Terminal Repeat-driven Gene Expression Research Articles

In Vitro Antiviral Activity of Mentha cordifolia Plant Extract in HIV-1 Latently Infected Cells Using an Established Human Cell Line.

Emergence of drug resistance demands new therapeutic strategies against the human immunodeficiency virus (HIV). Currently, there is an increasing research focus on targeting gene expression-the crucial step wherein new viruses and new viral strains are amplified. Moreover, natural products are also being considered as potential candidates for new antivirals. We screened the extract obtained from a Philippine medicinal plant, Mentha cordifolia (Mc). In this study, we demonstrated that Mc ammonium sulfate extract has antiretroviral activity against HIV. HIV-1 latently infected cells (OM10.1) were pretreated with Mc extract and activated with TNFα. In treated cells, viral replication was inhibited in both cell culture supernatant and whole cell lysates. The level of viral production, as measured by the viral p24 protein concentration, was very much inhibited under noncytotoxic concentrations to the similar level without addition of TNFα. Luciferase assays, however, showed that Mc does not inhibit the HIV-1 long terminal repeat-driven gene expression. IκBα degradation and p65 nuclear translocation was also not affected as visualized through Western blot and immunofluorescence. These observations demonstrated that Mc possessed an antiviral component against HIV-1 and warrant further work to explore its target of action at a later step of gene expression. Our study introduces a potential source of a lead compound that targets steps in the HIV life cycle.

Identification of novel inhibitors of human immunodeficiency virus type 1 replication by in silico screening targeting cyclin T1/Tat interaction.

Human immunodeficiency virus type 1 (HIV-1) transcription is essential for viral replication and the only step for viral genome amplification. Cyclin T1 (CycT1) interacts with HIV-1 Tat and transactivation-responsive (TAR) RNA, leading to the activation of viral transcription through the hyperphosphorylation of RNA polymerase II (RNAPII). Thus, the CycT1/Tat/TAR RNA interaction represents a novel target for inhibition of HIV-1 replication. In this study, we conducted in silico screening of compounds targeting the CycT1/Tat/TAR RNA complex and found that two structurally related compounds (C1 and C2) had high docking scores for a model of the complex. These compounds proved inhibitory to HIV-1 replication in tumor necrosis factor alpha-stimulated chronically infected cells. In addition, C3, a derivative of C1 and C2, was found to be a more potent inhibitor of HIV-1 replication in chronically infected cells. C3 also inhibited HIV-1 replication in acutely infected cells. The compound could suppress Tat-mediated HIV-1 long terminal repeat-driven gene expression and phosphorylation of RNAPII through inhibition of Tat binding to CycT1. Furthermore, the docking pose of C3 was defined by analyses for its in silico docking energy and in vitro antiviral activity, which indicates that C3 interacts with Tat-binding amino acids of CycT1. Thus, a series of compounds described herein are novel inhibitors of HIV-1 transcription through inhibition of CycT1/Tat interaction.

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising cyclin-dependent kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.

Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal repeat activation.

HIV-1 gene regulation is greatly dependent on the presence of the -104/-81 enhancer region which is regulated by both NF-kappaB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters from the IL-2 and TNF-alpha genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient cell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-sensitive. Mechanistic studies revealed that CD3zeta and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulation in turn affects HIV-1 long terminal repeat activation.

Attachment of Human Immunodeficiency Virus-1 (HIV-1) Particles Bearing Host-encoded B7-2 Proteins Leads to Nuclear Factor-κB- and Nuclear Factor of Activated T Cells-dependent Activation of HIV-1 Long Terminal Repeat Transcription

