Two strains of SIVmac show differential transactivation mediated by sequences in the promoter.
Two infectious molecular clones of simian immunodeficiency virus, SIVmac251 and SIVmac239, have very different in vivo properties, SIVmac239 being much more pathogenic than SIVmac251. To assess whether the in vivo differences between the two viruses would be reflected in transcriptional rates in vitro, transcriptional activity in the presence of the transactivation protein tat was analyzed by transient transfection assays in HUT-78 and U937 cells. Whereas the two promoters had similar basal activities (Anderson and Clements, 1991, J. Virol. 65, 51-60) the promoter of SIVmac239 was transactivated to a greater extent. Removal of sequences 5' to -225 and 3' to +18 maintained the basal activity, yet made the promoter unresponsive to tat. Addition of bases +19 to +149 reconstituted transactivation and decreased basal activity. Analysis of deletion mutants with reconstituted transactivation response region determined that differences between the two strains were maintained even when only the proximal sequences, -225 to +18 of the U3 and R region were placed upstream of the TAR sequences. This region contains four nucleotide differences and the potential Sp-1-binding sites, where there are an additional 11 bases in SIVmac239 that create a third potential Sp-1 site, compared to only 2 in SIVmac251. Transactivation in this assay system was found to correlate better to RNA differences shortly after transfection (12 hr) than later (46 hr).
Anderson
M G
MG
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218.
Clements
J E
JE
D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat; D014644:Genetic Variation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015302:Simian Immunodeficiency Virus / Q000145:classification* / Q000235:genetics*; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D014162:Transfection
D015696:Gene Products, tat; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase