Fig 1.
Serotype-dependent reovirus infection in vivo is reproduced in primary neuron cultures.
Cultured rat cortical neurons (CNs) or dorsal root ganglion neurons (DRGNs) were adsorbed at an MOI of 500 or 50 PFU per cell, respectively, with the reovirus strains shown, and cells were fixed at 24 h post-adsorption. (A, C) Neurons were immunostained using an antibody to detect a neuronal protein (MAP2) and reovirus-specific antiserum. Nuclei were stained with DAPI (blue). Representative micrographs display infected CNs (A) or DRGNs (C). Scale bars, 50 μm. (B, D) Infectivity of CNs (B) or DRGNs (D) was scored as the mean number of cells stained by reovirus antiserum per field-of-view. Bars indicate means from at least three independent experiments, each with duplicate or triplicate samples, normalized to T3SA+ infectivity. For reference, ~ 26% of CNs are infected by T3SA+ reovirus under the experimental conditions used.) Error bars indicate SEM. Individual data points are normalized averages from 8 to 12 fields-of-view from each sample. Values that differ significantly from T3SA+ by ANOVA and Dunnett's test are indicated (****, P < 0.0001).
Fig 2.
Anterograde spread of reovirus is limited in DRGNs.
(A) Schematic of a microfluidic device with two compartments connected by 450-μm-long microgrooves. (B) L929 cells were cultivated in both compartments (red and blue) of the microfluidic device. Cells in the left compartment were adsorbed with T3SA+ virions at an MOI of 10 PFU/cell, and viral titers in culture supernatants from both inoculated and opposing compartments were determined at the indicated times post-adsorption. Bars indicate mean titers of duplicate devices from one representative experiment. Error bars indicate SEM. (C) Representative micrographs show somal and axonal compartments of a microfluidic device with DRGNs cultivated for 7 days and stained with markers for nuclei (DAPI) and axons (non-phosphorylated neurofilament H, NF). (D-E) DRGNs cultivated in microfluidic devices were adsorbed with T3SA+ virions in the somal or axonal compartment as indicated in the schematics (D, top). L929 cells were cultivated in the axonal compartment to amplify virus released by anterograde spread following inoculation of the somal compartment. Viral titers in the culture supernatant in the compartment opposing the inoculated compartment at the indicated times post-adsorption are shown (D). Bars indicate means, and error bars indicate SEM. Individual data points represent titers from five devices in total from three independent experiments. Devices were fixed at 72 h post-adsorption and immunostained with reovirus antiserum and a neuronal marker (NF). Representative micrographs of somal and axonal compartments corresponding to the experimental setup in (D) are shown in (E). Scale bars, 50 μm. In B and D, dashed lines mark the limit of detection.
Fig 3.
Reovirus undergoes microtubule-dependent fast axonal transport in neurons.
(A) CNs were treated with the drugs shown, adsorbed with T3SA+ virions, and viral puncta transported per field-of-view were enumerated following treatment. Bars indicate mean values from at least two independent experiments, each with one to two samples, normalized to untreated control. Error bars indicate SEM. Individual data points are averages from 8 to 12 fields-of-view from each sample. Values that differ significantly from control by ANOVA and Dunnett's test are indicated (****, P < 0.0001). (B-E) CNs or DRGs were adsorbed with fluorescently-labeled T1L, T3SA+, or T3SA- virions for 30 min. The inoculum was removed, and the motion of virions was recorded using live-cell fluorescence imaging. (B) Time-lapse images of representative fields are shown for T3SA+ with positions of individual puncta tracked over time with discrete arrows or arrowheads. (C) Kymographs corresponding to B show the positions (x) of individual puncta along an axon over time (t). Scale bars, 5 μm. (D) Track speed was calculated for the reovirus strains shown as the overall distance traveled by each fluorescent reovirus puncta during the tracked interval (n > 300 tracks). MF refers to neurons cultured in microfluidic devices. Bars indicate medians, and error bars indicate 95% confidence intervals. (E) Fluorescent puncta transported for distances greater than 5 μm in each 82 x 82 μm field-of-view during 1 min of live-cell imaging were enumerated. Bars indicate means normalized to T3SA+, and error bars indicate SEM. Individual data points are averages from 8 to 12 fields-of-view for each sample. Values that differ significantly from T3SA+ by ANOVA and Dunnett's test are indicated (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).
Fig 4.
Multiple reovirus particles are transported together in axons.
(A) CNs were adsorbed with Alexa Fluor 647-labeled T3SA+ for 45 min. The distribution of intensities of individual transported puncta and a multi-peak Gaussian fit of intensities are shown (n = 265 puncta). (B-C) Cortical neurons were adsorbed with an equal number of Alexa Fluor 488- (green) and Alexa Fluor 647-labeled (red) T3SA+ virions. Puncta transported in axons that contain virions labeled with one (green or red) or both (yellow) fluorophores are indicated in representative time-lapse images and the corresponding kymograph (B) and the percentage of each is quantified (C). Scale bar, 5 μm. Bars indicate mean values. Error bars indicate SEM. Data are representative of three independent experiments each with duplicate samples and a total of 1072 puncta. Individual data points are averages from 8 to 12 fields-of-view from each sample.