Previous studies have shown that human immunodeficiency virus type-1 (HIV-1) can incorporate several surface proteins of host origin. Recent findings indicate that host-encoded cell surface constituents retain their functionality when found embedded into the viral envelope. The primary objective of the current study was to define whether interaction between some specific virion-bound host proteins with their natural cognate ligands present on target cells could mediate intracellular signaling cascade(s). For this purpose, we have generated a whole series of isogenic virus stocks (NL4-3 backbone) bearing or not bearing on their surface foreign CD28, CD54 (ICAM-1), CD80 (B7-1) or CD86 (B7-2) proteins. Our results indicate that incubation of human T lymphoid cells with virions bearing host-derived B7-2 proteins and anti-CD3 antibody can potently activate HIV-1 long terminal repeat-driven gene expression. This up-regulating effect necessitates the involvement of nuclear factor-kappa B (NF-kappa B) and nuclear factor of activated T cells (NFAT) as revealed by the use of vectors coding for dominant negative versions of both transcription factors (i.e. I kappa B alpha S32A/36A and dnNFAT) and band shift assays. The increase of NF-kappa B activity was abolished when infection with B7-2-bearing HIV-1 particles was performed in the presence of the fusion protein CTLA-4 Ig suggesting that the interaction between virally embedded B7-2 and CD28 on the target cell is responsible for the observed NF-kappa B induction. The findings presented here provide the first demonstration that host-encoded proteins acquired by HIV-1 can mediate signal transduction events.

Acridone derivatives are selective inhibitors of HIV-1 replication in chronically infected cells

In our extensive screening of anti-HIV-1 agents in chronically infected cell lines, we have found acridone derivatives to be selective inhibitors of HIV-1 replication. Among the acridone derivatives, 1-hydroxy-10-methyl-9,10-dihydroacrid-9-one (RD6-5071) suppressed tumor necrosis factor (TNF)-α-induced HIV-1 expression in the latently infected cell line OM-10.1, U1, and ACH-2. Its 50% effective concentration for HIV-1 p24 antigen production was 2.0 μg/ml in OM-10.1 cells, while its 50% cytotoxic concentration was 18 μg/ml. The compound also inhibited phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 expression in these cell lines. Furthermore, RD6-5071 was inhibitory to HIV-1 replication in acutely infected U937 and peripheral blood mononuclear cells. The compound was found to suppress TNF-α-induced HIV-1 long terminal repeat-driven gene expression. An inhibition assay for protein kinase C (PKC) revealed that RD6-5071 could reduce the enzyme activity. Furthermore, the compound was a moderate inhibitor of PMA-induced nuclear factor κB (NF-κB) activation, as determined by a gel mobility shift analysis. These results suggest that the acridone derivatives suppress HIV-1 replication at the transcriptional level primarily through a mechanism of PKC inhibition.

EM2487, a novel anti-HIV-1 antibiotic, produced by Streptomyces sp. Mer-2487: taxonomy, fermentation, biological properties, isolation and structure elucidation.

For the purpose of discovering novel agents that inhibit HIV-1 replication at the transcriptional level, we have established cell lines reflecting the HIV-1 long terminal repeat-driven gene expression. Using these cell lines, we have screened approximately 10,000 microorganism products and found that the culture supernatant of Streptomyces sp. Mer-2487 suppresses the HIV-1 Tat-induced gene expression without affecting the basal or tumor necrosis factor-alpha-induced transcription. The purified active component has a unique structure, as shown in Fig. 1. This compound has an inhibitory effect on HIV-1 replication in chronically infected cells as well as acutely infected cells, suggesting that the inhibition occurs at a postintegration step of HIV-1 proviral DNA in the HIV-1 replication cycle.

The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression.

