Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection.

BACKGROUND: The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1. RESULTS: Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites. CONCLUSION: The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.

IRF-1 is required for full NF-kappaB transcriptional activity at the human immunodeficiency virus type 1 long terminal repeat enhancer.

Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-kappaB transcriptional activity. We provide evidence that IRF-1 and NF-kappaB form a functional complex at the long terminal repeat (LTR) kappaB sites, which is abolished by specific mutations in the two adjacent kappaB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-kappaB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

Duplication of peri-kappaB and NF-kappab sites of the first human immunodeficiency virus type 2 (HIV-2) transmission in Brazil.

After the identification of HIV-2 in 1986, most of the cases reported have been concentrated in West Africa. We identified a case of HIV-2 infection in São Paulo, Brazil of a 45-year-old female who presented with Pneumocystis carinii pneumonia, with a CD4 count of 22 cells/ml. DNA samples from this patient were subjected to end-point PCR amplification of the LTR region. Clones were sequenced and subjected to phylogenetic analyses. All clones were subtype A related, and four presented an insertion, corresponding to an extra NF-kappaB site. This is the first confirmed case report of an HIV-2-infected subject identified in Brazil whose transmission occurred within the country. Furthermore, the NF-kappaB duplication would potentially be associated with an increase in viral cytopathogenicity. This raises concern for the need for permanent monitoring of the spread of HIV-2 in different areas of the world, even considering its lower rate of transmission and pathogenicity when compared to HIV-1.

Differential regulation of human immunodeficiency virus type 2 and simian immunodeficiency virus promoter activity.

Promoter activity of the HIV-1 long terminal repeat (LTR) is largely dependent on intact NF-kB and SpI binding sites in the U3 region. In contrast, upstream LTR sequences allow efficient simian immunodeficiency virus (SIVmac) transcription in the absence of the core enhancer promoter region. In the present study, we investigated whether the regulation of HIV-2 Rod LTR activity is more reminiscent of HIV-1 having the same host or of SIVmac239 belonging to the same phylogenetic group. Viral promoter activity was studied in the context of the integrated provirus using both single cycle assays with pseudotyped luciferase reporter viruses and replication-competent HIV-2 LTR mutants. Our results demonstrate that intact SpI binding sites are important for both HIV-2 and SIVmac LTR activity in T cells and monocyte-derived macrophages. In contrast, deletion of the NF-kB binding site or of upstream regulatory sequences impaired HIV-2 Rod LTR activity but had little effect on SIVmac239 promoter function. Thus, similar to HIV-1, regulation of HIV-2 LTR promoter activity shows a low degree of functional redundancy possibly suggesting a specific adaptation to the human host.

PU.1 binding to ets motifs within the equine infectious anemia virus long terminal repeat (LTR) enhancer: regulation of LTR activity and virus replication in macrophages.

Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the role of these three sites in EIAV LTR activity. While DNase I footprinting studies indicated that all three sites within the enhancer were bound by recombinant PU.1, reporter gene assays demonstrated that the middle motif was most important for basal levels of LTR activity in macrophages and that the 5' motif had little impact. The impact of the 3' site became evident in Tat transactivation studies, in which the loss of the site reduced Tat-transactivated expression 40-fold. In contrast, elimination of the 5' site had no effect on Tat-mediated activity. Binding studies were performed to determine whether differences in PU.1 binding affinity for the three sites correlated with the relative impact of each site on LTR transcription. While small differences were observed in the binding affinities of the three sites, with the promoter-proximal site having the strongest binding affinity, these differences could not account for the dramatic differences observed in the transcriptional effects. Instead, the promoter-proximal position of the 3' motif appeared to be critical for its transcriptional impact and suggested that the PU.1 sites may serve different roles depending upon the location of the sites within the enhancer. Infectivity studies demonstrated that an LTR containing an enhancer composed of the three PU.1 sites was not sufficient to drive viral replication in macrophages. These findings indicate that while the promoter-proximal PU.1 site is the most critical site for EIAV LTR activity in the presence of Tat, other elements within the enhancer are needed for EIAV replication in macrophages.

