The transcriptional regulation of the human immunodeficiency virus (HIV) type I involves the interaction of both viral and cellular proteins. The viral protein tat is important in increasing the amount of viral steady‐state mRNA and may also play a role in regulating the translational efficiency of viral mRNA. To identify distinct functional domains of tat, oligonucleotide‐directed mutagenesis of the tat gene was performed. Point mutations of cysteine residues in three of the four Cys‐X‐X‐Cys sequences in the tat protein resulted in a marked decrease in transcriptional activation of the HIV long terminal repeat. Point mutations which altered the basic C‐domain of the protein also resulted in decreases in transcriptional activity, as did a series of mutations that repositioned either the N or C termini of the protein. Conservative mutations of other amino acids in the cysteine‐rich or basic regions and in a series of proline residues in the N terminus of the molecule resulted in minimal changes in tat activation. These results suggest that several domains of tat protein are involved in transcriptional activation with the cysteine‐rich domain being required for complete activity of the tat protein.

Regulation of human immunodeficiency virus (HIV) gene expression is dependent on specific regulatory regions in the long terminal repeat. These regions include the enhancer, SP1, "TATA," and trans-activating (TAR) regions. In addition, viral regulatory proteins such as tat and rev are important in regulating HIV gene expression. The mechanism of tat activation remains the subject of investigation, but effects at both transcriptional and posttranscriptional levels seem likely. Previous mutagenesis of the tat protein revealed that the amino terminus, the cysteine-rich domain, and the basic domain were all required for complete tat activation. Mutants of other viral trans-acting regulatory proteins, including ElA, tax, and VM65, have been identified that were capable of antagonizing the activity of their corresponding wild-type proteins. We wished to determine whether mutants of the tat protein could be identified that exhibited a similar phenotype.

The 5' flanking region ofthe human a-globin gene is highly G+C rich and contains multiple copies of the consensus sequence for the Spl binding site. We investigated the role of this G+C-rich region in augmenting a-globin promoter activity in the presence of the far-upstream a-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the a-globin G+C-rich 5' flanking region has no effect on a-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an a-globin promoter retaining this region. These results suggest that the a-globin G+C-rich 5' flanking region augments a-globin promoter activity in a chromatin-dependent manner. We further show that this G+C-rich region is required for the activation of a-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G+C-rich region to increase ax-globin promoter activity from a stably integrated a-globin gene is mediated by its multiple binding sites for the transcription factor Spl.The human a-globin gene cluster lies within an early replicating G+C-rich isochore close to the telomere of the short arm of chromosome 16. It consists of three functional genes arranged 5'-;2-a2-a1-3'. As with the ,B-globin genes, the a-like globin genes are erythroid specific. However, they are structurally quite different to the f3-globin genes in that they are highly G+C rich, associated with CpG islands, and lack scaffold-associated regions (for review, see ref.

RNA-protein complexes isolated following a gel retardation assay can be footprinted within the gel matrix using the chemical nuclease activities of 4,7-dimethyl-, 5,6-dimethyl-, and 3,4,7,8-tetramethyl-1,10-phenanthroline-copper. These complexes are more reactive than 1,10-phenanthroline-copper but share its reaction preference for bulges and loops. The interaction of the coat protein of R-17 with its viral RNA target and tat- and tat-derived peptides with HIV TAR RNA have been studied. In both cases, the RNA sequence opposite a 2-3 nucleotide bulge are protected. Tat-derived peptides inhibit cleavage at sites which intact tat does not protect. These results are consistent with transcription studies which have suggested that truncation of tat increases nonspecific binding.

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus ElA and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the ElA and ElB proteins. To determine which regions of the HIV LTR were important for complete ElA/ElB activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. Sl nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivationresponsive element were each required for complete ElA/ElB-mediated activation of the HIV LTR. These same promoter elements were required for both basal and ElA/ElB-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with d1434, an ElA/ElB deletion mutant, or wild-type adenovirus. No mutations were found that reduced only ElA/ElB-induced expression without proportionally reducing basal levels of transcription-, suggesting that ElA/ElB-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for ElA/ElB-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction.