Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons
- Address correspondence to Lynn W. Enquist, lenquist{at}princeton.edu.
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Editor Terence Dermody, Vanderbilt University Medical Center
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FIG 1Construction and characterization of the GFP-Us9 fusion. (A) Schematic representations of Us9-GFP and GFP-Us9 enriched in lipid raft microdomains in relation to intracellular membranes. (B) Schematic design of the compartmentalized chamber SCG neuronal culture. Dissociated SCG neurons are seeded into the S compartment. Neurons are cultured for 3 weeks and extend neurites into the N compartment. PK15 cells are seeded into the N compartment 1 day prior to S compartment infection. All cells are harvested by scraping prior to viral titer determination. (C) Viral titers of wild-type (Wt) Us9 (PRV 328), Us9-GFP (PRV 164), and GFP-Us9 (PRV 340) in the S and N compartments of compartmentalized neuronal cultures were determined 24 h postinfection of the S compartment. Titers represent two biological replicates with three chambers for each virus. Median values are plotted as solid lines. (D) Single-step growth curves for wild-type Us9-expressing (PRV 328) or GFP-Us9-expressing (PRV 340) virus on PK15 cells were determined. Cells and medium were harvested at 0, 3, 6, 12, and 24 h after infection at an MOI of 10 PFU per cell.
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FIG 2Subcellular localization of GFP-Us9 in PRV-infected SCG neurons. (A) An SCG cell body infected with PRV 341 for 18 h. Differential interference contrast and GFP, mRFP, and merged GFP-mRFP fluorescence images are shown. Inset of a region of neuronal cytoplasm (white box in merged image) containing VP26-mRFP and GFP-Us9 puncta. Scale bar, 2 µm. (B) Representative frames from Movie S1 in the supplemental material with GFP-Us9, mRFP-capsid (VP26-mRFP), and an overlay of the two fluorescence channels are displayed as indicated. PRV 341 was used to infect dissociated SCG neurons. Axons at sites distal from the cell body were imaged by epifluorescence microscopy at 8.5 h postinfection. Scale bars, 5 µm. (C) Quantitation of fluorescent puncta from multiple movies with isolated axons. Initially, a total of 253 anterograde-directed fluorescent puncta were analyzed for mRFP or GFP fluorescence or if both fluorophores were present on the same migrating structure. Another 258 anterograde-moving, VP26-mRFP-positive structures were analyzed for detectable GFP content.
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FIG 3Us9 function is necessary for anterograde transport of capsids. (A) Analysis of anterograde transport capacity of PRV strains expressing mutated forms of Us9 or GFP-Us9. Viral titers of wild-type (Wt) Us9 (PRV 328), the Us9 Y49-50A mutant (PRV 172), GFP-Us9 (PRV 340), and the GFP-Us9 Y49-50A mutant (PRV 440) in the S and N compartments of compartmentalized neuronal cultures were determined at 24 h postinfection of the S compartment. (B) Representative images of SCG axons infected with either GFP-Us9/VP26-mRFP (PRV 341) or GFP-Us9 Y49-50A/VP26-mRFP (PRV 442) and fixed with 4% paraformaldehyde at 8 h postinfection. Closed arrowheads indicate doubly positive structures, whereas open arrowheads indicate singly positive structures. Scale bars, 5 µm. (C) Quantitation of dually and singly labeled VP26-mRFP puncta in SCG axons infected with PRV 341 or PRV 442. Quantitation was performed on 5 cells per infection and covered the initial 150 to 225 nm of the SCG axon. The numbers of doubly positive (yellow) and singly positive (red) VP26-mRFP puncta are expressed as percentages of the total structures counted across all of the images.
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FIG 4GFP-Us9 and gM-mCherry are cotransported in axonal anterograde-directed structures. (A) Representative images from Movie S3 in the supplemental material with GFP-Us9, gM-mCherry, and an overlay of the two fluorescence channels displayed as indicated. PRV 348 was used to infect dissociated SCG cultures. At 6 To 8 h postinfection, distal axon sites were imaged by epifluorescence microscopy. Scale bars, 5 µm. (B) Quantitation of eight movies was performed where anterograde-directed viral structures were scored for visible labeling with GFP-Us9, gM-mCherry, or both. A total of 214 anterograde-directed punctate structures were counted, the majority of which were colabeled with visible amounts of GFP-Us9 and gM-mCherry.
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FIG 5Schematic model of Us9 incorporation into viral particles. Three classes of viral particles are potentially labeled with GFP-Us9 as they undergo anterograde-directed transport within the axon: virions containing capsid assemblies, viral membrane proteins, and GFP-Us9. Subvirion assemblies that lack capsids but contain tegument proteins known as light particles would also associate with GFP-Us9. Finally, intracellular membranes containing viral membrane proteins (membrane protein vesicles) that would topologically resemble secretory vesicles would also be marked by GFP-Us9.
- Copyright © 2012 Taylor et al.
