Fig 1.
Characteristics of Neuronal Maturation of the Superior Cervical Ganglia.
A: Maturation of SCG neurons is characterized by building a neuronal-network. Phase contrast images comparing immature and mature SCG neurons. Mature neurons develop dense axon bundles (straight lines) and clusters of soma (dark grey). B: Mass per dissociated SCG at various DIV (days in vitro). Bars represent standard deviation. C: Mature neurons express NaV1.2 Immunofluorescence staining in axon. Axon of an immature (1 DIV) SCG neuron does not localize NaV1.2 compared to mature (20 DIV) axon localizing Nav1.2. Map2 serves as a neuronal marker and Dapi for nucleus. D: SCG maturation is acquired by 5 DIV. At 1.5 DIV, the maturation marker pNF (in red) is restricted to the cell body (round) and absent from the axon. At 5 DIV pNF localization spreads to the proximal axon. At 30 DIV pNF is expressed throughout the whole axon as seen by the colocalization of pNF and Map2. Map2 (in green) is a neuronal marker that does not change with age.
Fig 2.
Axonal sorting of Pseudorabies virus depends on neuronal age.
A: Confocal image of SCG neuron infected with PRV expressing mRFP-VP26 (capsid) and GFP-Us9 at 10 MOI for 12 hours. The number of PRV particles, represented by mRFP-VP26 puncta, that sorted into the proximal 30um of axon (white box) are measured. B: Quantification of particles sorted into immature and mature SCG axons. Bars represent standard deviation. C: Live-microscopy quantification measuring the dynamics of particle sorting. Sorted particles were categorized as moving in the anterograde direction (away from cell-body) or retrograde direction (towards cell body).
Fig 3.
Membrane Trafficking Proteome is Acquired After Maturation.
A: Workflow of the tandem mass tagging (TMT) based quantitative mass-spectrometry experiment. B: Maturity markers, Nav1.2 and pNfH are detected with higher abundances in mature SCG neurons. C: Gene Set Enrichment Analysis of the whole proteome (blue) reveals that immature SCG neurons are enriched (FDR 2.21×10−12) in transcription factors (red), and mature neurons are enriched (FDR 1.38×10−7) in membrane-trafficking associated proteins (yellow). D: Host proteins altered by infection. The heatmap graphs the log2 fold-change of host protein abundance values. Values are normalized to mock signal of the same age. All identified host proteins, that were found to be significantly differential (adjusted P-value ≤0.05) in a background-based ANOVA analysis in at least one comparison, were clustered with k means = 7. Cluster numbers correspond to the following enrichment GO-terms: 1-RNA splicing, 2-Metabolism and axon guidance, 3-Myelin sheath, 4-No enrichments, 5-RNA splicing. E: Viral proteins are more abundant in immature SCG neurons. The heatmap represents TMT reporter ion log2 fold-change values for PRV proteins. PRV proteins are temporally organized as IE (immediate early), E (early) and L (late) expressing.
Fig 4.
Identification of Us9 neuronal protein interaction networks.
A: Workflow describing the experimental setup. The 8 samples include immature and mature SCG neurons that are mock/uninfected or infected for 12 h with PRV 151 (GFP control), PRV 341 (GFP-Us9WT) or PRV 442 (GFP-Us9YY). Samples were lysed in detergent-resistant-membrane (DRM) preserving lysis buffer, followed by co-IP with GFP-conjugated magnetic beads and LC-MS/MS analysis. The resulting dataset was specificity filtered using the SAINT algorithm to identify high confidence interacting proteins. B: Principal Component Analysis (PCA) of the specificity-filtered data revealed clustering driven by neuronal developmental age rather than the virus state of infection. The immature neurons (red) clustered together, and the mature neurons (blue) clustered together. C and D: Volcano-Plot representation of the immature (C) and mature (D) neuronal interactome that is associated with Us9WT (left-half of each plot) or Us9YY (right-half of each plot). Grey dots represent novel interactions and blue dots represent proteins previously reported to interact with Us9. Proteins labeled with gene names are significantly (p-value ≤0.05) differential in relative association between US9WT and US9YY.
Fig 5.
SMPD4 knockdown facilitates PRV Spread.
A: Tri-chamber Anterograde Spread Assay workflow–Dissociated SCG neurons are seeded in the soma-S-compartment (left), growing axons penetrate through the middle-M-compartment into the neurite-N-compartment (right). siRNA are administered in the S-compartment for 3 days, followed by infection in the S-compartment. The spread of virus particles into the N-compartment can be detected by fluorescent expression of GFP-Us9 or mRFP-VP26 in the N-compartment. B: SMPD4 siRNA knockdown. Dissociated SCG neurons were transfected with 50uM of siRNA against SMPD4 (+) or Non-Target controls (-). At 3 days post siRNA transfection (labeled pre-infection), samples were collected and assayed on SDS-PAGE western blot to confirm protein knockdown. After the anterograde sorting assay, Soma form the S-compartment were collected again to measure knockdown for the duration of the assay. Each lane represents a different chamber. C: Robust spread detected after SMPD4 knockdown. At 48 hpi, the N-compartment of chamber treated with siRNA-SMPD4 (left) displayed greater GFP-Us9 (top) and mRFP-VP26 (bottom) signal, in comparison to the si:NonTarget negative-control (right chamber). D: Virus titers after anterograde sorting assay. N-sup represents virus particles that have sorted into the N-compartment and released into the supernatant. N-cells represents particles sorted into the N-compartment but confined inside the axons or PK-15 cells. S-Sup represent particles released into the supernatant of the S-compartment. Titer was measured by counting plaques on a monolayer of PK-15 cells. Statistics were performed using 2way-ANOVA test. E: SMPD4 localization after PRV infection. Confocal microscopy of siRNA transduced SCG cell body infected with virus expressing GFP-Us9WT and mRFP-VP26 capsids (PRV 341). After 12 hpi, cells were fixed for immunofluorescence staining of SMPD4. White arrow indicates foci of mRFP-VP26 and SMPD4 colocalization. Arrowhead indicates foci of mRFP-VP26 and GFP-Us9 colocalization. F: Quantification of mRFP-VP26 capsid distribution. All cytoplasmic mRFP-VP26 capsid foci were quantified for colocalization with GFP-Us9 and/or SMPD4 foci.
Fig 6.
A: Neuronal Maturation is required for efficient and robust anterograde spread of virus. Immature neurons lack the proteome necessary to regulate spread of virus particles, thus both Us9WT and the spread deficient Us9YY PRV particles can sort. Neuronal maturation is accompanied with establishing an Axon and expression of proteins specialized to regulate anterograde spread. B: SMPD4 blocks anterograde spread. Capsids (red) that assemble into membranes containing Us9 (green) can recruit transport machinery, such as the Kif1a microtubule motor, to facilitate anterograde spread along the axon. Capsids that colocalizing with SMPD4 foci (blue) do not assemble into Us9 membranes and thus fail to recruit transport machinery necessary for anterograde spread.