T-cell activation leads to poor activation of the HIV-1 clade E long terminal repeat and weak association of nuclear factor-kappaB and NFAT with its enhancer region.
The enhancer region in the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) is very important for viral transcription. This promoter sequence binds both nuclear factor-kappaB and NFAT, two important modulators of HIV-1 gene expression. Previous studies have indicated that the enhancer regions of the different HIV-1 clade LTRs differ in their number of NF-kappaB-binding sites. In this study, we have compared the activation potential of the different HIV-1 clade and HIV-2 LTRs and assessed their interaction with NFAT and NF-kappaB. In T-cell lines and primary CD4(+) T-cells, the results showed that the HIV-1 clade E LTR (with a single NF-kappaB-binding site) was the weakest LTR regardless of the tested activators, whereas the HIV-2 LTR was the most responsive LTR. The clade E enhancer region was also demonstrated to be the weakest enhancer region in transfection experiments with luciferase reporter-based vectors. Electrophoretic mobility shift assays with extracts from activated CD4(+) T-cells indicated that, although NF-kappaB and NFAT bound all enhancers, HIV-1 clade E and HIV-2 LTR enhancers were poor binding targets for these two factors. Weak NFAT binding to clade E enhancers was also confirmed using NFAT1-expressing 293T cells in competition experiments. We have also shown the absence of interaction of NF-kappaB or NFAT with the third NF-kappaB repeat present in clade C. However, the clade C enhancer bound NFAT more efficiently than all other enhancer regions tested. Our results hence demonstrate for the first time that differences in the binding of NF-kappaB and NFAT to the enhancer regions could be responsible for some of the observed variation in HIV-1 clade LTR activation, whereas HIV-2 LTR activation seems mostly independent of these interactions.
Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 Blvd. Laurier, Sainte-Foy, Quebec G1V 4G2, Canada.
Anne-Marie
AM
Lemieux
Marie-Eve
ME
Paré
Brigitte
B
Audet
Eric
E
Legault
Sylvain
S
Lefort
Nancy
N
Boucher
Sébastien
S
Landry
Tim
T
van Opijnen
Ben
B
Berkhout
Mojgan H
MH
Naghavi
Michel J
MJ
Tremblay
Benoit
B
Barbeau
D001665:Binding Sites; D001667:Binding, Competitive; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D008213:Lymphocyte Activation; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020079:Terminal Repeat Sequences; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; C494006:NFATC2 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D008156:Luciferases