Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat

Five regions of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) have been shown to be important in the transcriptional regulation of HIV in HeLa cells. These include the negative regulatory, enhancer, SP1, TATA, and TAR regions. Previous studies in which purified SP1 was used showed that the three SP1-binding sites in the HIV LTR were important in the in vitro transcription of this promoter. However, no studies to ascertain the role of each of these SP1-binding sites in basal and tat-induced transcriptional activation in vivo have been reported. To determine the role of SP1 sites in transcriptional regulation of the HIV LTR in vivo, these sites were subjected to oligonucleotide mutagenesis both individually and in groups. The constructs were tested by DNase I footprinting with both oligonucleotide affinity column-purified SP1 and partially purified HeLa extract and by chloramphenicol acetyltransferase assays in both the presence and absence of the tat gene. Mutagenesis of each SP1-binding site resulted in minimal changes in basal and tat-induced transcriptional activation. Mutations involving alterations of SP1 sites I and II, I and III, or II and III also resulted in minimal decreases in basal and tat-induced transcriptional activation. However, mutagenesis of all three SP1-binding sites resulted in a marked decrease in tat induction. The latter mutation also greatly decreased DNase I protection over the enhancer, TATA, and TAR regions when partially purified HeLa nuclear extract was used. Mutagenesis of the HIV LTR SP1 sites which converted them to consensus high-affinity SP1-binding sites with the sequence GGGGCGGGGC resulted in increased tat-induced gene expression compared with the wild-type HIV LTR template. These results suggest that SP1, through its interaction with other DNA-binding proteins, is critical for in vivo transcriptional regulation of HIV.

[1]  P. Luciw,et al.  Structural arrangements of transcription control domains within the 5'-untranslated leader regions of the HIV-1 and HIV-2 promoters. , 1988, Genes & development.

[2]  R. Tjian,et al.  Positive and negative regulation of transcription in vitro: Enhancer-binding protein AP-2 is inhibited by SV40 T antigen , 1987, Cell.

[3]  P. Luciw,et al.  Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[4]  B. Howard,et al.  Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells , 1982, Molecular and cellular biology.

[5]  D. Capon,et al.  A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator , 1988, Molecular and cellular biology.

[6]  R. Tjian,et al.  Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II , 1983, Cell.

[7]  J. Chermann,et al.  Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). , 1983, Science.

[8]  S. Arya,et al.  Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). , 1985, Science.

[9]  R. Gaynor,et al.  Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus. , 1987, The EMBO journal.

[10]  M. Siekevitz,et al.  Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I. , 1987, Science.

[11]  R. Tjian,et al.  Affinity purification of sequence-specific DNA binding proteins. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Michael B. Mathews,et al.  Transcriptional but not translational regulation of HIV-1 by the tat gene product , 1988, Nature.

[13]  J. Sodroski,et al.  The trans-activator gene of the human T cell lymphotropic virus type III is required for replication , 1986, Cell.

[14]  R. Tjian,et al.  Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. , 1986, Science.

[15]  R. Tjian,et al.  Multiple specific contacts between a mammalian transcription factor and its cognate promoters , 1984, Nature.

[16]  Eric C. Holland,et al.  HIV-1 tat trans-activation requires the loop sequence within tar , 1988, Nature.

[17]  R. Tjian,et al.  Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. , 1985, Science.

[18]  H. Gendelman,et al.  Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[19]  R. Tjian,et al.  Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40 , 1987, Nature.

[20]  B. Cullen,et al.  Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. Nabel,et al.  An inducible transcription factor activates expression of human immunodeficiency virus in T cells , 1987, Nature.

[22]  D. Galas,et al.  DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. , 1978, Nucleic acids research.

[23]  Robert Tjian,et al.  Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro , 1985, Cell.

[24]  G. Pavlakis,et al.  Expression and characterization of the trans-activator of HTLV-III/LAV virus. , 1986, Science.

[25]  R. Tjian,et al.  The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter , 1983, Cell.

[26]  D. Capon,et al.  Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein , 1987, Cell.

[27]  J. Sodroski,et al.  A second post-transcriptional trans-activator gene required for HTLV-III replication , 1986, Nature.

[28]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[29]  R. Gaynor,et al.  Functional domains required for tat‐induced transcriptional activation of the HIV‐1 long terminal repeat. , 1988, The EMBO journal.

[30]  P. Luciw,et al.  Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product , 1987, Nature.

[31]  B. Cullen,et al.  Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat , 1988, Journal of virology.

[32]  R. Gaynor,et al.  Purification of the human immunodeficiency virus type 1 enhancer and TAR binding proteins EBP‐1 and UBP‐1. , 1988, The EMBO journal.

[33]  Bryan R. Cullen,et al.  Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism , 1986, Cell.

[34]  M. Feinberg,et al.  HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA , 1986, Cell.

[35]  M. Karin,et al.  In vitro activation of the HIV‐1 enhancer in extracts from cells treated with a phorbol ester tumor promoter. , 1987, The EMBO journal.

[36]  R. Roeder,et al.  Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region , 1985, Cell.

[37]  Phorbol ester enhances human immunodeficiency virus-promoted gene expression and acts on a repeated 10-base-pair functional enhancer element , 1987 .

[38]  J. Sodroski,et al.  The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat , 1985, Cell.

[39]  J. Sodroski,et al.  Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. , 1985, Science.

[40]  Masami Horikoshi,et al.  Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex , 1988, Cell.

[41]  R. Roeder,et al.  Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-kappa B) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[42]  P. Luciw,et al.  Human immunodeficiency virus long terminal repeat responds to T-cell activation signals. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Gaynor,et al.  Alterations in binding characteristics of the human immunodeficiency virus enhancer factor , 1988, Journal of virology.

[44]  R. Tjian,et al.  Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. , 1986, Science.

[45]  B. Haynes,et al.  Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. , 1984, Science.

[46]  J. Sodroski,et al.  Post-transcriptional regulation accounts for the trans-activation of the human T-lymphotropic virus type III , 1986, Nature.