Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1.

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription.

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

The human immunodeficiency virus type 1 TAR RNA upper stem-loop plays distinct roles in reverse transcription and RNA packaging.

The human immunodeficiency virus type 1 (HIV-1) RNA genome is flanked by a repeated sequence (R) that is required for HIV-1 replication. The first 57 nucleotides of R form a stable stem-loop structure called the transactivation response element (TAR) that can interact with the virally encoded transcription activator protein, Tat, to promote high levels of gene expression. Recently, we demonstrated that TAR is also important for efficient HIV-1 reverse transcription, since HIV-1 mutated in the upper stem-loop of TAR showed a reduced ability both to initiate and to complete reverse transcription. We have analyzed a series of HIV-1 mutant viruses to better defined the structural or sequence elements required for natural endogenous reverse transcription and packaging of virion RNA. Our results indicate that the requirement for TAR in reverse transcription is conformation dependent, since mutants with mutations that alter the upper stem-loop orientation are defective for reverse transcription initiation and have minor defects in RNA packaging. In contrast, TAR mutations that allowed the formation of alternative upper stem-loop structure greatly reduced RNA packaging but did not affect reverse transcription efficiency. These results are consistent with direct involvement of the upper stem-loop structure in packaging of genomic RNA and suggest that the TAR RNA stem-loop from nucleotide +18 to +42 interacts with other components of the reverse transcription initiation complex to promote efficient reverse transcription.

Functional domains of Tat required for efficient human immunodeficiency virus type 1 reverse transcription.

Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with a tat gene deletion (Deltatat). Cell lines that contained Deltatat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3Lys were seen in the HIV-1 Deltatat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functional tat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

Treponema pallidum, lipoproteins, and synthetic lipoprotein analogues induce human immunodeficiency virus type 1 gene expression in monocytes via NF-kappaB activation.

Syphilitic genital ulcers are cofactors for the bidirectional transmission of human immunodeficiency virus (HIV). U937 human promonocytic cells chronically infected with HIV-1 (U1 cells) or transiently transfected with wild type or mutant HIV long terminal repeat (LTR) reporter constructs were used to examine mechanisms that likely underlie Treponema pallidum-induced immune cell activation and consequent induction of HIV. Virulent T. pallidum, a representative native treponemal lipoprotein (NTp47), or synthetic lipoprotein analogues (lipopeptides) all induced HIV replication in U1 cells. These stimuli also induced HIV gene expression from a wild type HIV LTR. HIV gene expression correlated with the translocation of NF-kappaB, and mutations within the NF-kappaB binding sites of the HIV LTR abrogated HIV gene expression. This study implicates treponemal lipoproteins as key mediators of immune cell activation and provides insights into the cellular and molecular bases for enhanced HIV transmission in syphilitic persons.

Tat is required for efficient HIV-1 reverse transcription.

The ability of human immunodeficiency virus-1 (HIV-1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA(3)(Lys) to the HIV-1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV-1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV-1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV-1 reverse transcription. HIV-1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV-1 proteins which were incorporated into the virion. Following transfection of wild-type but not mutant tat genes into cell lines producing HIV-1 lacking tat, the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV-1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat. Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV-1 reverse transcription.

Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B.

There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses.

Inhibition of human immunodeficiency virus type 1 replication is enhanced by a combination of transdominant Tat and Rev proteins.

Mutation of either of two critical human immunodeficiency virus type 1 (HIV-1) regulatory proteins, Tat and Rev, results in marked defects in viral replication. Thus, inhibition of the function of one or both of these proteins can significantly inhibit viral growth. In the present study, we constructed a novel transdominant Tat mutant protein and compared its efficiency in inhibiting HIV-1 replication with that of transdominant mutant Rev M10 when these proteins were stably expressed either alone or in combination in T-lymphocyte cell lines. The transdominant Tat mutant protein alone resulted in a modest inhibition of HIV replication, but it was able to enhance the ability of the M10 Rev mutant protein to inhibit HIV-1 replication. These results suggest a possible synergistic effect of these transdominant mutant proteins in inhibiting HIV-1 replication.

