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The activation of the human immunodeficiency virus, type 1 (HIV-1) by the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, and mitomycin C was observed in human B lymphocytes transiently transfected with plasmids in which the HIV-1 long terminal repeat (LTR) directed the expression of the bacterial chloramphenicol acetyltransferase gene. Deletion of the two NF-kappa B-binding sites of LTR abolished the HIV-1 activation induced by the three mutagens, while deletion of the three Sp1-binding sites slightly reduced it. Electrophoretic mobility shift assays revealed an increased binding to the kappa B sites of HIV-1 LTR in the nuclear extracts of human B lymphocytes upon mutagen treatment, while binding to Sp1 sites was unaffected. The TAR region was also involved in the mutagen-mediated activation of HIV-1 LTR inasmuch as a small deletion in the TAR sequence (nucleotides +34 to +37) greatly decreased the induction of HIV-1 expression. Moreover, an enhanced binding activity to the TAR DNA sequence (nucleotides +24 to +47) was observed in nuclear extracts of mutagen-treated lymphocytes. Thus, both the enhancer and the 5'-untranslated region of HIV-1 functionally cooperate in the mutagen-mediated induction of HIV-1 expression.

Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.

The 5'-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-kappaB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-kappaB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-alpha. The TAR NF-kappaB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-kappaB enhancer upstream of the TATA box in the NF-kappaB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-kappaB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-kappaB inducers; (ii) deletion or base pair substitutions of the TAR NF-kappaB site significantly reduce the HIV-1 LTR activation by NF-kappaB inducers; (iii) deletions of both the NF-kappaB sites upstream of the TATA box and the TAR NF-kappaB site abolish the activation of HIV-1 LTR in response to NF-kappaB inducers. Moreover, the p50 p65 NF-kappaB complex binds to the TAR NF-kappaB sequence and trans-activates the TAR NF-kappaB-directed expression. The identification of an additional NF-kappaB site in the HIV-1 LTR points to the relevance of NF-kappaB factors in the HIV-1 life cycle.

Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma, A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases, The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5' deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5'-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter, This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation, In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBP beta, which is a major mediator of IL6 promoter function, By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.