Transduction of the human immunodeficiency virus type 1 promoter into human chromosomal DNA by adeno-associated virus: effects on promoter activity.
Transcription of the human immunodeficiency virus type 1 (HIV-1) genome takes place after integration of the provirus into human chromosomal DNA. HIV transcription is known to be modulated by viral and cellular factors but the influence of flanking chromosomal sequences on proviral gene expression has not been well defined. To investigate the activity of the integrated HIV promoter, we exploited the ability of recombinant adeno-associated virus (AAV-2) to transfer and stably integrate genes into the human genome at random or site-specifically. Chimeric AAV vectors were constructed containing an HIV-CAT reporter cassette; some vectors also contained the neomycin resistance gene to facilitate the isolation of positive clones. HeLa cells were infected with recombinant AAV, in some instances together with wild-type virus as a source of AAV rep function. We isolated 25 clones of G418-resistant cells which carried the integrated HIV-CAT cassette, generally occupying unique sites that did not correspond to the AAV-specific region of chromosome 19. The HIV promoter was transcriptionally active in most of the clones. Basal promoter activity varied substantially among the clones, and its responsivity to the HIV transactivator Tat was also variable. The integrated HIV promoter was transactivated to comparable degrees by the one-exon form and two-exon form of Tat. These findings provide evidence that the transcriptional activity of the HIV promoter can be greatly influenced by the site of proviral insertion.
Nahreini
P
P
Cold Spring Harbor Laboratory, New York 11724, USA. nahreip@rpi.com
Mathews
M B
MB
D002461:Cell Line, Transformed; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002875:Chromosomes; D004247:DNA; D000229:Dependovirus; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D014407:Tumor Cells, Cultured; D016662:Virus Integration; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase