Characterization of the behavior of chimeras between GFP and N in RABV infected cells.

A-C) BSR-T7/5 cells were infected at an MOI of 0.5 by RABV (CVS strain) and transfected 1h post adsorption with pTit plasmids encoding the indicated N constructs. Cells were fixed 14 h post transfection and the localization of viral proteins and ectopically expressed N mutants was assessed by fluorescence confocal microscopy. A) N protein (either virally encoded or in the GFP-N chimera) was revealed with a mouse monoclonal anti-N antibody followed by incubation with Alexa-488 goat anti-mouse IgG. Scale bars: 10 μm and 2μm for the magnified image. B) Localization of viral P protein (revealed with a rabbit polyclonal anti-P antibody followed by incubation with Alexa-568 donkey anti-rabbit IgG) and GFP-N chimeras in RABV infected cells. Scale bar: 10μm. C) Effect of GFP-N and N-106GFP107 expression on RABV cell infection. P was revealed with a rabbit polyclonal anti-P antibody followed by incubation with Alexa-568 donkey anti-rabbit IgG. Asterisks indicate cells that are infected and express GFP-N. Note that GFP-N localization is diffuse in the cells that are not infected. Infected cells expressing N-106GFP107 are rare and absent in the displayed field. Scale bar: 10μm. D) Percentages of infected, transfected, as well as both infected and transfected cells (for GFP-N and N-106GFP107 chimeras) were assessed from experiments such as those presented in Fig 2C. Values are the average of 3 independent experiments ± the standard deviation. The total number of cells analyzed was 1555 (353 + 407 + 795) for GFP N and 1804 (241 + 615 + 948) for N-106GFP107. p-values were calculated using two-tailed Student’s t test. E) NBs containing P-mCherry, GFP-N or N-112GFP113 constructs were photobleached and the recovery of fluorescence was imaged with a spinning disk microscope. Whole NBs as well as NBs’ subvolumes were photobleached. Scale bars: 5 μm. For the plots on the right, FRAP data (obtained after whole NB photobleaching) were corrected for background, normalized to the minimum and maximum intensity. The mean is shown with error bars representing the SD.