El Correo gallego : diario político de la mañana: Ano LVIII Número 20110 - 1936 xullo 26

<p>Reverse transcriptase (RT)-associated DNA polymerase (RDDP) and ribonucleaser H (RNase H) functions are both essential for HIV-1 genome replication, and the identification of new inhibitors to block both of them is a goal actively pursued by the scientific community. In this field, natural extracts have shown a great potential as source of new antivirals. In the present work, we investigated the effect of <i>Uvaria angolensis</i> extracts on the HIV-1 reverse transcriptase-associated DNA polymerase and ribonuclease H activities. The <i>U. angolensis</i> stem bark methanol extract inhibit both HIV-1 RNase H function and RDDP activity with IC₅₀ values of 1.0 ± 0.2 and 0.62 ± 0.15 μg/mL, respectively and, after been fractionated with different solvents, its solid residue showed an IC₅₀ of 0.10 ± 0.03 and of 0.23 ± 0.04 μg/mL against RNase H and RDDP, respectively, hence laying the bases for further studies for identification of single active components.</p

ISG15 controls apoptosis in macrophages after VACV infections.

<p><b>A–B.</b> Time course of apoptosis markers during VACV infection in ISG15+/+ or ISG15<b>−/−</b> MEFs (<b>A</b>) or peritoneal macrophages (<b>B</b>). ISG15+/+ or ISG15−/− cells were mock-infected (M) or infected with VACV (WR strain, 10 PFU/cell). At the indicated times post-infection, cells were harvested and total proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-PARP-1, anti-caspase-9, anti-activated-capsase-3 and anti bcl-2 specific antibodies. Actin was measured as protein loading control. Protein standards are indicated. <b>C–D.</b> Apoptosis activation in ISG15+/+ or ISG15−/− MEFs (<b>C</b>) or peritoneal macrophages (<b>D</b>). Apoptosis was measured using the Caspase-Glo 3/7 assay kit at 24 hpi Results represent the mean ± the standard deviation of three independent experiments. P-values from a two-tailed t test assuming non-equal variance were determined. In all the cases, P<0.05.</p

Effect of ISG15 on cytotoxicity and infection progression in ISG15+/+ or ISG15−/− MEFs or macrophages infected with influenza virus (FluV).

<p><b>A–B. </b><i>De novo</i> protein synthesis in FluV- infected ISG15+/+ or ISG15−/− MEFs (<b>A</b>) or peritoneal macrophages (<b>B</b>). ISG15+/+or ISG15<b>−/−</b> cells were infected (10⁶ cells/time post-infection; 3 PFU/cell) with FluV (A/<i>WSN</i>/1933 strain) and labeled with ³⁵S-methionine (50 µCi/ml, 30 min) at the times indicated. Cellular lysates were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membranes and visualized by autoradiography. Protein standards are indicated. Uninfected cells (Mock) served as control. In the same membranes, the expression of ISG15 or tubuline (protein loading control) was detected by Western blot using specific antibodies. <b>C.</b> ISG15+/+ or ISG15<b>−/−</b> peritoneal macrophages were infected with FluV (A/<i>WSN</i>/1933 strain, 5 PFU/cell) and CPE was visualized by phase-contrast microscopy at indicated times post-infection. A selected cell is indicated with an arrow.</p

Phagocytosis of GFP-latex beads by ISG15 +/+ or ISG15 −/− macrophages.

<p><b>A–C.</b> Peritoneal macrophages isolated for ISG15+/+ or ISG15<b>−/−</b> mice were seeded in 8-well tissue culture plates and treated with type I IFN (100units/ml) for 16 hours (<b>A</b>) or not (<b>C</b>). After that, the cells were incubated with 1<b>-</b>µm-diameter latex beads conjugated to GFP in a ratio of 10 latex beads per cell. Phagocytized beads and cells were visualized with the time by fluorescent and phase contrast microscopy. <b>B.</b> Quantification of phagocytic cells in type I IFN alpha treated or not treated macrophages. Cells were quantified by IF in three independent experiments. Black bars represent ISG15+/+ macrophages, white bars represents ISG15<b>−/−</b> macrophages. Results represent the mean ± the standard deviation of three independent experiments. P values from a two-tailed t-test assuming non-equal variance were determined. In all the cases, P<0.05. <b>D.</b> Phagocytosis of GFP-latex beads by ISG15+/+ and ISG15<b>−/−</b> macrophages detected by immunofluorescence. Peritoneal macrophages were cultured on coverslips and incubated for 1 h with GFP-latex beads (20 beads per cell) and then washed three times with phosphate-buffered saline (PBS) and incubated with Dulbecco's medium (DMEM) medium for an additional hour. Cells were fixed with 4% PFA and processed for immunofluorescence analysis. Phagocytized beads and cells were visualized by confocal fluorescent microscopy. Representative fields are shown at a magnification of 40× (left panels) and 100× (right panels).</p

Wortmannin treatment inhibits phagocytosis of GFP-latex beads by ISG15+/+ macrophages.

<p>Peritoneal macrophages isolated from ISG15+/+ mice were seeded in 8-well tissue culture plates and treated with type I IFN (100 units/ml) for 16 hours. After that, were untreated or treated with wortmannin (100 nM) or rapamycin (100 nM) for 1 hour and subsequently incubated with 1<b>-</b>µm-diameter latex beads conjugated to GFP in a ratio of 20 latex beads per cell. Phagocytized beads and cells were visualized with the time by fluorescent and phase contrast microscopy. Representative fields are shown at a magnification of 40× (left panels) and 100× (right panels).</p

VACV protein synthesis on ISG15+/+ or ISG15−/− MEFs or macrophages.

