HIV-1 Tat-induced bystander apoptosis in Jurkat cells involves unfolded protein responses

ABSTRACTThe HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three isoforms of TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also controls HIV-1 replication by repressing TNPO3. TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Manipulation of miR-128 levels in HIV target cell lines and in primary human CD4 T-cells by over-expression or knockdown showed that modulation of TNPO3 by miR-128 affects HIV-1 replication but not MLV infection. In addition, we found that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus and in cells depleted of CPSF6, suggesting that miR-128-indued TNPO3 repression is partly required for miR-128-induced inhibition of HIV-1 replication. Finally, challenging miR-modulated Jurkat cells or primary CD4 T-cells with wildtype, replication-competent HIV-1 shows that miR-128 significantly delays spreading infection. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication partly by targeting TNPO3.

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A stronger transcription regulatory circuit of HIV-1C drives the rapid establishment of latency with implications for the direct involvement of Tat

10.1101/2020.02.20.958892 ◽

2020 ◽

Author(s):

Sutanuka Chakraborty ◽

Manisha Kabi ◽

Udaykumar Ranga

Keyword(s):

Transcription Factor ◽

Viral Infection ◽

Viral Vectors ◽

Transcription Factor Binding ◽

Jurkat Cells ◽

Subtype C ◽

Viral Promoter ◽

Factor Binding ◽

Transcriptional Silence ◽

Hiv 1

AbstractThe magnitude of transcription factor binding site variation emerging in HIV-1C, especially the addition of NF-κB motifs by sequence duplication, makes the examination of transcriptional silence challenging. How can HIV-1 establish and maintain latency despite having a strong LTR? We constructed panels of sub-genomic reporter viral vectors with varying copy numbers of NF-κB motifs (0 to 4 copies) and examined the profile of latency establishment in Jurkat cells. We found surprisingly that the stronger the viral promoter, the faster the latency establishment. Importantly, at the time of commitment to latency and subsequent points, Tat levels in the cell were not limiting. Using highly sensitive strategies, we demonstrate the presence of Tat in the latent cell, recruited to the latent LTR. Our data allude, for the first time, to Tat establishing a negative feedback loop during the late phases of viral infection, leading to the rapid silencing of the viral promoter.ImportanceOver the past 10-15 years, HIV-1C has been evolving rapidly towards gaining stronger transcriptional activity by sequence duplication of major transcription factor binding sites. The duplication of NF-κB motifs is unique and exclusive for HIV-1C, a property not shared with any of the other eight HIV-1 genetic families. What mechanism(s) does HIV-1C employ to establish and maintain transcriptional silence despite the presence of a strong promoter and a concomitant strong, positive transcriptional feedback is the primary question we attempted to address in the present manuscript. The role Tat plays in latency reversal is well established. Our work with the most common HIV-1 subtype C (HIV-1C) offers crucial leads towards Tat possessing a dual-role in serving both as transcriptional activator and repressor at different phases of the viral infection of the cell. The leads we offer through the present work have significant implications for HIV-1 cure research.

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An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1883-1893.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1883-1893

Author(s):

Y C Li ◽

J Ross ◽

J A Scheppler ◽

B R Franza

Keyword(s):

Human Immunodeficiency Virus ◽

Protein Synthesis ◽

Early Time ◽

In Vitro Transcription ◽

Jurkat Cells ◽

Time Points ◽

Immunodeficiency Virus ◽

Hiv 1

In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.

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Effect of alpha-1 antitrypsin Portland variant (α1-PDX) on HIV-1 replication

Biochemical Journal ◽

10.1042/bj3520091 ◽

2000 ◽

Vol 352 (1) ◽

pp. 91-98 ◽

Cited By ~ 8

Author(s):

Bouchaib BAHBOUHI ◽

Mourad BENDJENNAT ◽

Denise GUÉTARD ◽

Nabil Georges SEIDAH ◽

Elmostafa BAHRAOUI

Keyword(s):

Viral Replication ◽

Potential Role ◽

Serine Proteinase ◽

Recombinant Vaccinia Virus ◽

Jurkat Cells ◽

Serine Proteinase Inhibitor ◽

Cdna Insert ◽

Alpha 1 Antitrypsin ◽

Hiv 1

The present work investigated the potential role of alpha-1 antitrypsin Portland variant (α1-PDX), a bioengineered serine proteinase inhibitor (serpin), in the interference with the viral replication of HIV-1, induction of syncytia and maturation of envelope glycoprotein gp160 to gp120 and gp41. A Jurkat lymphoid cell line transfected with a plasmid containing the α1-PDX cDNA (J-PDX) and expressing the protein in a stable manner was infected with HIV-1Lai. Controls were Jurkat cells transfected with the same vector pcDNA3 without the cDNA insert (J-pcDNA3). The results showed that viral replication of HIV-1 was significantly inhibited with a delay in replication kinetics in J-PDX cells as compared with J-pcDNA3 cells. In addition, a comparison of the infectious capacity of viruses produced in the presence and absence of α1-PDX revealed that this capacity differed. It was found that α1-PDX exerts its effect by interfering with the formation of syncytia between J-PDX cells infected with gp160 recombinant vaccinia virus, or after infection by HIV-1 and co-culture with uninfected Molt-4 cells. In contrast, when the same experiments were performed with J-pcDNA3 cells, a large number of syncytia was obtained. Analysis of viral proteins by Western blotting and densitometry showed that the inhibition of the cytopathic effect of HIV-1 and viral replication was correlated with the capacity of α1-PDX to interfere with the maturation of gp160 to gp120 and gp41.