Cellular specificity of HIV-1 replication can be controlled by LTR sequences.

Two well-established determinants of retroviral tropism are envelope sequences that regulate entry and LTR sequences that can regulate viral expression in a cell-specific manner. Studies with human immunodeficiency virus-1 (HIV-1) have demonstrated that tropism of this virus maps primarily to variable envelope sequences. Studies have demonstrated that T cell and macrophage-specific transcription factor binding motifs exist in the upstream region of the LTR U3; however, the ability of the core enhancer/promoter proximal elements (two NF-kappaB and three Sp1 sites) to function well in macrophages and T cells have led many to conclude that HIV LTR sequences are not primary determinants of HIV tropism. To determine if cellular specificity could be imparted to HIV by the core enhancer elements, the enhancer/promoter proximal region of the HIV LTR was substituted with motifs that control gene expression in a myeloid-specific manner. The enhancer region from equine infectious anemia virus (EIAV) when substituted for the HIV enhancer/promoter proximal region was found to drive expression in a macrophage-specific manner and was responsive to HIV Tat. The addition of a 5' methylation-dependent binding site (MDBP) and a promoter proximal Sp1 motif increased expression without altering cellular specificity. Spacing between the promoter proximal region and the TATA box was also found to influence LTR activity. Infectivity studies using chimeric LTRs within the context of a dual-tropic infectious molecular clone established that these LTRs directed HIV replication and production of infectious virions in macrophages but not primary T cells or T cell lines. This investigation demonstrates that cellular specificity can be imparted onto HIV-1 replication at the level of viral transcription and not entry.

Structural and functional evolution of human immunodeficiency virus type 1 long terminal repeat CCAAT/enhancer binding protein sites and their use as molecular markers for central nervous system disease progression.

The appearance and progression of human immunodeficiency virus type 1 (HIV-1)-associated pathogenesis in the immune and central nervous systems is dependent on the ability of the virus to replicate in these compartments, which is, in turn, controlled by numerous factors, including viral binding and entry, receptor and coreceptor usage, and regulation of viral expression by the long terminal repeat (LTR). The LTR promotes viral expression in conjunction with viral and cellular regulatory proteins, including members of the CCAAT/enhancer binding protein (C/EBP) family, which modulate LTR activity through at least two cis-acting binding sites. Previous studies have shown that these sites are necessary for HIV-1 replication in cells of the monocyte/macrophage lineage, but dispensable in T lymphocytes. To establish potential links between this important family of transcription factors and HIV-1-associated pathogenesis, C/EBP site I and II sequence variation in peripheral blood mononuclear cell (PBMC)-derived LTRs from HIV-1-infected patients with varying degrees of disease severity was examined. A high prevalence of C/EBP site variants 3T (site I) and consensus B (site II) within PBMC-derived HIV-1 LTRs was shown to correlate with late stage disease in HIV-1-infected patients. These results suggest that the increased prevalence in the PBMCs of HIV-1 LTRs containing the 3T C/EBP site I variant and the consensus B site II variant may serve as a molecular marker for disease progression within the immune system. The relative low or high binding affinity of C/EBP beta to sites I and II in electrophoretic mobility shift (EMS) analyses correlated with low or high LTR activity, respectively, in transient expression analyses during both early and late disease stages. The 3T C/EBP site I was the only variant examined that was not found in LTRs derived from PBMCs of patients at early stages of HIV-1 disease, but was found at increasing frequencies in patients with late stage disease. Furthermore, the 3T C/EBP site I was not found in brain-derived LTRs of patients without HIV-1-associated dementia (HIVD), but was found in increasing numbers in brain-derived LTRs from patients diagnosed with HIVD. The C/EBP site I 3T variant appears to be exclusive to patients progressing to increasingly severe HIV-1-associated immunologic and neurologic disease.

