Gene expression in adeno-associated virus vectors: The effects of chimeric mRNA structure, helper virus, and adenovirus VA, RNA

Abstract

We used a recombinant plasmid containing an adeno-associated virus (AAV) genome to construct several vectors which express the gene for chloramphenicol acetyltransferase[dCAT[tiWe transfected four different AAV-CAT vectors into human 293[dadenovirus-transformed) cells and analyzed CAT activity. We show that, for vectors using the AAV p 40 and p 19 promoter, the chimeric AAV-CAT transcripts began from the correct 5' position but the basal level of CAT expression depended in part on the structure of the transcript. We also examined the effects of coinfection of the cells with the helper adenovirus or cotransfection with a plasmid which expressed the adenovirus translational control RNA, VA I RNA. Cotransfection with plasmids containing the gene for VA I RNA resulted in elevated levels of CAT activity. VA I RNA stimulated translation of the chimeric mRNA. However, in two cases, the VA I RNA apparently decreased the level of mRNA. These results suggest that in addition to its function in translation, VA, RNA acts at a second site to alter cytoplasmic accumulation of some mRNAs. Infection with adenovirus increased CAT activity several-fold by increasing the cytoplasmic levels of the chimeric AAV-CAT transcript. When the CAT gene is inserted down stream of the AAV intron, adenovirus and not VA, RNA alone increased CAT activity by promoting accumulation of a spliced transcript.