Fig 1.
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).
Fig 2.
RABV infects sympathetic neurons in tri-chambers via axonal infection.
(A) Retrograde infection in tri-chambers (S, soma (left); M, methocel (middle); N, neurite (right)). (B) Cell bodies in S at 24 h post RABV P-mCherry infection (105 ffu) in N. White arrow indicates an infected cell body with axonal connection to N. White asterisks indicate etched grooves (scale bar = 500 μm). (C) Quantification of % infected cell bodies at 72 hpi when axons were infected with different numbers of viral particles (100 ffu–106 ffu). (D) Quantification of % infected cell bodies from 20 h to 168 h post axonal infection with 105 ffu. Black dots in (C) and (D) represent individual tri-chambers (from three (C) and two (D) independent replicates). Horizontal lines and errors bars represent mean ± SD for each condition with **p = 0.01, ***p = 0.0006, ****p < 0.0001 using one-way ANOVA (ns = not significant). (E) Anti-mCherry western blot of protein lysate from S compartment cell bodies at 72 h post axonal infection (105 ffu). Arrowhead indicates band for P-mCherry fusion protein (~60 kDa). Asterisk indicates expected size of unfused mCherry protein (~27 kDa), which is not present in infected cells.
Fig 3.
Retrograde RABV infection is unaffected by axonal interferon treatment.
(A) Cell bodies in S at 24 h post RABV P-mCherry infection in N. IFNβ or IFNγ was added to N for 24 h prior to axonal infection (scale bars = 250 μm). (B) Quantification of % infected cell bodies at 24 hpi (+/-) IFNβ or IFNγ in N or S. Black dots represent individual tri-chambers (from three independent replicates). Horizontal lines and errors bars represent mean ± SD for each condition with *p = 0.0114, ****p < 0.0001 using one- way ANOVA (ns = not significant).
Fig 4.
Axonal interferon treatment does not alter retrograde axonal transport of RABV particles.
(A) Live particle tracking setup (see also S3 Fig). IFNβ or IFNγ was added to N for 24 h prior to infection in N. (B) Motile RABV particle tracks (black lines) visualized by maximum intensity projections from each field of view (FOV) along the M compartment barrier at 2–4 hpi in N (scale bars = 20 μm). One FOV is shown per condition. Blue boxes highlight tracks of particles that moved retrograde for the entire duration of the 15 sec movie (163 frames). (C) For tracks in blue boxes, kymographs show the displacement of each particle over time. Diagonal lines indicate particles moving retrograde. (D) Quantification of the number of RABV particles moving retrograde per FOV (+/-) IFNβ or IFNγ pretreatment in N. Each open circle represents an individual FOV. Vertical lines and error bars represent the mean ± SD for each condition (ns = not significant using one-way ANOVA). Total FOVs counted were 308 (no treatment), 297 (IFNβ), and 309 (IFNγ) across 3 independent replicate chambers per condition.
Fig 5.
Axonal emetine treatment blocks retrograde RABV infection by a mechanism that does not depend on protein synthesis inhibition.
(A) Cell bodies in the S compartment at 24 h post RABV P-mCherry infection in N. Emetine (100 μM) was added to N, 1 h prior to axonal infection and washed out at 5 hpi (scale bar = 250 μm). (B) Quantification of % infected cell bodies at 24, 48, and 72 hpi (+/-) 100 μM emetine in N. (C) Quantification of % infected cell bodies at 24 hpi (+/-) 100 μM emetine, 100 μg/ml CHX, or 10 μg/ml puromycin added to N or S, 1 h prior to infection in N. Inhibitors were washed out at 5 hpi. Black dots in (B) and (C) represent individual tri-chambers (from four (B) and three (C) independent replicates). Horizontal lines and errors bars represent mean ± SD for each condition with ****p < 0.0001 using two-way (B) or one-way (C) ANOVA (ns = not significant). (D) Levels of phosphorylated eIF2α (P-eIF2α) in dissociated SCG (+/-) 100 μM emetine, 100 μg/ml CHX or 10 μg/ml puromycin (6 h post treatment).
Fig 6.
Emetine restricts axonal transport of RABV particles but does not limit transport of Rab5- or Rab7-positive vesicles.
(A) 100 μM Emetine was added to N, 1 h prior to RABV P-mCherry infection in N. (B) Quantification of the number of RABV particles moving retrograde per FOV (+/-) emetine pretreatment in N. Open circles represent individual FOVs along the M compartment barrier. Vertical lines and error bars represent the mean ± SD for each condition with ****p < 0.0001 using unpaired t-test. Total FOVs counted were 224 (no treatment) and 166 (emetine) across 3 independent replicate chambers per condition. (C) S compartment cell bodies were transduced with adenoviruses expressing either Venus-Rab5 or Venus-Rab7 for 4 days. (D) Quantification of % moving Rab5 or Rab7 particles per axon (+/-) 100 μM emetine. Each dot represents one axon in the N compartment. A minimum of 22 axons were imaged per condition. Horizontal lines and error bars represent the mean ± SD for each condition (ns = not significant using two-way ANOVA).
Fig 7.
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.