstein-Barr Virus (EBV), a member of the gamma herpesvirus family, is a human tumor virus that is associated with various neoplastic diseases, such as Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s lymphoma, and opportunistic lymphomas in immunosuppressed transplantation recipients [1]
Various repetitive sequences are scattered throughout the EBV genome, and the Family of Repeats (FR) sequence is in the oriP region (Fig. 1A)
The FR sequence of the B95-8 strain of EBV has a 128-bp palindromic sequence in its 39 end [5], and the 252-bp sequence containing this palindromic sequence tends to be deleted when subcloned in E. coli-based plasmid vectors (Fig. 1B)
Summary
Epstein-Barr Virus (EBV), a member of the gamma herpesvirus family, is a human tumor virus that is associated with various neoplastic diseases, such as Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s lymphoma, and opportunistic lymphomas in immunosuppressed transplantation recipients [1]. The EBV genome is a linear, double-stranded, 175-kb genome, in which more than 80 viral genes are encoded [2,3]. The EBV genomes have another group of repetitive sequences, designated as Family of Repeats (FR) [2,5,6], which are located within the oriP region of the genome [7]. These repetitive sequences (IRs, TR, and FR) consist of unique sequences within loci made up of varied numbers of repeats
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