The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

To establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection.

The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1-3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity.

Epigenetic control of cytokine gene expression: regulation of the TNF/LT locus and T helper cell differentiation.

Epigenetics encompasses transient and heritable modifications to DNA and nucleosomes in the native chromatin context. For example, enzymatic addition of chemical moieties to the N-terminal "tails" of histones, particularly acetylation and methylation of lysine residues in the histone tails of H3 and H4, plays a key role in regulation of gene transcription. The modified histones, which are physically associated with gene regulatory regions that typically occur within conserved noncoding sequences, play a functional role in active, poised, or repressed gene transcription. The "histone code" defined by these modifications, along with the chromatin-binding acetylases, deacetylases, methylases, demethylases, and other enzymes that direct modifications resulting in specific patterns of histone modification, shows considerable evolutionary conservation from yeast to humans. Direct modifications at the DNA level, such as cytosine methylation at CpG motifs that represses promoter activity, are another highly conserved epigenetic mechanism of gene regulation. Furthermore, epigenetic modifications at the nucleosome or DNA level can also be coupled with higher-order intra- or interchromosomal interactions that influence the location of regulatory elements and that can place them in an environment of specific nucleoprotein complexes associated with transcription. In the mammalian immune system, epigenetic gene regulation is a crucial mechanism for a range of physiological processes, including the innate host immune response to pathogens and T cell differentiation driven by specific patterns of cytokine gene expression. Here, we will review current findings regarding epigenetic regulation of cytokine genes important in innate and/or adaptive immune responses, with a special focus upon the tumor necrosis factor/lymphotoxin locus and cytokine-driven CD4+ T cell differentiation into the Th1, Th2, and Th17 lineages.

The transcription factor NFATp plays a key role in susceptibility to TB in mice.

In T cells, the transcription factor nuclear factor of activated T cells p (NFATp) is a key regulator of the cytokine genes tumor necrosis factor (TNF) and interferon-γ (IFN-γ). Here, we show that NFATp-deficient (NFATp(-/-)) mice have a dramatic and highly significant increase in mortality after Mycobacterium tuberculosis (MTb) infection as compared to mortality of control animals after MTb infection. Animals deficient in NFATp have significantly impaired levels of TNF and IFN-γ transcription and protein expression in naïve or total CD4(+) T cells, but display wild-type levels of TNF mRNA or protein from MTb-stimulated dendritic cells (DC). The rapid mortality and disease severity observed in MTb-infected NFATp(-/-) mice is associated with dysregulated production of TNF and IFN-γ in the lungs, as well as with increased levels of TNF, in their serum. Furthermore, global blocking of TNF production by injection of a TNF neutralizaing agent at 6 weeks, but not 12 weeks, post-MTb-infection further decreased the survival rate of both wild-type and NFATp(-/-) mice, indicating an early role for TNF derived from cells from the monocyte lineage in containment of infection. These results thus demonstrate that NFATp plays a critical role in immune containment of TB disease in vivo, through the NFATp-dependent expression of TNF and IFN-γ in T cells.

Arc of a vicious circle: pathways activated by Mycobacterium tuberculosis that target the HIV-1 long terminal repeat.

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.

Transcriptional control of the TNF gene.

The cytokine TNF is a critical mediator of immune and inflammatory responses. The TNF gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving the recruitment of distinct sets of transcription factors to a compact and modular promoter region. In this review, we describe our current understanding of the mechanisms through which TNF transcription is specifically activated by a variety of extracellular stimuli in multiple cell types, including T cells, B cells, macrophages, mast cells, dendritic cells, and fibroblasts. We discuss the role of nuclear factor of activated T cells and other transcription factors and coactivators in enhanceosome formation, as well as the contradictory evidence for a role for nuclear factor kappaB as a classical activator of the TNF gene. We describe the impact of evolutionarily conserved cis-regulatory DNA motifs in the TNF locus upon TNF gene transcription, in contrast to the neutral effect of single nucleotide polymorphisms. We also assess the regulatory role of chromatin organization, epigenetic modifications, and long-range chromosomal interactions at the TNF locus.

A dimer-specific function of the transcription factor NFATp.

