[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) disease in HIV co-infected patients contributes to increased mortality by activating innate and adaptive immune signaling cascades that stimulate HIV-1 replication, leading to an increase in viral load. Here, we demonstrate that silencing of the expression of the transcription factor nuclear factor of activated T cells 5 (NFAT5) by RNA interference (RNAi) inhibits Mycobacterium tuberculosis (MTb)-stimulated HIV-1 replication in co-infected macrophages. We show that NFAT5 gene and protein expression are strongly induced by MTb, which is a Toll-like receptor (TLR) ligand, and that an intact NFAT5 binding site in the viral promoter of R5-tropic HIV-1 subtype B and subtype C molecular clones is required for efficent induction of HIV-1 replication by MTb. Furthermore, silencing by RNAi of key components of the TLR pathway in human monocytes, including the downstream signaling molecules MyD88, IRAK1, and TRAF6, significantly inhibits MTb-induced NFAT5 gene expression. Thus, the innate immune response to MTb infection induces NFAT5 gene and protein expression, and NFAT5 plays a crucial role in MTb regulation of HIV-1 replication via a direct interaction with the viral promoter. These findings also demonstrate a general role for NFAT5 in TLR- and MTb-mediated control of gene expression.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT:
Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.
Here, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.
Use of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.
BMC Research Notes 09/2011; 4(1):340. DOI:10.1186/1756-0500-4-340
[Show abstract][Hide abstract] ABSTRACT: In this review, we examine how a subset of signal transduction cascades initiated by Mycobacterium tuberculosis (Mtb) infection modulates transcription mediated by the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). We describe two distinct phases of signaling that target transcription factors known to bind the HIV-1 LTR, and thus drive viral transcription and replication, in cells of the Mtb-infected host. First, Mtb-derived molecules, including cell wall components and DNA, interact with a number of host pattern recognition receptors. Second, cytokines and chemokines secreted in response to Mtb infection initiate signal transduction cascades through their cognate receptors. Given the variation in cell wall components among distinct clinical Mtb strains, the initial pattern recognition receptor interaction leading to direct LTR activation and differential cytokine and chemokine production is likely to be an important aspect of Mtb strain-specific regulation of HIV-1 transcription and replication. Improved understanding of these molecular mechanisms in the context of bacterial and host genetics should provide key insights into the accelerated viral replication and disease progression characteristic of HIV/TB coinfection.
American Journal of Respiratory Cell and Molecular Biology 08/2011; 45(6):1116-24. DOI:10.1165/rcmb.2011-0186TR · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Tuberculosis (TB) is the largest cause of death in human immunodeficiency virus type 1 (HIV-1) infection, having claimed an estimated one third to one half of the 30 million AIDS deaths that have occurred worldwide. Different strains of Mycobacterium tuberculosis (MTb), the causative agent of TB, are known to modify the host immune response in a strain-specific manner. However, a MTb strain-specific impact upon the regulation of HIV-1 replication has not previously been established.
We isolated normal human peripheral blood mononuclear cells (PBMC) and co-infected them with HIV-1 and with either the well characterized CDC1551 or HN878 MTb clinical isolate. We show that HIV-1 co-infection with the CDC1551 MTb strain results in higher levels of virus replication relative to co-infection with the HN878 MTb strain ex vivo. Furthermore, we show that the distinct pattern of CDC1551 or HN878 induced HIV-1 replication is associated with significantly increased levels of TNF and IL-6, and of the transcription and nuclear translocation of the p65 subunit of the transcription factor NF-κB, by CDC1551 relative to HN878.
These results provide a precedent for TB strain-specific effects upon HIV-1 replication and thus for TB strain-specific pathogenesis in the outcome of HIV-1/TB co-infection. MTb strain-specific factors and mechanisms involved in the regulation of HIV-1 during co-infection will be of importance in understanding the basic pathogenesis of HIV-1/TB co-infection.
PLoS ONE 02/2009; 4(7):e6116. DOI:10.1371/journal.pone.0006116 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor-viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5-LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS.
[Show abstract][Hide abstract] ABSTRACT: The transactivator of transcription (Tat) protein is essential for efficient HIV type 1 (HIV-1) replication and is involved in the transcriptional regulation of the host immune response gene, TNF. In this study, we demonstrate that Tat proteins from representative HIV-1 subtype E isolates, but not from subtypes B or C, selectively inhibit TNF gene transcription and protein production in CD4(+) Jurkat T cells. Strikingly, we show that this repression is due to a tryptophan at residue 32 of Tat E and is secondary to interference with recruitment of the histone acetyltransferase P/CAF to the TNF promoter and with chromatin remodeling of the TNF locus. This study presents a novel mechanism by which HIV-1 manipulates a host immune response gene that is important in its own replication. Moreover, these results demonstrate a new mechanism by which the TNF gene is regulated via chromatin remodeling secondary to viral infection.
The Journal of Immunology 05/2006; 176(7):4182-90. DOI:10.4049/jimmunol.176.7.4182 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis (MTb) is the leading cause of death in the setting of AIDS. MTb enhances the pathogenicity and accelerates the course of HIV disease and, furthermore, infection with HIV-1 increases the risk of reactivation or reinfection with MTb. In this study, we show that host-specific recall responses to one pathogen, MTb, has a direct effect upon the regulation of a second pathogen, HIV-1. Using cells from immunocompetent former tuberculosis (TB) patients who displayed either a persistently positive (responsive) or negative (anergic), delayed-type hypersensitivity (DTH) reaction to intradermal injection of purified protein derivative (PPD), we investigated the effect of recall Ags to MTb upon the replication of HIV-1 primary isolates in vitro. We show that HIV-1 replication of a T cell-tropic isolate was significantly impaired in MTb-stimulated PBMC from PPD-anergic donors. Furthermore, these donors displayed a significant increase in CD8(+) T cells and IL-10 levels and lower levels of IL-2 and TNF-alpha relative to PPD-responsive donors in response to PPD stimulation. Strikingly, CD8(+) T cell depletion and blocking of IL-10 significantly increased HIV-1 replication in these PPD-anergic donors, indicating that an immunosuppressive response to MTb recall Ags inhibits HIV-1 replication in PPD-anergic individuals. Therefore, immunotherapeutic approaches aimed at recapitulating Ag-specific MTb anergy in vivo could result in novel and effective approaches to inhibit HIV-1 disease progression in MTb/HIV-1 coinfection.
The Journal of Immunology 03/2004; 172(3):1953-9. DOI:10.4049/jimmunol.172.3.1953 · 4.92 Impact Factor