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Analysis of rescue efficiency, replication, and matrix localization of recombinant Sendai virus bearing matrix nuclear export mutants.

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posted on 17.03.2015, 05:18 by Mickey Pentecost, Ajay A. Vashisht, Talia Lester, Tim Voros, Shannon M. Beaty, Arnold Park, Yao E. Wang, Tatyana E Yun, Alexander N. Freiberg, James A. Wohlschlegel, Benhur Lee

(A) Comparison of virus-like particle (VLP) budding between 3X-Flag-tagged NiV-M, HeV-M, MuV-M, and SeV-M. Anti-Flag immunoblots of cell lysate and purified VLPs from HEK 293T cells 24 h post transfection with the indicated constructs. Normalized budding index was calculated from immunoblot integrated intensities. (B) Quantification of rescue events (GFP+ cells) from three independent rescues of rSeV-eGFP containing WT, L102 L103 mutant, or K254R mutant SeV-M at day 2 post rescue in HEK 293T cells. (C) Quantification of viral titers from the rescues of rSeV-eGFP containing WT, L102 L103 mutant, or K254R mutant SeV-M at day 6 post rescue in HEK 293T cells. (D) Extended Focus (maximum intensity projection) view of 3D confocal micrographs of HEK 293T cells at day 6 post rescue of rSeV-eGFP containing WT, L102 L103 mutant, or K254R mutant SeV-M. Cells were counterstained with anti-SeV-M antibodies, red. (E) Extended Focus (maximum intensity projection) view of 3D confocal micrographs of HEK 293T cells at day 6 post rescue of rSeV-eGFP containing WT, L102 L103 mutant, or K254R mutant SeV-M. Cells were counterstained with anti-SeV-F antibodies, red. (F) XYZ Planes View of 3D confocal micrographs of HEK 293T cells at day 6 post rescue of rSeV-eGFP containing WT, L102 L103 mutant, or K254R mutant SeV-M. Cells were counterstained with DAPI to visualize nuclei, blue, anti-SeV-M antibodies, red, and anti-Fibrillarin antibodies to visualize nucleoli, grayscale.

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