Differential regulation of HIV-1 clade-specific B, C, and E long terminal repeats by NF-kappaB and the Tat transactivator.

The major group of human immunodeficiency viruses (HIV-1) that comprise the current global pandemic have diversified during their worldwide spread and may be divided into at least 10 distinct subtypes or clades, A through J. Subtype B predominates in North America and Europe, subtype E predominates in Southeast Asia, and subtype C predominates in sub-Saharan Africa. Functional distinctions in long terminal repeat (LTR) architecture among HIV subtypes have been identified, thus raising the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. In addition to the transcriptional specificity of the HIV-1 LTR, productive HIV-1 replication is also dependent upon the viral Tat protein. Therefore, we sought to investigate whether interactions between host signaling pathways and the NF-kappaB regions of different HIV-1 subtypes, together with subtype-specific interactions between Tat, TAR, and cellular proteins, modulate the efficiency of HIV-1 clade-specific gene transcription. We demonstrate that the NF-kappaB sites of subtypes B and E both bind NF-kappaB-related complexes. However, the duplicated kappaB sites of the C subtype do not compete for NF-kappaB binding. Also, clade E Tat protein possesses the highest transactivation capacity, regardless of the LTR context. Furthermore, preliminary evidence suggests that the acetylation of subtype-specific Tat proteins may correlate with their transactivation efficiency.

Protein phosphatase 2A activates the HIV-2 promoter through enhancer elements that include the pets site.

Human immunodeficiency virus type 2 (HIV-2) gene expression is regulated by upstream promoter elements, including the peri-Ets (pets) site, which mediate enhancer stimulation following treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We previously showed that the oncoprotein DEK binds to the pets site in a site-specific manner. In this report, we show that binding to the HIV-2 pets site is modulated by treatment of U937 monocytic cells with TPA, an activator of protein kinase C. TPA treatment resulted in a reduction in the levels of DEK and the formation of a faster migrating pets complex in gel shift assays. We show further that the actions of TPA on pets binding can be duplicated by phosphatase treatment of nuclear proteins and is blocked with okadaic acid, a protein phospatase-2A (PP2A) inhibitor. Finally, we demonstrate that ectopic expression of the catalytic domain of PP2A can activate the HIV-2 enhancer/promoter alone or in synergy with TPA, an effect mediated in part through the pets site. These results suggest that, through an interaction with the protein kinase C pathway, PP2A is strongly involved in regulating HIV-2 enhancer-mediated transcription. This is a consequence of its effects on DEK expression and binding to the pets site, as well as its effects on other promoter elements. These findings have implications not only for HIV-2 transcription but also for multiple cellular processes involving DEK or PP2A.

Multiorgan nuclear factor kappa B activation in a transgenic mouse model of systemic inflammation.

We utilized a line of transgenic mice expressing Photinus luciferase complementary DNA (cDNA) under the control of a nuclear factor kappa B (NF-kappaB)-dependent promoter (from the 5' human immunodeficiency virus-1 [HIV-1] long terminal repeat) to examine the role of NF-kappaB activation in the pathogenesis of systemic inflammation induced by bacterial endotoxin (lipopolysaccharide [LPS]). After intraperitoneal injection of E. coli LPS, these mice displayed a time- and dose-dependent, organ-specific pattern of luciferase expression, showing that NF-kappaB-dependent gene transcription is transiently activated in multiple organs by systemic LPS administration. Luciferase expression in liver could be specifically blocked by intravenous administration of replication-deficient adenoviral vectors expressing a dominant inhibitor of NF-kappaB (IkappaB-alphaDN), confirming that luciferase gene expression is a surrogate marker for NF-kappaB activation in this line of mice. After treatment with intraperitoneal LPS, the mice were found to have increased lung tissue messenger RNA (mRNA) expression of a variety of cytokines that are thought to be NF-kappaB-dependent, as well as elevated serum concentrations of presumed NF-kappaB-dependent cytokines. In lung tissue homogenates, a close correlation was identified between luciferase activity and KC levels. These studies show that systemic treatment with LPS orchestrates a multiorgan NF-kappaB-dependent response that likely regulates the pathobiology of systemic inflammation.

