We have studied extensively the directional, transneuronal spread of PRV in the rat visual system (6
). In these studies, virus is injected into the vitreous humor of the rat eye where it infects retinal ganglion cells. Infection then spreads in an anterograde fashion (presynaptic to postsynaptic neurons) to all regions of the brain that receive retinal input. Immunohistochemical staining of viral antigens is then performed on sliced, fixed tissue from infected animals, typically a tedious process. Feierbach et al. recently reported the use of a facile in vitro chamber system that recapitulates the transneuronal spread phenotypes of several PRV mutants (15
). Ganglion explants are plated and allowed to extend axons for 1 week. A nonseptated, Teflon chamber disk is placed on top of the axons thereby capturing a subpopulation of axon ends. Dissociated neurons are then plated inside the chamber ring and allowed to form connections with the explant axon termini. Thus, an isolated population of both presynaptic and postsynaptic neurons can be established.
We have reported that deleting Us9 precludes anterograde, transneuronal spread through anterograde circuits in vivo, while trafficking through retrograde circuits is unaffected (3
). To test whether a Us9-null mutant was unable to spread from primary to second-order neurons using the in vitro chamber system, we infected the SCG explant on the outside of the chamber either with wild-type PRV Becker or with Us9-null mutants. After 24 h, the dissociated SCG neurons inside the chambers were examined by indirect IF (illustrated in Fig. ). After infection with PRV Becker, >70% of second-order neurons inside the chamber reacted readily with antiserum specific for viral glycoproteins gE and gB, as well as the major capsid protein VP5 (Fig. , top row). This was determined by calculating the number of immunopositive cell bodies as a ratio to the total number of cell bodies in a field of view (n
= 20). In contrast, when explants were infected with the Us9-null virus, dissociated neurons inside the chamber ring showed no reactivity with gE, gB, or VP5 (Fig. , middle row). However, Us9-null mutants were capable of efficient infection of explant neurons as determined by strong reactivity with antiserum against gE, gB, and VP5 (Fig. , bottom row). We did note a uniform, nonspecific “speckle” pattern in some samples labeled with the anti-gE antibody. This pattern was present in mock-infected cells and corresponded to areas of high cell density (e.g., near the SCG explant). However, it was not a confounding factor in the interpretation of our results. Overall, these findings are consistent with the inability of Us9-null infections to spread through anterograde circuitry of the rat visual system (3
FIG. 1. Us9 is essential for transneuronal spread in vitro. (A) Diagram illustrating the isolator chamber system in which one-half of an SCG explant is plated on Aclar and allowed to extend neurites. A Teflon chamber ring is then placed on top of preformed axons, (more ...)
Tomishima and Enquist speculated that the Us9 transneuronal spread phenotype reflected the lack of sorting of viral glycoproteins into the axon of infected neurons. However, they reported that in Us9-null infections, capsids, and tegument proteins entered the axon unimpeded (28
). To confirm the finding in our in vitro chamber system, we imaged the axon shafts emanating from the SCG explant physically isolated inside the chamber ring (Fig. , arrowhead). Axons originating from Becker-infected explants showed strong reaction with antisera specific for gE, gB, and VP5 (Fig. , top row). Some puncta labeled with VP5 antibody also costained with gE and gB antisera, suggesting that capsids were transported together with viral glycoproteins (Fig. , inset). In contrast, axons originating from explants infected with the Us9-null mutant did not react with antisera against gE or gB, which is consistent with previous observations (28
). Surprisingly, we did not detect any reaction with VP5 antibodies, suggesting that sorting of viral capsids into axons is, in fact, dependent on Us9 (Fig. , bottom row).
FIG. 2. Axonal sorting of viral capsid and membrane proteins is dependent on Us9. All samples were prepared and imaged in duplicate. (A) Presynaptic axon shafts were imaged directly inside the chamber ring (denoted by the red arrowhead). (B) Explants were infected (more ...)
