Schematic description of EBNA1 expression constructs containing identical mRNA sequences whilst encoding alternative repeat reading frames.
A, The EBNA1 frameshift constructs were generated in either pcDNA3 for in vitro translation studies or in pEGFP-N1 for EBNA1-GFP expression and immunological studies. The overlapping DNA binding and dimerization domain, nuclear localization signal (NLS) and Glycine/Arginine (GR) repeat flanking sequences essential for genome maintenance functions are shown. The localizations of a model SIINFEKL epitope and the endogenous EBNA1 HPVGEADYFEY epitope used in the presentation assays are highlighted. B, Alignment of amino acid sequences of the EBNA1 (E1) repeat frameshift variants E1-GA(wild-type), E1-GQE(frameshift 1) and E1-GRS(frameshift 2). An asterisk indicates identical residues in all three proteins. Arrows denote nucleotide deletion positions at the start of the internal repeat and nucleotide insertion positions at the ends of the repeat in the E1-GA(wild-type) sequence to generate alternative repeat reading frames whilst maintaining wild-type EBNA1 sequence at both the N- and C-terminal domains flanking the internal repeat sequence.