<p><b>A–B.</b> Viral protein expression during VACV infection of ISG15+/+ or ISG15−/− MEFs or macrophages. ISG15+/+ or ISG15−/− MEFs (<b>A</b>) or macrophages (<b>B</b>) were infected (10⁶ cells/time post-infection; 3 PFU/cell) with VACV (WR strain) and, at the indicated times post-infection, cell extracts were prepared and equal amounts of proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, and detected with specific antibodies virus early (p25), intermediate (p39) and late proteins (p14). ISG15 expression levels were detected by Western blot using specific antibodies. Actin was measured as protein loading control. Protein standards are indicated. Uninfected cells (Mock) served as control. <b>C–D. </b><i>De novo</i> protein synthesis in VACV-infected ISG15+/+ or ISG15−/− MEFs (<b>C</b>) or macrophages (<b>D</b>). ISG15+/+ or ISG15<b>−/−</b> cells were infected (10⁶ cells/time post-infection; 3 PFU/cell) with VACV (WR strain) and labeled with ³⁵S-methionine (50 µCi/ml, 30 min) at the different times indicated. Cellular lysates were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membranes and visualized by autoradiography. Protein standards are indicated. Uninfected cells (Mock) served as control. In the same membranes, the expression of ISG15 or tubuline (protein loading control) was detected by Western blot using specific antibodies.</p

AKT phosphorylation levels are altered in ISG15+/+ or ISG15−/− macrophages after VACV infection.

<p><b>A.</b> ISG15+/+ or ISG15<b>−/−</b> macrophages were infected with VACV (WR strain, 60 PFU/cel) and, at the indicated times, cells were harvested and total proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phosphoS473<b>-</b>AKT. The expression of total AKT was evaluated using a specific antibody (AB). <b>B.</b> Phagocytosis of GFP-latex beads by macrophages treated with wortmannin, rapamycin or LPS by confocal microscopy. Peritoneal macrophages isolated from ISG15+/+ or ISG15−/− mice were seeded in 8-well tissue culture plates and treated with type I IFN (100 units/ml) for 16 hours. After that, cells were untreated or treated with wortmannin (100 nM) or rapamycin (100 nM) and, 1 hour after the treatment, the cells were incubated with 1<b>-</b>µm-diameter latex beads conjugated to GFP in a ratio of 20 latex beads per cell. Phagocytized beads and cells were visualized with the time by confocal fluorescent microscopy. <b>C.</b> AKT phosphorylation levels after the treatment with PI3K pathway inhibitors. ISG15+/+ macrophages were untreated or treated with wortmannin (100 nM) or rapamicyn (100 nM) and, 1 hour after the treatment, cells were incubated with latex bead (10 bead per cell) for 2 hours. Cells were consecutively harvested and total proteins were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phosphoSer473<b>-</b>AKT. The expression of AKT was evaluated using a specific antibody.</p

ISG15 control the VACV entry.

<p>Peritoneal macrophages isolated from ISG15+/+ or ISG15<b>−/−</b> mice were seeded in 8-well tissue culture plates and treated with type I interferon (IFN) (100units/ml) for 16 hours. After that, the cells infected with VACV-YFP (60 PFU/cell). Infected-cells were visualized with the time by fluorescent and phase contrast microscopy. Representative fields are shown at a magnification of 40× (left panels).</p

ISG15 regulates the phagocytosis and the clearance of VACV-infected MEFS regulating AKT phosphorylation.

<p><b>A.</b> ISG15−/− MEFS were infected with VACV-YFP at 1 PFU/cell for 8 hours, and added to the ISG15+/+ or ISG15−/− macrophages culture with a ratio of one MEF cells to four macrophages. The mixed culture was monitored for 2,5 hours at 37°C by time-lapse microscopy and representative images were illustrated. <b>B.</b> Inhibition of VACV growth in the presence of ISG15+/+ macrophages. (A) ISG15−/− MEFS were infected with VACV-YFP at 1 PFU/cell for 8 hours and further cultured with DMEM (as negative control) or macrophages (ISG15+/+ or ISG15<b>−/−</b>) with a ratio of 1∶2 using 10⁵ macrophages. Infected cells were harvested at different times postinfection and virus yields were determined by plaque assay. Results represent the mean ± the standard deviation of three independent experiments. P values from a two-tailed t test assuming non-equal variance were determined. In all the cases, P<0.05. <b>C-D-E.</b> Viral protein expression (<b>C</b>) ISG15 (<b>D</b>) and AKT phosphorylation levels (<b>E</b>) were measured in the above described co-cultures. Total proteins from cells described above were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-ISG15 or anti-phosphoSer473<b>-</b>AKT. The expression of AKT was evaluated using a specific antibody. Actin was measured as protein loading control. Protein standards are indicated. <b>F–G</b> Quantification of phagocytic cell, expressed as relative amount of the total number of macrophages, and the viral titer was determined in the absence (<b>F</b>) or presence of 100 nM wortmannin (<b>G</b>). Means and standard deviations of a typical example from three independent experiments are presented. P values from a two-tailed t test assuming non-equal variance were determined. In all the cases, P<0.05.</p