12 research outputs found

    Mining a Kröhnke Pyridine Library for Anti-Arenavirus Activity

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    Several arenaviruses cause hemorrhagic fever (HF) disease in humans and represent important public health problems in their endemic regions. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus is a neglected human pathogen of clinical significance. There are no licensed arenavirus vaccines, and current antiarenavirus therapy is limited to an off-label use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel therapeutics to combat human pathogenic arenaviruses, a task that will be facilitated by the identification of compounds with antiarenaviral activity that could serve as probes to identify arenavirus–host interactions suitable for targeting, as well as lead compounds to develop future antiarenaviral drugs. Screening of a combinatorial library of Krönhke pyridines identified compound KP-146 [(5-(5-(2,3-dihydrobenzo­[b]­[1,4] dioxin-6-yl)-4′-methoxy-[1,1′-biphenyl]-3-yl)­thiophene-2-carboxamide] as having strong anti-lymphocytic choriomeningitis virus (LCMV) activity in cultured cells. KP-146 did not inhibit LCMV cell entry but rather interfered with the activity of the LCMV ribonucleoprotein (vRNP) responsible for directing virus RNA replication and gene transcription, as well as with the budding process mediated by the LCMV matrix Z protein. LCMV variants with increased resistance to KP-146 did not emerge after serial passages in the presence of KP-146. Our findings support the consideration of Kröhnke pyridine scaffold as a valuable source to identify compounds that could serve as tools to dissect arenavirus–host interactions, as well as lead candidate structures to develop antiarenaviral drugs

    Antiviral T cell function and expansion is reduced in BST-2 deficient mice.

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    <p><b>A-D.</b> Naïve WT and BST-2 KO mice that received 2,000 Thy1.1+ P14 and 2,000 CD45.1+ SMARTA cells were infected with LCMV Cl-13. On day 8 post-infection, the absolute number of splenocytes (A), P14 cells (C), and SMARTA (C) cells was quantified. In addition, the percentage of endogenous (B) and transferred (D) IFNγ+ TNFα+ antiviral T cells was determined following a 5 hr <i>in vitro</i> stimulation with GP33-41 (CD8) or GP61-80 (CD4) peptide (n = 9 mice per group; 2 independent experiments). Data are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05).</p

    Endogenous BST-2 expression levels have a very limited impact on LCMV propagation <i>in vitro</i>.

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    <p><b>A.</b> Cell surface expression of BST-2 in HeLa-TKD and HeLa-pLKO cells. Cells were fixed and analyzed by FACS using either a control antibody (Ab) or anti-BST-2 conjugated to PE. <b>B.</b> Growth kinetics of LCMV in HeLa-pLKO and HeLa–TKD cells. Cells were infected with LCMV (moi = 0.01). At 24 and 48 hrs p.i, virus titers in TCS were determined by plaque assay (n = 3; 2 independent experiments). <b>C.</b> Propagation of LCMV in HeLa-pLKO and HeLa-TKD cells. Cells were infected with LCMV (moi = 0.1). At 24 and 48 h p.i., cells were fixed with 4% PFA and after permeabilization stained with a MAb to LCMV NP, followed with a second antibody conjugated to FITC. Nuclei were stained with DAPI. Mock infected cells were used as control. <b>D.</b>Similar Z protein expression levels in HeLa-TKD and HeLa-pLKO cells at 16 h p.i. Cells were infected (moi = 0.1) with rLCMV/Z-FLAG and at 16 h p.i. expression levels of Z protein determined by WB using a MAb to FLAG. Levels of actin were used as loading control. <b>E-F.</b> Production of virion particles by LCMV-infected HeLa-TKD and HeLa-pLKO cells. Cells were infected with rLCMV/Z-FLAG (moi = 0.1) and at 24 hrs p.i. virion particles present in TCS were recovered by ultracentrifugation, and cell lysates prepared. Levels of Z protein present in virion particle preps and cell lysates were determined by WB using an Ab to FLAG (<b>E</b>), and signals quantified with LAS3000 (Fuji Film) (<b>F</b>). The ratio of virus/cell Z protein levels in HeLa-pLKO was set to 1.0 (n = 3; 3 independent experiments). <b>G.</b> Growth kinetics of VSV in HeLa-pLKO and HeLa–TKD cells. Cells were infected with VSV (moi = 0.01). At 8, 18 and 24 h p.i, virus titers in TCS were determined by plaque assay (n = 3; 2 independent experiments). Data correspond to mean + SD. Asterisks (*) denote statistical significance (<i>P</i> < 0.05).</p

    BST-2 contains LCMV within the splenic marginal zone.

