Journal: Virology

Article Title: Modulation of Apoptosis and Immune Signaling Pathways by the Hantaan Virus Nucleocapsid Protein

doi: 10.1016/j.virol.2010.02.018

Figure Lengend Snippet: Illustration of HTNV N protein deletion mutants and summary of their localization and caspase activities. (A) N-terminal deletion mutants of the HTNV N protein included NΔ270, NΔ300, NΔ330, and NΔ360, which were lacking 270, 300, 330 and 360 amino acids from the N-terminus respectively. C-terminally deleted HTNV N protein mutants included CΔ99, CΔ129, CΔ159, and CΔ189, lacking 99, 129, 159, and 189 amino acids from the C-terminus, respectively. Deletion mutant 271–300 contained both N- and C-terminal deletions. Intracellular localization was determined and noted as perinuclear (P), cytoplasmic (C), or indeterminate (I) (see panel B, data not shown for all constructs). We summarize caspase activities for each construct from experiments presented in Figure 3 and Figure 4. Caspase activation was scored with increasing activity correlating with increasing number of plus-signs; ++++ (high) and + (low). Data for these results are shown in subsequent figures. (B) Vero E6 cells were transfected with plasmid DNA expressing the fusion proteins indicated at the top of each panel. After incubation at 37 °C, 5% CO2 for 24 h, N protein expression was detected by fluorescence generated by GFP. Slides were acetone fixed and processed, and examined for fluorescence at excitation and emission spectra of 470 and 509 nm, respectively, using a Zeiss Axiovert 200 microscope. Scale bar, 50 µm, with 40X objective at 1.6 optovar/tubelens (Zeiss Axiovert 200).

Article Snippet: In one sample, the caspase extract of STR treated cells (6-well plate) was supplemented with 15 μl of 100 μM caspase inhibitor Z-D(OMe)E(OMe)VD(OMe)– FMK [Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK] (Imgenex, San Diego, CA) to determine the effect of inhibitors on caspase extract activity.

Techniques: Mutagenesis, Construct, Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Expressing, Incubation, Fluorescence, Generated, Microscopy

Journal: Virology

Article Title: Modulation of Apoptosis and Immune Signaling Pathways by the Hantaan Virus Nucleocapsid Protein

doi: 10.1016/j.virol.2010.02.018

Figure Lengend Snippet: Deletion of amino acids 270 to 330 results in the activation of caspase-7 and -8, but not of caspase-9 in HeLa cells. (A) Quantification of caspase-3/7-like activity, expressed as luminescence unit (LMU) in 2 × 105 cells, using Caspase-Glo® 3/7 Assay in Vero E6 cells after transfection with 0.8 µg plasmid DNA at 24 h in 24 well plates. *p < 0.002 versus cells expressing GFP-HTNS. #p < 0.003 versus Lipofectamine treated cells. (B) Caspase-3/7, -8, and -9-like activity in Vero E6 cells after transfection with 5 µg plasmid DNA at 24 h in 6 well plates as measured by Ac-DEVD-AMC, Ac-IETD-AMC, or Ac-LEHD-AMC cleavage, respectively, expressed as the fluorescence unit (FSU) in 1 × 106 cells, after transfection with plasmid DNA for 24 h. *p < 0.003 versus cells expressing GFP-HTNS. #p < 0.04 versus mock cells. For A and B, each value is the mean of three different samples, and the vertical bars represent the standard deviation. (C) Examination of caspase-3, -7, and -8, TNRF-1, and Fas-R levels in HTNV N protein or NΔ360 expressing cells. (D) Examination of caspase-3, -7, and -8 levels in ANDV N protein or NΔ360 expressing cells. (E) Examination of cleaved PARP, NF-κB (Rel-A), and p-NF-κB (p-Rel-A) in HTNV N protein or NΔ360 expressing cells. For western blots, HeLa cells were transfected with 5 µg plasmid DNA expressing HTNV or ANDV N protein, HTNV or ANDV NΔ360, or mock, or treated with 1 µM staurosporine (STR). Proteins were transferred onto nitrocellulose membranes and probed with antibodies against caspase-3, -7, and -8, cleaved PARP, NF-κB (Rel-A), p-NF-κB (p-Rel-A), or actin (loading control). (F) HeLa cells were transfected with 0.8 µg of plasmid DNA expressing HTNV N protein, NΔ360, or mock, or treated with 1 µM STR for 3 h. Slides were examined by indirect immunofluorescence for N protein or NΔ360 (Green) as GFP expression, or cleaved PARP (Red) with polyclonal antibodies as described in materials and methods with 63× objectives using a Leica confocal microscope.

Article Snippet: In one sample, the caspase extract of STR treated cells (6-well plate) was supplemented with 15 μl of 100 μM caspase inhibitor Z-D(OMe)E(OMe)VD(OMe)– FMK [Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK] (Imgenex, San Diego, CA) to determine the effect of inhibitors on caspase extract activity.

Techniques: Activation Assay, Activity Assay, Caspase-Glo Assay, Transfection, Plasmid Preparation, Expressing, Fluorescence, Standard Deviation, Western Blot, Control, Immunofluorescence, Microscopy

Journal: Virology

Article Title: Modulation of Apoptosis and Immune Signaling Pathways by the Hantaan Virus Nucleocapsid Protein

doi: 10.1016/j.virol.2010.02.018

Figure Lengend Snippet: Analysis of HTNV N protein interaction with NF-κB, and the examination the cytoplasmic fraction from cells expressing HTNV N protein has on caspase activity. (A) HeLa cells were transfected with 75 µg of plasmid expressing myc-tagged HTNV N protein or empty vector. Immunoprecipitations (IP) were performed with anti-myc antibody bound to magnetic Dynabeads. Immunoprecipitations and whole cell extracts were analyzed by western blot for the levels of HTNV N protein, TRADD, TRAF-2, NF-κB (Rel-A), p-NF-κB (p-Rel-A), or IκB. (B) A modified protocol was used with a longer incubation time of 24 hrs to facilitate immune complex formation. (C) HeLa cells were transfected with 5 µg or 10 µg of plasmid DNA in 6-well plates or T-25 flasks, respectively. Whole cell extracts were made from cells lysed with caspase extract buffer in 6-well plates that were untreated or treated with 5 µM STR and the caspase-3/7 activity was measured. Caspase activity was also measured in the presence of caspase inhibitor (Ci) Z-D(OMe)E(OMe)VD(OMe)–FMK. The cytoplasmic fraction of cells expressing HTNV N protein or mock, untreated or treated with 20 ng/ml of TNF-α for 20 min was extracted and concentrated from cells in T-25 flasks. Caspase activity in cells treated with STR (6-well plates) was measured that were supplemented with 15 µl of cytoplasmic extracts from T-25 flasks. Each value is the mean of three different samples, and the vertical bars represent the standard deviation.

Article Snippet: In one sample, the caspase extract of STR treated cells (6-well plate) was supplemented with 15 μl of 100 μM caspase inhibitor Z-D(OMe)E(OMe)VD(OMe)– FMK [Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK] (Imgenex, San Diego, CA) to determine the effect of inhibitors on caspase extract activity.

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Modification, Incubation, Standard Deviation