Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells
Key findings
Chapman et al. cloned and sequenced a 2.4 kb fragment of the human cytomegalovirus Towne strain immediate-early gene containing intron A (823 bp), revealing a perfect consensus NF1 binding site at nucleotides 1488-1501 and an 18 bp regulatory element in exon A. The sequence analysis extended the known hCMV Towne IE1 gene by 533 bases and showed 97% homology with the AD169 strain in both coding and non-coding regions.
Expression vectors containing intron A demonstrated 2.3 to >300-fold increases in secreted glycoprotein levels compared to intron-deleted vectors in transiently transfected COS7 cells. Factor VIII heavy chain (gp92) expression increased from 43% to 100% of maximum, HIV-1 gpl20 expression rose from <0.3% to 100%, and full-length Factor VIII increased from 31% to 100% when intron A was present, demonstrating broad enhancement across diverse mammalian glycoproteins.
Site-directed mutagenesis of three base pairs in the intronic NF1 consensus sequence reduced Factor VIII gp92 expression to 69% and Factor VIII gp80 expression to 59% of wild-type intron A levels. Gel shift assays confirmed reduced NF1 protein binding to the mutant oligonucleotide compared to wild-type sequence, demonstrating that the NF1 binding site partially but not completely accounts for intron A-mediated expression enhancement in monkey kidney cells.