Domain Structure of Lassa Virus L Protein ▿

The 200-kDa L protein of arenaviruses plays a central role in viral genome replication and transcription. This study aimed at providing evidence for the domain structure of L protein by combining bioinformatics with a stepwise mutagenesis approach using the Lassa virus minireplicon system. Potential interdomain linkers were predicted using various algorithms. The prediction was challenged by insertion of flexible sequences into the predicted linkers. Insertion of 5 or 10 amino acid residues was tolerated at seven sites (S407, G446, G467, G774, G939, S1952, and V2074 in Lassa virus AV). At two of these sites, G467 and G939, L protein could be split into an N-terminal and a C-terminal part, which were able to trans-complement each other and reconstitute a functional complex upon coexpression. Coimmunoprecipitation studies revealed physical interaction between the N- and C-terminal domains, irrespective of whether L protein was split at G467 or G939. In confocal immunofluorescence microscopy, the N-terminal domains showed a dot-like, sometimes perinuclear, cytoplasmic distribution similar to that of full-length L protein, while the C-terminal domains were homogenously distributed in cytoplasm. The latter were redistributed into the dot-like structures upon coexpression with the corresponding N-terminal domain. In conclusion, this study demonstrates two interdomain linkers in Lassa virus L protein, at G467 and G939, suggesting that L protein is composed of at least three structural domains spanning residues 1 to 467, 467 to 939, and 939 to 2220. The first domain seems to mediate accumulation of L protein into cytoplasmic dot-like structures

Decreased expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4⁺ T cells and peripheral blood from tuberculosis patients.

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4(+) T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4(+) T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease

Decreased expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells and peripheral blood from tuberculosis patients

CITATION: Kleinsteuber, K. et al. 2013. Decreased expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells and peripheral blood from tuberculosis patients. PLoS ONE, 8(4): e61609, doi:10.1371/journal.pone.0061609.The original publication is available at http://journals.plos.org/plosoneThe vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4+ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4+ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061609Publisher's versio

Target genes and overlaps for miR-21, miR-26a, miR-29a and miR-142-3p.

<p>(<b>A</b>) A Venn diagram indicates the overlap of target genes for miR-21, miR-26a, miR-29a and miR-142-3p. Common targets of at least three candidate miRNAs are listed by name. (<b>B</b>) Comparison between expression miR-21 (upper left graph), miR-26a (upper right graph), miR-29a (lower graph) and protein expression of the common target PTEN in lymphocytes from children with tuberculosis (n = 19) and LTBI (n = 3) is shown. We indicate relative miRNA expression to house keeping gene RNU48 by quantitative PCR and compare this to PTEN protein expression determined by flow cytometry using median fluorescence intensity (MFI) analysis. Each symbol represents data from an individual donor.</p

MiRNA candidate expression of peripheral blood cells from children with tuberculosis and LTBI.

<p>Expression of miR-21 (triangles), miR-26a (squares), miR-29a (circles), and miR-142-3p (diamonds) in whole blood is shown for children with tuberculosis (TB, black symbols), healthy latently <i>M. tuberculosis</i> children (LTBI, grey symbols), and PPD negative contacts (PPD_neg, open symbols) (<b>A</b>) as well as for children with tuberculosis under therapy and recovery (<b>B</b>). Each symbol indicates miRNA candidate expression for an individual donor relative to ‘housekeeping’ control RNU48. Significant differences are indicated as asterisks (* for P<0.05; ** for P<0.01, Mann-Whitney U-test). Exact P-values (Mann-Whitney U-test) are indicated for tendencies.</p

Median Expression of miRNAs in PPD_neg donors, LTBI and tuberculosis patients.

1<p>control; nd: miRNA not detectable in at least two donors.</p

Four miRNAs are differentially expressed in CD4⁺ T cells from tuberculosis patients and LTBI.

<p>Expression of miR-21 (triangles), miR-26a (squares), miR-29a (circles), and miR-142-3p (diamonds) in CD4⁺ T cells from tuberculosis patients (TB) (black symbols, n = 6), LTBI (grey symbols, n = 7), and PPD negative healthy controls (PPDneg) (open symbols, n = 3). Median expression of candidate miRNAs relative to the housekeeping gene RNU48 is shown. Significant differences are indicated as asterisks (* for p<0.05; ** for p<0.01, Mann-Whitney U-test).</p

IFNγ and miR-29a expression of infected children and ectopic miR-29a suppression in T cells.

<p>(<b>A</b>) IFNγ expression after short-term <i>in vitro</i> re-stimulation of PBMC from children with tuberculosis (upper left graph) prior to chemotherapy (black symbols), three months under chemotherapy (grey symbols), after recovery (open symbols), as well as children with LTBI (upper right graph) with <i>M. tuberculosis</i>-specific PPD is shown. Proportions of IFNγ expressing CD4⁺ T cells (y-axis) for different time points after treatment onset of patients (x-axis) are depicted. Each symbol represents data from an individual patient with tuberculosis (hexagon) or LTBI (filled trigon). <i>Ex vivo</i> whole blood miR-29a expression (x-axis) in comparison to the proportions of IFNγ expressing CD4⁺ T cells (x-axis) for individual children with TB under therapy (circles) is shown in the lower graph. (<b>B</b>) Modulation of miR-29a expression (upper left graph) and control miR-16 (upper right graph) of CD4⁺ T cells by specific antagomirs (Homo sapiens (Hsa)-29a, Hsa-16, and Caenorhabditis elegans (Cel)-67) and effects on IFNγ expression (lower graphs) are depicted. IFNγ expression of activated CD4⁺ T cells after miR-29a or miR-16 suppression is shown as proportions of IFNγ⁺ cells (lower left graph) or as the IFNγ expression level per cell (indicated by median fluorescence intensities (MFI)) (lower right graph).</p

Expression of candidate miRNAs in T_H1 and T_H17 clones.

<p>Expression of miR-21 (triangles), miR-26a (squares), miR-29a (circles), miR-142-3p (diamonds), and miR-155 (hexagons) was compared between IFNγ-expressing T_H1 (open symbols, n = 11) and IL-17-expressing T_H17 clones (black symbols, n = 9) of healthy donors prior to (<b>A</b>) and after <i>in vitro</i> activation (<b>B</b>). Expression of candidate miRNAs was normalized to the housekeeping gene RNU48. Median expression and range is shown. Significant differences are indicated as asterisks (* for P<0.05, Mann-Whitney-U test). (<b>B</b>) MiRNA expression after 12 h <i>in vitro</i> re-stimulation with PMA/Ionomycin. Relative expression to the respective non-activated control is shown (dotted line).</p