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

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1654-1664 ◽  
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.

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

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1654-1664
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

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.

scholarly journals The Human Immunodeficiency Virus Type 1 Tat Protein Up-Regulates the Promoter Activity of the Beta-Chemokine Monocyte Chemoattractant Protein 1 in the Human Astrocytoma Cell Line U-87 MG: Role of SP-1, AP-1, and NF-κB Consensus Sites

2000 ◽  
Vol 74 (4) ◽  
pp. 1632-1640 ◽  
Author(s):  
Siew Pheng Lim ◽  
Alfredo Garzino-Demo
Keyword(s):  
Human Immunodeficiency Virus ◽  
Promoter Activity ◽  
Tat Protein ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein ◽  
Astrocytoma Cell ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT It has been shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can specifically enhance expression and release of monocyte chemoattractant protein 1 (MCP-1) from human astrocytes. In this study, we show evidence that Tat-induced MCP-1 expression is mediated at the transcriptional level. Transient transfection of an expression construct encoding the full-length Tat into the human glioblastoma-astrocytoma cell line U-87 MG enhances reporter gene activity from cotransfected deletion constructs of the MCP-1 promoter. HIV-1 Tat exerts its effect through a minimal construct containing 213 nucleotides upstream of the translational start site. Site-directed mutagenesis studies indicate that an SP1 site (located between nucleotides −123 and −115) is critical for both constitutive and Tat-enhanced expression of the human MCP-1 promoter, as mutation of this SP1 site significantly diminished reporter gene expression in both instances. Gel retardation experiments further demonstrate that Tat strongly enhances the binding of SP1 protein to its DNA element on the MCP-1 promoter. Moreover, we also observe an increase in the binding activities of transcriptional factors AP1 and NF-κB to the MCP-1 promoter following Tat treatment. Mutagenesis studies show that an upstream AP1 site and an adjacent NF-κB site (located at −128 to −122 and −150 to −137, respectively) play a role in Tat-mediated transactivation. In contrast, a further upstream AP1 site (−156 to −150) does not appear to be crucial for promoter activity. We postulate that a Tat-mediated increase in SP1 binding activities augments the binding of AP1 and NF-κB, leading to synergistic activation of the MCP-1 promoter.

scholarly journals Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

2001 ◽  
Vol 75 (17) ◽  
pp. 7944-7955 ◽  
Author(s):  
Noriko Nakajima ◽  
Richard Lu ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Dna Recombination ◽  
Cell Types ◽  
Class Ii ◽  
Class I ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

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

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.

scholarly journals Tat Protein of Human Immunodeficiency Virus Type 1 Subtype C Strains Is a Defective Chemokine

2004 ◽  
Vol 78 (5) ◽  
pp. 2586-2590 ◽  
Author(s):  
Udaykumar Ranga ◽  
Raj Shankarappa ◽  
Nagadenahalli B. Siddappa ◽  
Lakshmi Ramakrishna ◽  
Ramalingam Nagendran ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
The United States ◽  
Tat Protein ◽  
Subtype C ◽  
Monocyte Migration ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.

scholarly journals Human Immunodeficiency Virus Env-Independent Infection of Human CD4− Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 10994-11000 ◽  
Author(s):  
Shen Pang ◽  
Duan Yu ◽  
Dong Sung An ◽  
Gayle C. Baldwin ◽  
Yiming Xie ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Alternative Pathway ◽  
Neutralizing Antibody ◽  
Green Fluorescent Protein Gene ◽  
Immunodeficiency Virus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT CD4− epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4− human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4− cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.

scholarly journals A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4737-4745 ◽  
Author(s):  
G Furlini ◽  
M Vignoli ◽  
E Ramazzotti ◽  
MC Re ◽  
G Visani ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Human Herpesvirus ◽  
Human Herpesvirus 6 ◽  
Hematopoietic Progenitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

In human immunodeficiency virus type-1 (HIV-1) infected individuals, CD34+ hematopoietic stem/progenitor cells are profoundly impaired in their proliferation/differentiation capacities. The bulk of the available experimental evidence seems to indicate that hematopoietic progenitors are not susceptible to HIV-1 infection and their defects seem rather the consequence of direct or indirect negative influences of HIV-1-specific soluble proteins released by productively infected accessory cells. We have now shown that in the presence of a concurrent human herpesvirus-6 infection, two hematopoietic (TF-1 [erythromyeloid] and KG-1 [lymphomyeloid]) progenitor cell lines and human CD34+ hematopoietic progenitors isolated from the bone marrow of normal donors, became susceptible to HIV-1 infection and permissive to HIV-1 replication, although with a limited virus yield. These results suggest a further possible mechanism leading to hematopoietic derangement in HIV-1-infected subjects and may help to clarify the controversial issue of the susceptibility of human hematopoietic progenitors to HIV-1 infection.

scholarly journals The Human Immunodeficiency Virus Type 1 Tat Protein Enhances Cryptosporidium parvum-Induced Apoptosis in Cholangiocytes via a Fas Ligand-Dependent Mechanism

2006 ◽  
Vol 75 (2) ◽  
pp. 684-696 ◽  
Author(s):  
Steven P. O'Hara ◽  
Aaron J. Small ◽  
Jeremy B. Nelson ◽  
Andrew D. Badley ◽  
Xian-Ming Chen ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Cryptosporidium Parvum ◽  
Fas Ligand ◽  
Tat Protein ◽  
Immunodeficiency Virus ◽  
Induced Apoptosis ◽  
Hiv 1 ◽  
Dependent Mechanism

ABSTRACT While Cryptosporidium parvum infection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediated C. parvum cytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation on C. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found that C. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P < 0.05) increased C. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced both C. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blocked C. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhances C. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, and C. parvum infection synergistically increase cholangiocyte apoptosis and thus jointly contribute to AIDS-related cholangiopathies.

scholarly journals A functional genetic approach suggests a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Tat protein and HIV-1 TAR RNA in vivo

2003 ◽  
Vol 84 (3) ◽  
pp. 603-606 ◽  
Author(s):  
Lars H. Lund ◽  
Britta Wahren ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Tat Protein ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Tar Rna ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) Tat and human Cyclin T1 form a complex and together recognize the viral TAR RNA element with specificity. Using HIV-1/equine infectious anaemia virus TAR chimeras, we show that in addition to the well-characterized interaction with the bulge, Tat recognizes the distal stem and the loop of TAR. These data support previously proposed, but unproven, molecular models.

scholarly journals gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue

2009 ◽  
Vol 83 (17) ◽  
pp. 8596-8603 ◽  
Author(s):  
Earl Stoddard ◽  
Houping Ni ◽  
Georgetta Cannon ◽  
Chunhui Zhou ◽  
Neville Kallenbach ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Genital Tract ◽  
Heterosexual Transmission ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.

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