In addition to their role in reverse transcription, the R-region sequences of some retroviruses affect viral transcription. The first 28 nucleotides of the R region within the long terminal repeat (LTR) of the murine type C retrovirus SL3 were predicted to form a stem-loop structure. We tested whether this structure affected the transcriptional activity of the viral LTR. Mutations that altered either side of the stem and thus disrupted base pairing were generated. These decreased the level of expression of a reporter gene under the control of viral LTR sequences about 5-fold in transient expression assays and 10-fold in cells stably transformed with the LTR-reporter plasmids. We also generated a compensatory mutant in which both the ascending and descending sides of the stem were mutated such that the nucleotide sequence was different but the predicted secondary structure was maintained. Most of the activity of the wild-type SL3 element was restored in this mutant. Thus, the stem-loop structure was important for the maximum activity of the SL3 LTR. Primer extension analysis indicated that the stem-loop structure affected the levels of cytoplasmic RNA. Nuclear run-on assays indicated that deletion of the R region had a small effect on transcriptional initiation and no effect on RNA polymerase processivity. Thus, the main effect of the R-region element was on one or more steps that occurred after the template was transcribed by RNA polymerase. This finding implied that the main function of the R-region element involved RNA processing. R-region sequences of human immunodeficiency virus type 1 or mouse mammary tumor virus could not replace the SL3 element. R-region sequences from an avian reticuloendotheliosis virus partially substituted for the SL3 sequences. R-region sequences from Moloney murine leukemia virus or feline leukemia virus did function in place of the SL3 element. Thus, the R region element appears to be a general feature of the mammalian type C genus of retroviruses.

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

C-C chemokine RANTES and HIV long terminal repeat-driven gene expression.

The C-C chemokines RANTES, MIP-1alpha, and MIP-1beta have been characterized as constituents of an HIV- and SIV-suppressive factor released by CD8+ cells. Furthermore, it has been demonstrated that chemokine receptors cooperate in HIV entry. However, these proteins are also known to have an effect on multiple intracellular signaling cascades that may affect the process of transcription. In the present study we demonstrate that treatment of CD4+ T cells with these chemokines or with cell supernatants from HTLV-I-immortalized CD8+ T cells results in significant reduction in the abundance of HIV-1-specific RNA as analyzed by Northern blot hybridization. To examine the possibility that such suppressive factors may inhibit HIV RNA transcription, we studied the effect of RANTES, the most effective HIV-suppressive chemokine, on basal and Tat-induced HIV-directed LTR expression of a reporter gene. Neither recombinant RANTES nor conditioned medium from CD8+ cells significantly altered HIV-1 LTR-directed chloramphenicol acetyltransferase expression in either transiently or stably transfected CD4+ T cell lines, either in the presence or in the absence of Tat. These results suggest that C-C chemokines do not inhibit viral RNA transcription.

CD8+ T-cell-mediated suppression of HIV-1 long terminal repeat-driven gene expression is not modulated by the CC chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta.

To assess the role of RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta in modulation of HIV-1 long terminal repeat (LTR)-mediated gene expression and determine whether these chemokines share identity with CD8+ T-lymphocyte-derived HIV-1 LTR-suppressive factors. HIV-1 LTR-directed reporter gene expression is a model for transcription that is susceptible to inhibition by factors produced by CD8+ lymphocytes of HIV-1-infected individuals. The effect of recombinant chemokines on LTR-directed gene expression was examined. The ability of chemokines found to be present in CD8 supernatants to suppress HIV-1 LTR-mediated gene expression was determined by antibody inhibition assays. The concentrations of RANTES, MIP-1 alpha and MIP-1 beta in a panel of CD8+ T-lymphocyte-derived supernatants were determined by enzyme-linked immunosorbent assay. Recombinant chemokines were added to freshly transfected (pLTR-CAT and pSV40-tat) human Jurkat T cells. Excessive polyclonal neutralizing antibodies to these chemokines were added to transfected Jurkat T cells cultured in the presence of strongly inhibitory CD8+ T-cell-derived supernatants with known chemokine concentrations. The concentrations of RANTES, MIP-1 alpha and MIP-1 beta in a panel of CD8+ lymphocyte-derived supernatants were found to correlate with their relative ability to suppress the LTR-mediated gene expression (r = 0.679, 0.764 and 0.48, respectively). The addition of recombinant CC chemokines had no effect over a broad range of doses on HIV-1 LTR-mediated gene expression. The CD8-suppressive effect on HIV-1 LTR-driven gene expression was not abrogated by a combination of antibodies of RANTES, MIP-1 alpha and MIP-1 beta. RANTES, MIP-1 alpha and MIP-1 beta do not alter HIV-1 LTR-directed gene expression at doses up to 100 ng/ml. Although present in varying concentrations in supernatants derived from CD8+ lymphocytes from HIV-positive individuals, these chemokines are not responsible for the powerful CD8-derived suppressive effect on HIV-1 LTR-mediated gene expression observed in our system.