To confirm and extend these unexpected findings, we first infected SCG explants on the outside of the chamber with PRV GS443, a recombinant PRV strain that expresses GFP fused to VP26, a capsid protein (25
). This fusion protein efficiently assembles into capsids that can be visualized as uniform green puncta in live imaging and fixed preparation studies. We and others have used PRV GS443 for assessing capsid trafficking kinetics in axons (25
), virus particle composition during anterograde and retrograde transport (1
), and actin/assembly body formation in the nucleus of infected neurons (16
). After 24 h, capsid puncta had traveled from the explant and were readily detected in axons inside the chamber (Fig. ). Importantly, when explants were infected with PRV 368, a GFP-VP26-expressing mutant with Us9 deleted, no green puncta were detected inside the chamber, although an extensive network of axons was visible (Fig. ). This was determined by analyzing multiple fields of view (n
= 20) of duplicate samples at high magnification.
FIG. 3. GFP-tagged capsids do not enter the axon in the absence of Us9. (A) A chamber ring was placed on top of preformed axons emanating from the SCG explant to physically separate the site of infection from the site of imaging. No dissociated SCG neurons were (more ...)
We next used transmission electron microscopy to determine whether capsids were present in the distal axons of infected explants. Explants on the outside of the chamber were infected with PRV Becker or a Us9-null mutant and then imaged directly inside the chamber ring 24 h postinfection. Capsids were detected readily in the distal axons of explants infected with PRV Becker. All were found within a membranous vesicle and contained a proteinaceous tegument layer juxtaposed to the capsid (Fig. ). This morphology is consistent with other ultrastructural studies examining PRV infection of primary neurons and tissue culture cells (15
). Importantly, when explants were infected with the Us9-null mutant, no capsid structures could be detected inside the chamber ring despite an extensive search (Fig. ).
FIG. 4. Axons are devoid of enveloped virus particles during a Us9-null infection. Explants on the outside of the chamber were infected for 24 h with PRV Becker or PRV 160 (Us9-null), and axons inside the chamber were visualized by transmission electron microscopy. (more ...)
We next used a live-cell imaging approach in order to quantify the number of capsids moving in the axons of dissociated SCG neurons (as deduced by GFP-VP26 puncta visible in the confocal microscope). This technique enabled us to distinguish GFP-VP26 puncta moving by fast-axonal transport in the anterograde or retrograde direction from those that were not moving or from background fluorescence. Dissociated neurons were infected with PRV GS443 or PRV 368 and imaged between 13 and 14 h postinfection. A total of 128 GFP-VP26 puncta were observed moving in the anterograde direction in PRV GS443-infected neurons, entering the field of view by, on average, 4.6 ± 1.6 capsids/min (Fig. ; see also Movie S1 in the supplemental material). Despite extensive analyses, no capsid puncta were observed in the axon of neurons infected with PRV 368, although GFP-VP26 signal was clearly visible in the soma (Fig. ; see also Movie S2 in the supplemental material). To test whether the axonal sorting defect in PRV 368 was due to the lack of Us9 protein, we coinfected neurons with PRV 368 and PRV 180 (a Becker recombinant expressing mRFP-VP26) (11
). We could easily detect green, red, and yellow puncta moving in the anterograde direction in coinfected neurons (Fig. and see also Movie S3 in the supplemental material). We conclude that PRV 368-infected neurons can sort and move GFP-VP26 capsid puncta in their axons when complemented in trans
by a Us9-expressing virus recombinant.
FIG. 5. Live-cell imaging of GFP-tagged capsid viruses. Dissociated SCG neurons were plated on glass-bottom MatTek dishes and allowed to differentiate for 2 weeks. Images are merged overlays of differential interference contrast and GFP. A minimum of 30 infected (more ...)
FIG. 6. Complementation of the Us9-null axonal sorting defect with PRV 180. Dissociated SCG neurons were coinfected with PRV 368 (GFP-VP26, Us9 null) and PRV 180 (RFP-VP26) and imaged on the Leica SP5 confocal microscope between 13 and 14 h postinfection. Images (more ...)