Fig 5.
Clathrin-mediated endocytosis is not required for reovirus entry into neurons.
(A) CNs were treated with PitStop2 at he concentrations shown, and the uptake of transferrin-Alexa Fluor 488 was quantified following treatment. Data are presented in a box plot with horizontal bars marking 25th and 75th percentiles and median values, and the whiskers marking extreme values. Individual data points represent means normalized to DMSO-treated controls from three independent experiments, each with duplicate samples. Values that differ significantly from control by ANOVA and Dunnett's test are indicated (***, P < 0.001; ****, P < 0.0001). (B) Representative micrographs show reduced uptake of transferrin following treatment with 25 μm PitStop2 compared with a DMSO-treated control. Scale bar, 10 μm. (C) CNs or DRGNs were treated with 25 μm PitStop2 (Table 1), and T3SA+ puncta transported per field-of-view were enumerated following treatment. Bars indicate means of four samples normalized to DMSO-treated control. Error bars indicate SEM. Individual data points are averages from 8 to 12 fields-of-view per sample. Values did not differ significantly from control as determined by t-test (P > 0.05). (D-E) CNs were incubated with fluorescently-labeled transferrin or dextran (10 kDa) and T3SA+ virions. The percentage of reovirus puncta that co-traffic with transferrin or dextran is shown (D) along with representative kymographs (E). **, P < 0.01 determined by t-test. Bars indicate means of duplicate samples from two independent experiments. Error bars indicate SEM. Individual data points represent averages from 8 to 12 fields-of-view from each sample (n = 184 and 335 reovirus puncta analyzed for co-transport with transferrin and dextran, respectively). Scale bar, 5 μm.
Fig 6.
Macropinocytosis-mediated entry is required for reovirus neuronal transport and infection.
(A) Schematic shows host factors mediating endocytic pathways and corresponding small molecule inhibitors (red) (RTK, receptor tyrosine kinase). (B, D) CNs and DRGNs (B) or CNs (D) were treated with the drugs shown (Table 1), adsorbed with T3SA+ virions, and viral puncta transported per field-of-view were enumerated following treatment. (C) CNs were treated with the drugs shown (Table 1), adsorbed with T3SA+ virions or ISVPs at an MOI of 50,000 particles/cell, and infectivity was quantified 24 h post-adsorption. In B-D, bars indicate means normalized to untreated controls. Error bars indicate SEM. Data are representative of at least two independent experiments each with one to three samples. Individual data points are averages from 8 to 12 fields-of-view per sample. Values that differ significantly from control by ANOVA and Dunnett's test are indicated (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 7.
Disassembly of reovirus occurs in neuronal soma following axonal transport.
(A-B) DRGNs cultivated in microfluidic devices were adsorbed in the axonal compartment with fluorescently-labeled T3SA+ virions (green) for 1 h. LysoTracker (red) was added to both compartments during the last 20 min of virus adsorption, and cells were imaged at the times shown after virion addition. Representative images show LysoTracker and reovirus distribution in a DRG soma (A—left, micrograph) and motion in axons within a microgroove (A—right, kymograph). Scale bars, 5 μm. The percentage of reovirus puncta co-transported with LysoTracker in axons within the microgrooves or colocalized with LysoTracker in the soma were quantified (B). Bars indicate means from at least two independent experiments with a total of four devices. Error bars indicate SEM. Individual data points are averages from at least 16 microgrooves containing axons or 8 to 12 fields-of-view containing soma from each device. (C) CNs were treated with E64 (Table 1), and T3SA+ puncta transported per field-of-view were enumerated following treatment. (D) CNs were treated with E64, adsorbed with T3SA+ virions or ISVPs at an MOI of 50,000 particles/cell, and infectivity was quantified at 24 h post-adsorption. In C and D, bars indicate means normalized to untreated controls. Error bars indicate SEM. Data are representative of two independent experiments, each with duplicate samples. Individual data points are averages from 8 to 12 fields-of-view from each sample. Values that differ significantly from control by t-test are indicated (****, P < 0.0001). (E-F) DRGNs were adsorbed with fluorescently-labeled T3SA+ virions (red) for 30 min and incubated at 37°C for 1–3 h before fixation and staining with anti-core polyclonal serum (green). Representative fluorescence micrographs (E) and quantification of the number of reovirus core puncta per soma (F) are shown. Scale bar, 5 μm. Bars indicate means from two samples each with > 23 soma. Error bars indicate SEM. (G) Representative micrographs show distribution of T3SA+ replication factories stained by σNS antiserum and axons stained by an antibody against non-phosphorylated neurofilament H (NF). Scale bar, 10 μm.
Fig 8.
Schematic of reovirus entry and transport in neurons.
Reovirus entry in neurons is mediated by macropinocytosis and requires actin, dynamin, PI(3)K, and receptor tyrosine kinase (RTK). Following entry, multiple intact virions are transported together in non-acidified vesicles by dynein motors along microtubule tracks in the retrograde direction. Vesicle acidification in the soma triggers proteolytic disassembly of virions, which enables release of cores into the cytoplasm. Transcription of viral RNAs from cores leads to the establishment of viral replication factories or inclusions.
Table 1.
Treatment of neurons with small molecule inhibitors.