Microarray analysis of RABV-infected neurons isolated by FACS 3 months after infection indicates dysregulation of genes involved in nervous system function and cellular assembly.

<p>Cell suspensions prepared from whole mouse brains 3 months post-infection were sorted on a MoFlo cell sorter for EGFP+ (previously infected) and EGFP- (uninfected) cell populations. The 1248 transcripts differentially expressed between infected and uninfected cells (≥1.5 fold change, p<0.05) were analyzed by Ingenuity Pathway Analysis (IPA) to identify biological functions most significantly affected by the infection (significance predicted by p-value). Shown are the top ten most significant biological systems affected by the gene dysregulation, with the horizontal bars representing the negative log of their p-value (greatest significance at the top). Below each bar is the top three sub-categories affected by gene dysregulation in the respective categories. Each category/sub-category has the number of genes involved (up or down-regulated).</p

Emetine restricts axonal transport of RABV particles but does not limit transport of Rab5- or Rab7-positive vesicles.

<p><b>(A)</b> 100 μM Emetine was added to N, 1 h prior to RABV P-mCherry infection in N. <b>(B)</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. <b>(C)</b> S compartment cell bodies were transduced with adenoviruses expressing either Venus-Rab5 or Venus-Rab7 for 4 days. <b>(D)</b> 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).</p

Axonal emetine treatment blocks retrograde RABV infection by a mechanism that does not depend on protein synthesis inhibition.

<p><b>(A)</b> 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>(B)</b> Quantification of % infected cell bodies at 24, 48, and 72 hpi (+/-) 100 μM emetine in N. <b>(C)</b> 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). <b>(D)</b> 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).</p

EGFP+ neurons are positive for RABV antigen.

<p>Brains were collected from Cre reporter mice fifteen days post-infection, cryosectioned, and EGFP+ regions compared to cell-specific labeling, A) NeuN (blue, neuronal nuclei antibody, 20× fluorescence imaging), B) GFAP (blue, astrocyte antibody, 40× confocal imaging), or C) RABV P antigen (purple) and DAPI nuclear stain (blue, 63× confocal imaging). White arrows in (C) indicate regions positive for RABV P.</p

<i>In vivo</i> analysis of RABV-infected cells using Cre reporter mouse model.

<p>A) Timeline of mouse experiment. Cre reporter mice were infected intranasally (IN) with 10⁵ ffu RABV-Cre and sacrificed at the specified times. B) Weights of infected mice were monitored as a measure of disease throughout the experiment and demonstrate productive infection in all mice within this experiment. C) Brains collected at different time points post-infection were analyzed for the presence of EGFP-expressing cells in the following anatomical regions: olfactory bulb (OB), cerebral cortex (CC), cerebrum (CR), hippocampus (HIP), cerebellum (CB) and midbrain/hindbrain (MB-HB).</p

Retrograde RABV infection is unaffected by axonal interferon treatment.

<p><b>(A)</b> 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>(B)</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).</p

Characterization of Cre-expressing RABV.

<p>A) Genome of rabies virus (RABV) and the recombinant RABV expressing Enterobacteria phage P1 Cre recombinase (RABV-Cre) with a 5′ nuclear localization signal (shown in orange). B) Cre expression was confirmed by western blot analysis. Neuroblastoma cells were infected with either RABV-Cre, RABV expressing HIV-1 Gag (RABV-Gag), or mock infected (uninfected). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blotting with antibodies specific for Cre, RABV P or N, and actin. C) Viral growth kinetics were evaluated by multi-step growth curve assay in which BSR cells were infected at MOI 0.01 with either RABV or RABV-Cre, and viral titers determined from samples taken at the indicated time points post-infection. D) A schematic depicting the Cre-specific expression cassette in the Cre reporter mouse. In the absence of Cre, the chicken β-actin core promoter with a CMV enhancer (CAG) drives constitutive expression of membrane-targeted tandem dimer tomato (tdTomato) expression; EGFP is not expressed. After Cre-mediated excision of the tdTomato gene at the loxP sites, membrane-targeted enhanced green fluorescent protein (EGFP) is expressed. pA denotes polyadenylation sites. E) Cre functionality was evaluated <i>in vitro</i> by infecting primary fibroblasts isolated and cultured from Cre reporter mice for 96 hours with either RABV-Cre or RABV at MOI 20. EGFP and tdTomato labeling were detected by fluorescence microscopy (top) and flow cytometry (bottom) (using FITC and PE channels, respectively).</p

Neurons survive RABV infection and viral clearance.

<p>RNA was isolated from brains of mice at the indicated time points post-infection and assayed by real-time quantitative PCR for RABV genomic RNA (blue circles and line; left y-axis), RABV messenger RNA (black squares and line; left y-axis), and relative EGFP expression (green bars; right y-axis). Gene expression of all targets was normalized to the RPL13A (L13A) housekeeping gene. Data displayed was collected from 3 to 4 mice at each time point.</p

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

<p><b>(A)</b> 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>(B)</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). <b>(C)</b> 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. <b>(D)</b> 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) <b>(E)</b> 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. <b>(F)</b> 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.</p

Dysregulated genes and predicted effect on cell functions using Ingenuity Pathway Analysis.

*<p>Neur: growth of neurites; Cskel: organization of cytoskeleton; Cplsm: organization of cytoplasm; Micr: microtubule dynamics.</p>**<p>Increased: gene expression pattern predicts an increase in the specific function; Decreased: gene expression pattern predicts a decrease in the specific function; Affected: gene is involved in specific function, but unclear how it would influence it.</p