Long terminal repeat promoter/enhancer activity of different subtypes of HIV type 1.

Transcription of the HIV-1 provirus genome is regulated by a complex interplay between viral regulatory proteins and cellular transcription factors that interact with the viral long terminal repeat (LTR) region of HIV-1. However, several cellular transcription factors have been identified that can interact with the HIV-1 LTR; the significance of all of these factors is not clearly understood. In this study we have characterized the LTR region of different subtypes of HIV-1 with regard to nucleotide sequence and promoter activity. The LTR regions of HIV-1 from peripheral blood mononuclear cells of 29 infected individuals originating from 10 different geographical regions were sequenced and further analyzed for promoter/enhancer activity in transient transfection of HeLa cells, in the context of a reporter gene and in the context of the complete virus genome. We found several subtype-specific LTR sequences of the various HIV-1 strains, such as an insertion that created a potential third NF-kappaB site in the LTR of the subtype C strains. The USF-binding site in the NRE also contained subtype-specific sequences. Interestingly, the promoter/enhancer activities of the subtype C LTRs were higher than the activities of the other subtypes analyzed here (subtypes A, B, D, E, and G), suggesting that the potential third NF-kappaB site may confer higher LTR activity or that the subtype C NRE may be less potent. Thus, our data suggest that genetic diversity of the LTR may result in HIV-1 subtypes with different replicative properties.

Activation of HIV-1 enhancer sequence by vaccinia virus.

To investigate whether vaccinia virus (VV) can augment gene expression of human immunodeficiency virus type 1 (HIV-1), co-transfection experiments were carried out in which recombinant plasmids containing various portions of the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into cultured cells. A high level of enhancement in CAT activity directed by the HIV-1 LTRs containing the enhancer sequence was observed in cells infected with VV, as in the cells infected with type 1 herpes simplex virus (HSV-1). The sequence responsible for this augmentation of CAT activity was different from that recognized by HIV-1 Tat. These data clearly demonstrated that VV transactivates HIV-1 LTR through a mechanism distinct from that of activation by HIV-1 Tat.

Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells.

The distal enhancer region of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) is known to be essential for HIV replication and to contain immediately adjacent E-box and Ets binding sites. Based on a yeast one-hybrid screen we have identified the E-box binding protein USF-1 as a direct interaction partner of Ets-1 and found that the complex acts on this enhancer element. The binding surfaces of USF-1 and Ets-1 map to their DNA-binding domains and although these domains are highly conserved, the interaction is very selective within the respective protein family. USF-1 and Ets-1 synergize in specific DNA binding as well as in the transactivation of reporter constructs containing the enhancer element, and mutations of the individual binding sites dramatically reduce reporter activity in T cells. In addition, a dominant negative Ets-1 mutant inhibits both USF-1-mediated transactivation and the activity of the HIV-1 LTR in T cells. The inhibition is independent of Ets DNA-binding sites but requires the Ets binding surface on USF-1, highlighting the importance of the direct protein-protein interaction. Together these results indicate that the interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells.

NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro.

NF-kappa B is a potent inducible transcription factor that regulates many genes in activated T cells. In this report we examined the ability of different subunits of NF-kappa B to enhance HIV-1 transcription in vitro with chromatin templates. We find that the p65 subunit of NF-kappa B is a strong transcriptional activator of nucleosome-assembled HIV-1 DNA, whereas p50 does not activate transcription, and that p65 activates transcription synergistically with Sp1 and distal HIV-1 enhancer-binding factors (LEF-1, Ets-1, and TFE-3). These effects were observed with chromatin, but not with nonchromatin templates. Furthermore, binding of either p50 or p65 with Sp1 induces rearrangement of the chromatin to a structure that resembles the one reported previously for integrated HIV-1 proviral DNA in vivo. These results suggest that p50 and Sp1 contribute to the establishment of the nucleosomal arrangement of the uninduced provirus in resting T cells, and that p65 activates transcription by recruitment of the RNA polymerase II transcriptional machinery to the chromatin-repressed basal promoter.