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    <p><b>A-B.</b> Representative confocal images from naïve, d3 WT, and d3 BST-2 KO mice depict the splenic distribution of LCMV Cl-13 (green; A) or CD169+ metallophillic macrophages (green; B) in relation to BST-2 expression (red) (n = 4 mice per group; 2 independent experiments). <b>C.</b> Representative confocal images captured in the spleens of WT vs. BST-2 KO mice at day 3 post-infection show the distribution of LCMV (red) in relation to the white pulp (WP), marginal zone (MZ), and red pulp (RP). Laminin staining is shown in green to delineate these anatomical regions. <b>D.</b> Quantification of the LCMV staining pattern shown in panel C (n = 8 mice per group; 2 independent experiments). Data are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05).</p

    BST-2 deficiency impedes control of acute and persistent strains of LCMV.

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    <p><b>A.</b> Serum viral titers were quantified by plaque assay in WT vs. BST-2 KO mice infected with LCMV Cl-13 (n = 5 mice per group; 2 independent experiments). <b>B.</b> Brain viral loads were quantified by Q-PCR [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007172#ppat.1007172.ref048" target="_blank">48</a>] in WT vs. BST-2 KO mice 237 days following infection with LCMV Cl-13 (n = 3–4 mice per group; 2 independent experiments). <b>C.</b> Serum viral titers were quantified by plaque assay in WT vs. BST-2 KO mice 5 days following infection with LCMV Arm (n = 3–4 mice per group; 2 independent experiments). All data in this figure are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05).</p

    Effect of BST-2 on LCMV Z-mediated VLP production and vRNP activity.

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    <p><b>A.</b> Effect of BST-2 expression on Z-mediated VLP production. 293T cells were transfected with pC-LCMV-Z-FLAG (0.2 μg) together with increasing amounts of pTeth-FL (BST-2). At 48 hrs post transfection, VLPs were recovered from TCS by ultracentrifugation and cell lysates prepared. Levels of Z protein present in VLPs and cell lysates, and BST-2 levels in cell lysates, were detected by WB using an Ab to FLAG. Normalized levels of VLP production were determined by assigning the value of 1 to the ratio of VLP/cell Z protein expression levels in cells transfected with pC-LCMV-Z-FLAG in the absence of BST-2 (n = 3; 2 independent experiments). <b>B.</b> Effect of BST-2 expression on LCMV vRNP activity. 293T cells were transfected with plasmids expressing the components of the LCMV MG-CAT, together with control BST-2 or GFP expression plasmids using the indicated plasmid DNA amounts. CAT expression was measured at 48 hrs post transfection. CAT expression levels were normalized by setting to 1 levels expressed by cells transfected with control plasmid. <b>C, D.</b> Effect of BST-2 over-expression on pol-II mediated expression. 293T cells were transfected with psiCHECK2 together with control plasmid (pcDNFL) or increasing amounts of BST-2 expression plasmid. At 48 hrs post transfection, cell lysates were prepared for detection of both Firefly luciferase (FL) (<b>C</b>) and Renilla luciferase (RL) (<b>D</b>) using a dual luciferase detection system. Expression levels of FL and RL were normalized by setting to 1 the expression levels obtained in cells transfected with FL or RL and the control plasmid. <b>E.</b> Effect of BST-2 expression on 293T cell viability. Cells were transfected with increasing amount of BST-2 expression plasmid, and at 48 hrs post transfection, cell viability was determined by Cell-Titer Glo (Material and Methods). Values were normalized by setting to 1 results from cells transfected with control plasmid (pcDNFL). Data correspond to mean + SD (three independent experiments). Asterisks denote statistical significance (*P < 0.05).</p

    Effect of LCMV infection on protein expression and subcellular distribution of endogenous BST-2.