A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression is dependent on the transactivator protein Tat and an RNA element extending from the transcription initiation site to +57 known as TAR. TAR forms a stable RNA secondary structure which is critical for high levels of HIV-1 gene expression and efficient viral replication. Using a genetic approach, we isolated HIV-1 mutants in TAR that were competent for high levels of gene expression but yet were markedly defective for viral replication. Single-cycle infections with these viruses demonstrated that they were defective in the initiation of reverse transcription. Additional mutational analysis revealed a variety of other HIV-1 TAR mutants with the same defective phenotype. Thus, in addition to the well-characterized role of the primer binding site, other RNA elements within the HIV-1 genome are also critical in the regulation of reverse transcription. These studies demonstrate that HIV-1 TAR RNA is a key regulator of the reverse transcription and illustrate how a unique RNA structure can modulate diverse regulatory processes in the HIV-1 life cycle crucial for efficient viral replication.

Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication.

The TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Jurkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression and replication. We previously demonstrated that viruses containing mutations of the TAR RNA stem, bulge, or loop structures have 200- to 5,000-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jurkat cell lines infected with TAR element mutant viruses which spontaneously produced culture supernatants with wild-type-like levels of reverse transcriptase activity. These viral supernatants were used to infect Jurkat cells, and following PCR amplification of the viral long terminal repeats, their DNA sequences were analyzed. This analysis demonstrated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR. In gel retardation analysis, recombinant Tat protein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs and assayed following transfection of Jurkat cells. Constructs containing revertant TAR element mutations were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary structure of TAR RNA sequences suggested that TAR RNA structures which differed from that of wild-type TAR were still capable of strong activation in response to Tat. These results further define critical sequences in TAR RNA that are required for tat activation. In addition, since TAR structures with lower free energy that preserve the loop and bulge structures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for tat activation.

Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs.

Human immunodeficiency virus type 1 (HIV-1) gene expression is modulated by both viral and cellular factors. A regulatory element in the HIV-1 long terminal repeat known as TAR, which extends from nucleotides -18 to +80, is critical for the activation of gene expression by the transactivator protein, Tat. RNA transcribed from TAR forms a stable stem-loop structure which serves as the binding site for both Tat and cellular factors. Although TAR RNA is critical for Tat activation, the role that TAR DNA plays in regulating HIV-1 gene expression is not clear. Several studies have demonstrated that TAR DNA can bind cellular proteins, such as UBP-1/LBP-1, which repress HIV-1 gene expression and other factors which are involved in the generation of short, nonprocessive transcripts. In an attempt to characterize additional cellular factors that bind to TAR DNA, a lambda gt11 expression cloning strategy involving the use of a portion of TAR DNA extending from -18 to +28 to probe a HeLa cDNA library was used. We identified a cDNA, designated TAR DNA-binding protein (TDP-43), which encodes a cellular factor of 43 kDa that binds specifically to pyrimidine-rich motifs in TAR. Antibody to TDP-43 was used in gel retardation assays to demonstrate that endogenous TDP-43, present in HeLa nuclear extract, also bound to TAR DNA. Although TDP-43 bound strongly to double-stranded TAR DNA via its ribonucleoprotein protein-binding motifs, it did not bind to TAR RNA extending from +1 to +80. To determine the function of TDP-43 in regulating HIV-1 gene expression, in vitro transcription analysis was performed. TDP-43 repressed in vitro transcription from the HIV-1 long terminal repeat in both the presence and absence of Tat, but it did not repress transcription from other promoters such as the adenovirus major late promoter. In addition, transfection of a vector which expressed TDP-43 resulted in the repression of gene expression from an HIV-1 provirus. These results indicate that TDP-43 is capable of modulating both in vitro and in vivo HIV-1 gene expression by either altering or blocking the assembly of transcription complexes that are capable of responding to Tat.

Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes.

The role of NF-kappa B-dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF-kappa B subunit and the induction by phorbol esters of bona fide (p50-p65) NF-kappa B complexes. In parallel, we observed clear enhancer-dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B-responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF-kappa B-dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants.

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.

Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs.

A primary site of infection by human adenoviruses is lymphoid cells. However, analysis of the viral control elements and the cellular factors that regulate adenoviral gene expression in lymphocytes has not been reported. The adenovirus early region 3 (ES) gene products are involved in the maintenance of viral persistence by complexing with the class I MHC antigens, thus preventing their cell surface expression with a resultant decrease in host immunologic destruction. To determine whether different cellular factors were involved in E3 regulation in lymphocytes as compared with HeLa cells, both DNA binding and transfection analysis with the E3 promoter in both cell types were performed. These studies detected two novel domains referred to as L1 and L2 with a variety of lymphoid but not HeLa extracts. Each of these domains possessed strong homology to motifs previously found to bind the cellular factor NF-kappa B. Transfections of E3 constructs linked to the chloramphenicol acetyltransferase gene revealed that mutagenesis of the distal NF-kappa B motif (L2) had minimal effects on promoter expression in HeLa cells, but resulted in dramatic decreases in expression by lymphoid cells. In contrast, mutagenesis of proximal NF-kappa B motif (L1) had minimal effects on gene expression in both HeLa cells and lymphoid cells but resulted in a small, but reproducible, increase in gene expression in lymphoid cells when coupled to the L2 mutation. Reversing the position and subsequent mutagenesis of the L1 and L2 domains indicated that the primary sequence of these motifs rather than their position in the E3 promoter was critical for regulating gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes.

Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus early region 3 promoter regulation by E1A/E1B is independent of alterations in DNA binding and gene activation of CREB/ATF and AP1.

Transcription of the adenovirus early region 3 promoter is strongly induced by the adenovirus E1A protein. Previous DNase I footprinting has indicated that four regions in this promoter serve as binding sites for HeLa nuclear proteins. These include binding sites for NF-1 (site IV), AP1 (site III), CREB/activating transcription factor (ATF) (site II), and TATA (site I). To determine the relative importance of these sites in both the in vivo and in vitro transcriptional regulation of the E3 promoter, oligonucleotide-directed mutagenesis of these sites was performed. Each of these constructs was assayed by transfection onto HeLa cells in the presence of either dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. Mutations of either the ATF- or AP1-binding sites but not the TATA- and NF1-binding sites resulted in severe decreases in both basal and E1A/E1B-induced transcriptional levels. These constructs were also assayed in in vitro transcription assays with cellular extracts prepared from dl434-infected or wild-type-adenovirus-infected HeLa cells. The wild-type E3 promoter was transcribed approximately 30 times more efficiently in extracts containing the E1A/E1B proteins compared with extracts lacking these proteins. Mutations of either the TATA element, the ATF site, or the AP1-binding site decreased both basal and E1A/E1B-induced transcriptional levels. Gel retardation analysis using these extracts indicated that the binding to ATF, AP1, or NF1 oligonucleotides was not altered in the presence of the E1A/E1B proteins compared with extracts lacking these proteins. Northern (RNA) blot analysis of c-jun and CREB RNA prepared from wild-type adenovirus and dl434-infected cells indicated that the levels of these RNAs were not altered by the E1A/E1B proteins. Immunoprecipitation of AP1 and CREB from both dl434- and wild-type-adenovirus-infected cells indicated that the amounts of these proteins were not significantly altered. These results suggest that E1A/E1B-induced activation of the E3 promoter does not involve activation of transcription factor genes nor a change in the DNA binding activity of important promoter-binding components. Our results are consistent with a model in which the E1A/E1B proteins either directly or indirectly alter the interactions of factors that bind to the basal E3 promoter transcription complex, thereby inducing transcription.

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.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression.

Repeated B motifs in the human immunodeficiency virus type I long terminal repeat enhancer region do not exhibit cooperative factor binding.

The enhancer element of the human immunodeficiency virus type I (HIV-I) long terminal repeat (LTR) contains two copies of nearly identical sequences AGGGACTTTCC (3G sequence) and GGGGACTTTCC (4G sequence) that are important in transcriptional regulation. A single copy of the 4G sequence is found in the NF-kappa B site of the immunoglobulin kappa-chain enhancer. Only the 4G motif in the HIV enhancer is bound by cellular proteins in extracts prepared from unstimulated HeLa cells, whereas the 3G and 4G motifs are bound by factors in extracts prepared from HeLa cells treated with phorbol esters [phorbol 12-myristate 13-acetate (PMA)] and lymphoid cells. To determine if this change in binding to the HIV enhancer was due to phosphorylation of a cellular protein, partially purified PMA-treated HeLa nuclear extracts were digested with calf intestinal phosphatase. Phosphatase digestion of nuclear extracts from PMA-treated HeLa cells markedly decreased factor binding to the HIV enhancer. Accordingly, phosphorylation of the DNA binding protein itself, or an inhibitor protein present in the partially purified extract, must mediate binding to the recognition sequence. Binding studies confirmed that each of the enhancer sequences was capable of binding factors independent of the activity of the other site and that the HIV enhancer was occupied by only one factor at any one time. Chloramphenicol acetyltransferase assays using mutants in either one or both HIV enhancer repeats revealed that each site was capable of functioning as a tat-inducible enhancer element in PMA-treated HeLa cells. These results suggest that the 3G and 4G motifs in the HIV enhancer function independently and that duplication in the HIV enhancer augments activity by a mechanism distinct from cooperative binding of NF-kappa B.