HIV genome transcription induced by polyoma virus middle T antigen through both enhancer- and promoter-dependent LTR activation.

In order to understand the regulation of HIV genome transcription induced by cell stimulation through transmembrane receptors, we have transfected cells with polyoma middle T antigen (PyMT) expression vectors, thus mimicking activated receptor-dependent cell stimulation. PyMT-expressing Cos7 cells provided an environment where transcription of an HIV provirus was activated. PyMT expression induced the activity of both enhancer- and promoter-dependent HIV-LTR luciferase vectors. Induction of the HIV promoter domain depended on Sp1-binding sites and could be blocked by Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K). This indicates that PyMT-induced HIV transcription and replication are controlled by both the enhancer and promoter domains of the HIV-LTR. The latter, but not the former, was induced in a PI3K-dependent way. Thus at least 2 different transduction pathways appear to collaborate for induction of full HIV genome transcription in activated cells.

Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro.

Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. Individually or collectively, none of the three enhancer-binding proteins (LEF-1, Ets-1, and TFE-3) could activate transcription in the absence of Sp1. A truncation mutant of LEF-1 (HMG-88), which contains the HMG box but lacks the trans-activation domain, did not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. We conclude that transcription activation by LEF-1 in vitro is a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain.

Multiple enhancer motifs in HIV type 1 strains from Ethiopia.

PIP: The authors determined the sequences of the LTR region of Ethiopian HIV-1 subtype C strains and compared them with Swedish HIV-1 subtype B strains and earlier published data. Peripheral blood mononuclear cells (PBMCs) were obtained from seven randomly chosen HIV-1 infected Ethiopian patients, all with pre-AIDS or AIDS. PBMCs were also obtained from three Swedish HIV-1 subtype B-infected patients. Extraction of HIV-1 DNA was performed with the phenol-chloroform plus ethanol precipitation method. In all the Ethiopian HIV-1 subtype C strains, the first five nucleotides were changed to (G/A)CAGA, a finding not observed in the Swedish subtype B strains sequenced at the same time. The most remarkable feature of the Ethiopian NF-KB region was the presence of what appears to be an extra site located upstream of the usual sites I and II. At the same time, the core enhancer sequence GGGACTTTCC at site I was modified by a deletion of the A nucleotide and a change of the first T to a G. The gross LTR organization may be radically different in "African" subtype HIV-1 isolates compared to the American/European HIV-1 subtype B prototype strains. These data from the Ethiopian strains reinforce the validity of this conclusion.^ieng

Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid.

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.

[Study on the specific binding site of NF-k B-like factor at the 5' upstream region of human lymphotoxin gene].

Lymphotoxin(LT) is a glycoprotein secreted by activated T lymphocytes. The expression of LT gene is primarily controlled at the level of transcription. By using human LT DNA as a probe, we have carried out a RNA dot blotting test and found that the longer the Jurkat cells exposed to the PMA and PHA, the more was the endogenous LT mRNA. Results of gel retardation assay showed that nuclear extract from Jurkat cells treated with PMA and PHA formed different complexes. Changes in the complex patterns were observed in different time of PMA and PHA induction. A specific protein-binding site was mapped to a 22bp sequence at the 5' upstream region of human LT gene by DNase I footprinting analysis. This region is similar to the sequence recognized by the proteins of NF-KB family. The results of fragment competition test and homology analysis indicated that the 22-bp sequence contains a KB-like motif only, which located at base pairs -100 to -90(5' -GGGGGCTTCCC-3'). Thus the NF-KB-like factors were involved in the protein-DNA interaction. Furthermore, there were more than one retarded bands appearing in the gel retardation assay. It suggested that there may be various kinds of NF-KB-like factors involved in the regulation of LT gene transcription at the same site.