Emetine acts after RABV entry to reduce the velocity and transport distance of virus particles moving retrograde in axons.

(A) Quantification of % infected cell bodies at 24 hpi (+/-) 100 μM emetine added to N either 1 h pre or 1 h post infection. Black dots represent individual tri-chambers (from two independent replicates). Horizontal lines and errors bars represent mean ± SD for each condition with ****p < 0.0001 using one-way ANOVA (ns = not significant). (B) Motile RABV particle tracks (black lines) visualized by maximum intensity projections from each FOV along the M compartment barrier at 2–4 hpi in N (scale bars = 20 μm). (C) For tracks in blue boxes, kymographs show the displacement of particles over time during the 15 sec movie (163 frames). The slope indicates the velocity of particle movement, where a vertical line has a slope of zero and indicates particle stalling. (D) Distribution of RABV particle velocity (μm/sec) across a population of retrograde moving RABV particles in the untreated (red; n = 10300 constant velocity segments from 1116 events) or emetine-pretreated (blue; n = 3363 constant velocity segments from 338 events) condition. Events were pooled from three independent replicates. Y axis indicates the frequency for each velocity on the x-axis. Positive and negative values indicate retrograde and anterograde directed motility, respectively. v = the mean velocity ± standard error of the mean (SEM) (E) Distribution of RABV particle track length for particles moving retrograde in the untreated (red; n = 637 particles) or emetine-pretreated (blue; n = 231 particles) conditions. Y axis indicates the frequency for each track length on the x-axis. x = the mean particle track length (± SEM). Lines on the histograms in (D) and (E) are cubic spline curves. (F) Our suggested model summarizing the effect of emetine on post-entry retrograde transport of RABV virions. RABV particles first attach to the cell surface receptors (step 1) to initiate entry through endocytosis (step 2). Step 1 and 2 induce signaling pathways including JNK, ERK, RhoA, stathmin and NfkB in axons (step 3). RABV-carrying endosomes must recruit dynein motors and adapters (step 4) to facilitate efficient retrograde transport on microtubules (step 5). We propose that emetine does not block step 1 or 2, but it interferes with step 5 by possibly inhibiting step 3 and/or 4.