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Upregulation of c-Fos in Activated T Lymphoid and Monocytic Cells by Human Immunodeficiency Virus-1 Tat Protein

Blood ◽

10.1182/blood.v89.5.1654 ◽

1997 ◽

Vol 89 (5) ◽

pp. 1654-1664 ◽

Cited By ~ 20

Author(s):

Davide Gibellini ◽

Antonella Caputo ◽

Silvano Capitani ◽

Michele La Placa ◽

Giorgio Zauli

Keyword(s):

Human Immunodeficiency Virus ◽

Cell Lines ◽

Promoter Activity ◽

Jurkat Cells ◽

U937 Cells ◽

Tat Protein ◽

Fos Protein ◽

Significant Difference ◽

Immunodeficiency Virus ◽

Hiv 1

Abstract The regulatory Tat protein of the human immunodeficiency virus type-1 (HIV-1) is essential for viral replication and also shows pleiotropic activities on various cell functions. To get further insights into the molecular mechanisms underlying the biological activity of Tat, we investigated the effect of endogenous and exogenous Tat protein on c-fos gene expression in T lymphoblastoid (Jurkat) and monocytic (U937) cell lines, as well as in primary peripheral blood mononuclear cells (PBMC). Transient cotransfection of tat cDNA in sense orientation (tat/S), together with a plasmid containing the c-fos promoter (FC3, from −711 to +42) in front of the bacterial chloramphenicol acetyltransferase (CAT) gene significantly enhanced CAT activity in Jurkat cells activated by the addition of 15% fetal calf serum (FCS) or 5 μg/mL phytohemagglutinin plus 10−7 mol/L phorbol myristate acetate (PMA) and U937 cells activated by 15% FCS or 10−7 mol/L PMA. This effect was specifically due to Tat, since Jurkat and U937 cells cotransfected either with tat cDNA in antisense orientation (tat/AS), tat carrying a mutation in the aminoacid cys22 - gly22 (tat 22/S) or with the backbone vector alone (pRPneo-SL3) did not show any significant difference in c-fos promoter activity as compared to cells transfected with FC3 plasmid alone. By using deletion mutants of the c-fos promoter, we found that the minimal DNA sequence required for Tat activity was located between nucleotides −404/−220 and that the serum responsive element (SRE, −317/−288), present within this region, was still responsive to Tat. A single point mutation in the SRE completely abrogated the responsiveness to tat/S. Exogenous recombinant Tat protein was also able to upregulate c-fos promoter activity in serum-activated Jurkat and U937 cells, as well as endogenous c-fos mRNA expression and c-Fos protein synthesis in both serum-activated cell lines and primary PBMC. c-Fos protein was shown essential for an optimal transactivation of the HIV-1 long terminal repeat (LTR) by Tat: incubation of Jurkat cells with antisense, but not sense, c-fos oligonucleotides significantly reduced either the Tat-enhanced expression of an LTR-CAT reporter construct or the levels of gag p24 in the culture supernatants of Jurkat cells and PBMC acutely infected with HIV-1. Our data suggest that the c-fos upregulation mediated by Tat might play a significant role in the control of viral gene transactivation.

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Plasma Membrane Proteomic Profile Discovers Macrophage-capping Protein Related to Latent HIV-1

Current HIV Research ◽

10.2174/1570162x17666190506155222 ◽

2019 ◽

Vol 17 (1) ◽

pp. 42-52

Author(s):

Yujiao Zhang ◽

Yinzhong Shen ◽

Lin Yin ◽

Tangkai Qi ◽

Xiaofang Jia ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Lines ◽

Western Blotting ◽

Jurkat Cells ◽

Differentially Expressed ◽

Hiv Latency ◽

Capping Protein ◽

Potential Biomarker ◽

Latent Hiv ◽

Hiv 1

Background:Due to the persistence of latent HIV-infected cellular reservoirs, HIV virus can not be eradicated completely.Objective:To identify proteins related to HIV latency, we performed a subcellular proteomic study in HIV latent cell lines.Method:An established HIV-1 latent cell model (J-Lat Tat-GFP Clone A7 cells, A7 cells) and its parental cell line (Jurkat cells) were used. The plasma membrane (PM) fraction from cultured cells was enriched through aqueous two-phase partition. PM proteins were extracted and then separated using two-dimensional electrophoresis (2DE). Differentially expressed proteins were identified by mass spectrometry, and verified by western blotting.Results:Thirteen non-redundant proteins were identified to be differentially expressed in the A7 PM fraction compared to those in the Jurkat PM. Eight had a PM location through Gene Ontology (GO) analysis. A differential protein network of CAPG-ACTR3-CD3D was detected to have interactions with HIV Vpr, Tat, gp160, etc. through STRING software analysis. One of the differential proteins (Macrophage-capping protein (CAPG)) was verified by western blotting to be down- regulated in two cell lines and HIV resting CD4+ T cells negatively selected from patients.Conclusion:We identified 13 proteins in A7 compared to Jurkat cells. CAPG may be a potential biomarker related to HIV latency.

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