The transcription factor NFATp integrates multiple signal transduction pathways through coordinate binding with basic-region leucine zipper (bZIP) proteins and other transcription factors. The NFATp monomer, even in the absence of its activation domains, recruits bZIP proteins to canonical NFAT-bZIP composite DNA elements. By contrast, the NFATp dimer and its bZIP partner bind noncooperatively to the NFAT-bZIP element of the tumor necrosis factor (TNF) gene promoter. This observation raises the possibility that the function of the activation domains of NFATp is dimer-specific. Here, we determine the consensus DNA binding site of the NFATp dimer, describe monomer- and dimer-specific NFATp-DNA contact patterns, and demonstrate that NFATp dimerization and dimer-specific activation subdomains are required for transcriptional activation from the TNF NFAT-bZIP element. We also show that these NFATp subdomains interact with the coactivator CBP (CREB-binding protein), which is required for NFATp-dependent TNF gene transcription. Thus, the context-specific function of the activation domains of NFAT can be potentiated by DNA-directed dimerization.

Activation-dependent intrachromosomal interactions formed by the TNF gene promoter and two distal enhancers.

Here we provide a mechanism for specific, efficient transcription of the TNF gene and, potentially, other genes residing within multigene loci. We identify and characterize highly conserved noncoding elements flanking the TNF gene, which undergo activation-dependent intrachromosomal interactions. These elements, hypersensitive site (HSS)-9 and HSS+3 (9 kb upstream and 3 kb downstream of the TNF gene, respectively), contain DNase I hypersensitive sites in naive, T helper 1, and T helper 2 primary T cells. Both HSS-9 and HSS+3 inducibly associate with acetylated histones, indicative of chromatin remodeling, bind the transcription factor nuclear factor of activated T cells (NFAT)p in vitro and in vivo, and function as enhancers of NFAT-dependent transactivation mediated by the TNF promoter. Using the chromosome conformation capture assay, we demonstrate that upon T cell activation intrachromosomal looping occurs in the TNF locus. HSS-9 and HSS+3 each associate with the TNF promoter and with each other, circularizing the TNF gene and bringing NFAT-containing nucleoprotein complexes into close proximity. TNF gene regulation thus reveals a mode of intrachromosomal interaction that combines a looped gene topology with interactions between enhancers and a gene promoter.

Primate TNF promoters reveal markers of phylogeny and evolution of innate immunity.

BACKGROUND: Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. CONCLUSIONS/SIGNIFICANCE: Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF.

Post-induction, stimulus-specific regulation of tumor necrosis factor mRNA expression.

The tumor necrosis factor (TNF) gene is activated by multiple extracellular signals in a stimulus- and cell type-specific fashion. Based on the presence of kappaB-like DNA motifs in the region upstream of the TNF gene, some have proposed a direct role for NF-kappaB in lipopolysaccharide (LPS)-induced TNF gene transcription in cells of the monocyte/macrophage lineage. However, we have previously demonstrated a general and critical role for a minimal TNF promoter region bearing only one of the kappaB-like motifs, kappa3, which is bound by nuclear factor of activated T cell proteins in lymphocytes and fibroblasts in response to multiple stimuli and Ets proteins in LPS-stimulated macrophages. Here, in an effort to resolve these contrasting findings, we used a combination of site-directed mutagenesis of the TNF promoter, quantitative DNase I footprinting, and analysis of endogenous TNF mRNA production in response to multiple stimuli under conditions that inhibit NF-kappaB activation (using the proteasome inhibitor lactacystin and using cells lacking either functional NF-kappaB essential modulator, which is the IkappaB kinase regulatory subunit, or the Nemo gene itself). We find that TNF mRNA production in response to ionophore is NF-kappaB-independent, but inhibition of NF-kappaB activation attenuates virus- and LPS-induced TNF mRNA levels after initial induction. We conclude that induction of TNF gene transcription by virus or LPS does not depend upon NF-kappaB binding to the proximal promoter; rather, a stimulus-specific post-induction mechanism involving NF-kappaB, yet to be characterized, is involved in the maintenance of maximal TNF mRNA levels.

NFAT5 regulates HIV-1 in primary monocytes via a highly conserved long terminal repeat site.

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.

Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein-protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

Global analysis of protein localization in budding yeast.

A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.