CD8+ T cell-mediated suppression of HIV long terminal repeat-driven gene expression is not associated with improved clinical status.

To determine the associations between the suppression of HIV-1 long terminal repeat (LTR)-mediated gene expression by CD8+ T-cell supernatants and clinical correlates of well-being, including CD4+ and CD8+ T-cell counts, beta-chemokine production and clinical stage of disease. Culture supernatants of activated CD8+ T cells derived from a panel of HIV-1-infected subjects were assessed for their ability to suppress HIV-1 LTR-mediated chloramphenicol acetyl transferase (CAT) expression. The percentage suppression of gene expression was correlated with CD4+ and CD8+ T-cell counts and clinical stage of infection. Some individuals within this group were followed at 2-3 month intervals over time to assess the consistency of the suppression. Selected CD8+ T-cell culture supernatants of diverse suppressive ability were screened for the levels of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta and RANTES. The ability of CD8+ T cells of HIV-1 infected subjects to suppress HIV-1 LTR-mediated gene expression did not show a dependence upon high CD4+ T-cell counts or on the clinical stage or duration of infection. The ability to suppress gene expression did show a relationship with higher CD8+ T-cell counts and correlated with the levels of beta-chemokines in the culture supernatants. In contrast, strong suppression was mediated by CD8+ T-cell supernatants from some subjects with very low CD8+ T-cell counts and relatively low chemokine levels. Although the suppression of gene expression by CD8+ T-cell culture supernatants showed statistical correlation with beta-chemokine levels and with higher CD8+ T-cell count, no correlation could be found with correlates of clinical well-being.

Repression of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by binding of the virus to its primary cellular receptor, the CD4 molecule

We have previously postulated that the binding of the human immunodeficiency virus type 1 (HIV-1) to cell surface CD4 induces signal transduction pathways that down-modulate production of progeny virions in acutely infected T cells (M. Tremblay, S. Meloche, S. Gratton, M. A. Wainberg, and R.-P. Sékaly, EMBO J. 13:774-783, 1994). To evaluate the possibility that CD4 cross-linking might indeed affect viral gene expression, we have introduced a molecular construct made of the luciferase reporter gene placed under the control of the regulatory elements of HIV-1 in several CD4-positive T-cell lines. We found that cross-linking of CD4 with defective HIV-1 particles and heat-inactivated viruses inhibits long terminal repeat-dependent luciferase expression. Experiments revealed that the gp120-CD4 interaction was necessary to repress HIV-1 long terminal repeat-dependent luciferase activity. The cytoplasmic domain of CD4 was also found to be required for this effect to occur. The virus-mediated signal transduction was shown to be mediated via p56lck-dependent and -independent pathways. These results indicate that the earliest event in the HIV-1 replicative cycle, namely, the binding of the virus to its cellular receptor, can lead to signal transduction culminating in down-modulation of viral gene expression. Thus we propose that defective viruses could regulate the pathogenesis of HIV disease as they constitute the vast majority of circulating HIV-1 particles.

Transcriptional silencing of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by the Krüppel-associated box repressor domain targeted to the transactivating response element.

The evolutionarily conserved protein domain, called the Krüppel-associated box (KRAB), present in the amino termini of a large number of Krüppel-type zinc finger proteins is a strong repressor domain. In order to develop novel strategies to control human immunodeficiency virus type 1 (HIV-1) gene expression, we constructed a series of expression vectors expressing the wild-type Tat or Tat transdominant negative mutants fused to the KRAB repressor domain. We found that the KRAB domain tethered to the transactivating response element is able to suppress both basal and Tat-mediated activity of HIV-1 long terminal repeat-driven gene expression. These results suggest that the KRAB repressor domain fused to the Tat transdominant negative mutants can be successfully employed to control HIV-1 gene expression.