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    <p><b>A.</b> HeLa cells were infected with LCMV (moi = 0.01). At 48 hrs p.i, cells were lysed and BST-2 expression was analyzed by WB using a polyclonal serum to α-BST-2. <b>B.</b> HeLa cells were infected with LCMV (moi = 0.01). At 48 hrs p.i, cells were reacted with either control antibody (Ab) or α-BST-2 Ab conjugated with PE and then fixed. Cell surface expression of BST-2 was analyzed by FACS Calibur (BD, San Jose, CA). <b>C.</b> Subcellular localization of BST-2 in HeLa cells infected with rLCMV/Z-FLAG. HeLa cells were infected with rLCMV/Z-FLAG (moi = 0.01). At 24 and 48 hrs p.i., cells were fixed with 4% PFA and stained with α-FLAG antibody and α-BST-2 antibody, followed with second antibodies conjugated with Alexa 568 or Alexa 488, respectively. Nuclei were stained with DAPI. White arrows indicate LCMV Z localized at the plasma membrane without BST-2 co-localization.</p

    BST-2 deficiency changes the splenic distribution and proliferative capacity of antiviral CD8+ T cells.

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    <p><b>A.</b> Representative confocal images were captured at day 4 post-infection in the spleens of WT and BST-2 KO mice seeded with mOrange+ P14 cells (green). The splenic distribution of P14 cells in relation to LCMV Cl-13 (red) is shown. The white dotted line demarcates the border between the white pulp (WP) and red pulp (RP). <b>B.</b> The bar graph shows quantification of the white vs. red pulp P14 percentage in WT vs. BST-2 KO mice (n = 4 mice per group; 2 independent experiments). Data are represented as mean ± SD. Asterisks denote statistical significance (*P < 0.05). <b>C.</b> The representative histogram depicts the dilution of CFSE in P14 cells from WT (blue) vs. BST-2 KO (red) mice at day 3 post-infection. <b>D.</b> The bar graph shows quantification of the CFSE dilution data in panel C (n = 4 mice per group; 2 independent experiments). Graphed are the percentage of P14 cells that divided more than 5 times. Data are represented as mean ± SD. Asterisks denote statistical significance (*P ≤ 0.05).</p

    rscVSVs used to study LASV-specific T cell responses.

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    A) Schematic of the LASV genome showing the four genes coded in an ambisense direction and non-coding regions (top). Schematic below shows NP and GPC antigens encoded by rscVSVs. B) BHK-21 cells were infected with each rscVSV encoding LASV NP and GPC antigens and GFP control. cDNA was made with poly-dT oligonucleotide primers to amplify only mRNA sequences. LASV gene specific and flag epitope primers were used to amplify cDNA. C) Protein expression of LASV genes and GFP from rscVSV infected BHK-21 cells was determined by Western blot using a flag epitope specific antibody.</p

    T cell responses to LASV antigens.

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    A) Percentages of LF survivors from both Sierra Leone and Nigeria with CD8+ and CD4+ T cell responses to LASV antigens. B) Percentage of LF survivors from Sierra Leone (white bars) and Nigeria (grey bars) harboring CD8+ and CD4+ T cells responding to rscVSVs encoding NP, GPC or both. Responses were defined by individuals expressing both IFN-γ and TNF-α at 1.2 log10 over the median fluorescence of negative controls. Samples were considered to respond to GPC if we observed T cell expression of IFN-γ and TNF-α upon stimulation with either rscVSVs encoding GP1, GP2, and/or SSP-GP2. Samples were considered to respond to both NP and GPC if we observed T cell expression of IFN-γ and TNF-α to upon stimulation with rscVSV-NP and any of the GPC encoding rscVSVs. Only individuals with CD8+ (left) or CD4+ T cell responses (right) were included. C) Percentages of CD3+ CD8+ T cells expressing IFN-γ and TNF-α from Sierra Leonian (left) and Nigeran (right) LF survivors. Reponses considered positive (black) and negative (red) are shown. Some responses were considered negative because they didn’t meet the threshold of >3 events in the double positive quadrant. Zero values are indicated by the numbers above the x-axis. No significance was found between any groups using mixed-effects analysis (Tukey’s multiple comparisons test). D) Percentages of CD3+ CD8+ T cells expressing IFN-γ and TNF-α from all LF survivors. Data was analyzed using mixed-effects analysis, **p<0.005. E) Percentages of CD3+ CD8+ T cells expressing IFN-γ and TNF-α from Sierra Leonian Nigeran LF survivors. Only values considered positive for a LASV-specific response are shown. No significance was found between Sierra Leonean and Nigerian responses using 2way ANOVA.</p
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