Construction of a CVS-N2c RABV recombinant for live-cell imaging in PNS neurons.

(A) Genome schematics of the parental strain and P-mCherry-expressing, spread-deficient RABV recombinant. (B) Protein composition of sucrose-purified RABV P-mCherry particles (lanes 1 and 3) using SDS-PAGE and western blotting with anti-RABV P, G, N, or M. N2c mCherry particles (G-complemented N2cΔG expressing diffusible mCherry) are included for comparison (lanes 2 and 4). Asterisk indicates P-mCherry fusion protein. (C) Pie chart shows the proportion of enveloped virions (dual colored; 888) to non-enveloped nucleocapsids (red-only; 426) in the RABV P-mCherry stock (n = 1314 particles total). P-mCherry-positive particles (red) from purified supernatants were stained with anti-RABV G and Alexa Fluor 488-conjugated 2° antibody (green) (scale bars = 10 μm) (see also S1 Fig). (D) Dissociated SCG were infected with RABV P-mCherry (10⁵ ffu) (red) and stained at 48 hpi with FITC-conjugated anti-RABV targeting the N protein (green). DAPI stains nuclei (blue). White arrow indicates a cytoplasmic inclusion body (scale bar = 25μm).