A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection

SummaryThe 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

The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells

<p>Abstract</p> <p>Background</p> <p>Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.</p> <p>Findings</p> <p>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-1_Lai-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.</p> <p>Conclusions</p> <p>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.</p

Regulation of Mycobacterium tuberculosis-Dependent HIV-1 Transcription Reveals a New Role for NFAT5 in the Toll-Like Receptor Pathway

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

Minigenome-Based Reporter System Suitable for High-Throughput Screening of Compounds Able To Inhibit Ebolavirus Replication and/or Transcription ▿

We describe an Ebolavirus minigenome-based system that is suitable for high-throughput screening of compounds able to impair Ebolavirus virus replication and/or transcription. The assay is robust (Z′ factor, >0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented

A Distal Locus Element Mediates IFN-γ Priming of Lipopolysaccharide-Stimulated TNF Gene Expression

Summary: Interferon γ (IFN-γ) priming sensitizes monocytes and macrophages to lipopolysaccharide (LPS) stimulation, resulting in augmented expression of a set of genes including TNF. Here, we demonstrate that IFN-γ priming of LPS-stimulated TNF transcription requires a distal TNF/LT locus element 8 kb upstream of the TNF transcription start site (hHS-8). IFN-γ stimulation leads to increased DNase I accessibility of hHS-8 and its recruitment of interferon regulatory factor 1 (IRF1), and subsequent LPS stimulation enhances H3K27 acetylation and induces enhancer RNA synthesis at hHS-8. Ablation of IRF1 or targeting the hHS-8 IRF1 binding site in vivo with Cas9 linked to the KRAB repressive domain abolishes IFN-γ priming, but does not affect LPS induction of the gene. Thus, IFN-γ poises a distal enhancer in the TNF/LT locus by chromatin remodeling and IRF1 recruitment, which then drives enhanced TNF gene expression in response to a secondary toll-like receptor (TLR) stimulus. : Interferon γ (IFN-γ) priming is a critical immune event that enhances the monocyte and macrophage response, particularly expression of the TNF gene, to toll-like receptor (TLR) signaling. Chow et al. demonstrate that IFN-γ priming requires a distal enhancer element within the TNF/LT locus, thereby expanding the role of distal regulatory elements in the innate immune response

ALIX/AIP1 Is Required for NP Incorporation into Mopeia Virus Z-Induced Virus-Like Particles▿

During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions

Production of Novel Ebola Virus-Like Particles from cDNAs: an Alternative to Ebola Virus Generation by Reverse Genetics

We established a plasmid-based system for generating infectious Ebola virus-like particles (VLPs), which contain an Ebola virus-like minigenome consisting of a negative-sense copy of the green fluorescent protein gene. This system produced nearly 10(3) infectious particles per ml of supernatant, equivalent to the titer of Ebola virus generated by a reverse genetics system. Interestingly, infectious Ebola VLPs were generated, even without expression of VP24. Transmission and scanning electron microscopic analyses showed that the morphology of the Ebola VLPs was indistinguishable from that of authentic Ebola virus. Thus, this system allows us to study Ebola virus entry, replication, and assembly without biosafety level 4 containment. Furthermore, it may be useful in vaccine production against this highly pathogenic agent

MTb induces NFAT5 gene expression and knockdown of NFAT5 impairs HIV-1 replication during MTb co-infection.

<p>(A) MTb but not HIV-1 increases NFAT5 mRNA synthesis in human macrophages. MDM were isolated from PBMC obtained from four normal donors. The cells were incubated for 24 or 48 hours with heat-inactivated or live R5-tropic HIV-1 subtype B, C, or E isolates, live MTb strain CDC1551 (1∶1 MDM∶bacilli), or left untreated. Quantitative real-time PCR analysis of NFAT5 mRNA expression levels revealed that the low, constitutively present level of NFAT5 mRNA expressed in untreated cells was not affected by exposure to live or inactivated HIV-1, but was significantly increased at both 24 (*, p<0.05) and 48 (**, p<0.01) hours following infection with MTb. (B) NFAT5-specific siRNA effectively suppresses NFAT5 expression in MDM in both the absence and presence of MTb co-infection. MDM (1×10⁶ cells/well in a 6-well plate) from four normal donors were transfected with siRNA specific for NFAT5 or, as a control, GFP, and were then infected with MTb (1∶1 MDM∶bacilli) or left uninfected. In the cells transfected with control siRNA, MTb infection significantly increased NFAT5 expression in comparison to uninfected cells (open bars). When MDM were transfected with NFAT5-specific siRNA, both constitutively expressed (p = 0.048) and MTb-induced (p = 0.021) NFAT5 mRNA were significantly suppressed (grey bars). (C) HIV-1 subtype B replication is significantly suppressed in the presence of MTb co-infection when NFAT5 expression is abrogated in human MDM. MDM from four normal donors were transfected with NFAT5-specific and control siRNAs as described in 2B. Cells were then infected with HIV-1_Lai/Bal-env followed by co-infection with MTb CDC1551. Virus replication was measured at days 6, 9, and 12 post-infection. At day 12, virus replication was significantly reduced in cells transfected with NFAT5-specific siRNA in comparison to cells transfected with control siRNA (*, p<0.05). We monitored the morphology of cells within the infected cultures and did not observe noticeable macrophage necrosis even at the final timepoint of viral harvest.</p

The MyD88-mediated signaling cascade is important for MTb induction of NFAT5 expression in human monocytes.

<p>(A) Engagement of TLR2 significantly enhances NFAT5 mRNA synthesis in monocytes. THP-1 cells were incubated with the TLR2-specific ligand Pam3Cys (5 µg/ml), infected with MTb strain CDC1551 (1∶1 cells∶bacilli), or left untreated. After 16 hours, NFAT5 mRNA levels were measured. NFAT5 mRNA expression was significantly increased when the cells were incubated with Pam3Cys (*, p<0.05) or infected with MTb (**, p<0.01). (B) MyD88 is important for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered shRNA targeting MyD88 or control shRNA targeting GFP were constructed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002620#s2" target="_blank">Materials and Methods</a>. Cells were infected with MTb strain CDC1551 (1∶1 cells∶bacilli) or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 mRNA levels were significantly inhibited in the cells transduced with MyD88-specific shRNA (black bars) compared with the cells transduced with control shRNA (open bars) in both, the absence or presence of MTb infection. TNF and CD86 mRNAs, the expression of which are MyD88-dependent and -independent, respectively, during MTb infection, were measured as controls. (C, D) IRAK1 and TRAF6 are required for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered IRAK1- or TRAF6-specific shRNA were constructed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002620#s2" target="_blank">Materials and Methods</a>. THP-1 cells expressing control shRNA were used as described in 7B. Cells were infected with MTb strain CDC1551 or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 expression was significantly inhibited in the MTb-infected cells expressing either IRAK1 (C) or TRAF6 (D) shRNA (black bars) compared to cells expressing control shRNA (open bars). TNF and CD86 mRNAs, both of which require IRAK1 and TRAF6 to be induced by PRR activation, were measured as controls. (*, p<0.05; **, p<0.01).</p

MTb-induced HIV-1 replication in PBMC depends on binding of NFAT5 and NF-κB to the LTR.

<p>Specific disruption of the NF-κB or NFAT5 binding sites in the HIV-1 subtype B LTR inhibits R5-tropic virus replication in human PBMC after co-infection with MTb. PBMC from four normal donors were infected with 1000 TCID₅₀ HIV-1_Lai/Bal-env-WT or the mutants HIV-1_Lai/Bal-env-κB I-Mut, HIV-1_Lai/Bal-env-κB II-Mut, HIV-1_Lai/Bal-env-κB I+II-Mut, or HIV-1_Lai/Bal-env-N5-Mut and either; (A) left untreated, or (B) co-infected with MTb isolate CDC1551 (10∶1 cells∶bacilli). Viral p24 levels in culture supernatants were measured at days 4, 7 and 12 post-infection. (C) Histograms show viral p24 levels at day 12 in the MTb uninfected (grey bars) and MTb co-infected (black bars) PBMC cultures. Replication of the mutant viruses was compared to wild-type virus replication under the same experimental conditions (without and with MTb co-infection, respectively). *, p<0.05; **, p<0.01, as compared to HIV-1_Lai/Bal-Env-WT.</p