In vivo footprinting analysis of constitutive and inducible protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1.
The regulation of the rate of transcription of human immunodeficiency virus type 1 is mainly exerted through the long terminal repeat (LTR) at the 5' end of the provirus. A large number of cis-acting regulatory elements have been identified in the LTR by in vitro binding studies; the biological role of these sites within living infected cells, however, is still not clear. We have studied the interactions of nuclear proteins with the LTR in the U1 monocytic cell line by in vivo dimethylsulfate footprinting, using the ligation-mediated polymerase chain reaction technique. In this cell line, transcription of the virus, which is very low under basal conditions, is highly inducible by treatment with phorbol esters; therefore, this system is likely to represent a suitable cellular model to study viral latency. Independently of the level of viral transcription, major in vivo footprints appear at the two Sp1 sites adjacent to the enhancer, the downstream-positioned enhancer repeat, the NFAT binding site, and one of the purine-rich sites of the negative regulatory element. Upon transcriptional activation by phorbol myristate acetate, the only perturbation in the footprinting pattern is a dramatic increase in dimethylsulfate sensitivity of guanine at position -92 in the downstream enhancer repeat. This modification is correlated with the transient induction of two enhancer-binding activities, as determined by gel retardation assays. While the transcriptional rate is still increasing and the in vivo footprinting pattern is unchanged at up to 24 h postactivation, these enhancer-binding factors are considerably reduced at this time. Therefore, further levels of regulation have to be considered to explain the maintenance of the induced state.
Demarchi
F
F
International Centre for Genetic Engineering and Biotechnology, Trieste.
D'Agaro
P
P
Falaschi
A
A
Giacca
M
M
D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D016655:HIV Core Protein p24 / Q000032:analysis; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D016133:Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014775:Virus Activation / Q000235:genetics; D017735:Virus Latency / Q000235:genetics
D004268:DNA-Binding Proteins; D016655:HIV Core Protein p24; D013755:Tetradecanoylphorbol Acetate