Pseudorabies virus Us9 directs axonal sorting of viral capsids

Abstract

Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.

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, capturing a subpopulation of axon ends. Dissociated SCG neurons are then plated inside the chamber, where they mature and form synaptic connections with axons emanating from the explant. Dissociated SCG neurons inside the chamber are equivalent to one-fourth of an SCG ganglia, which is approximately 5,000 neurons. All chambers were prepared in duplicate. The red arrowhead denotes where the images in panel B were taken in reference to the SCG explant. (B) Explants were infected on the outside of the chamber with wild-type PRV Becker or PRV 160 (Us9-null). At 24 h postinfection, both the explant and the dissociated neurons inside the chamber were fixed and labeled with antibodies that recognize the viral glycoproteins gE and gB, as well as the major capsid protein VP5. Postsynaptic neurons in contact with the Becker-infected SCG explant stained for viral glycoprotein and capsid proteins (top row). No labeling of viral antigen was detected in postsynaptic neurons in contact with the explant infected with PRV 160 (middle row), although the explant itself showed extensive infection (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 with PRV Becker and PRV 160 for 24 h and then fixed and labeled with antibodies that recognize gE, gB, and VP5. A minimum of 20 fields were analyzed at a high magnification (×60). Axons emanating from Becker-infected explants showed strong labeling for both viral capsid and glycoproteins (top row, inset). Explants infected with PRV 160 did not have any labeling above background fluorescence (bottom row). It is noteworthy that there were no observable gradations in staining with the Us9-null mutant; viral proteins were not detected inside the chamber.

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 plated inside the chamber. The arrowhead highlights the region where images were taken at high magnification within the chamber. (B and C) Explants were infected for 24 h with PRV GS443 (GFP-VP26) (B) or PRV 368 (GFP-VP26, Us9 null) (C) and fixed with 4% paraformaldehyde. Direct fluorescence of explants outside the chamber (×20 magnification) and capsids inside the chamber (×60 magnification) was visualized by using spinning-disk confocal microscopy.

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. Samples were examined in duplicate. A 1-mm square with high axonal density inside the chamber was selected for sectioning by the ultramicrotome. Six serial sections (70 nm apart) were scrutinized for the presence of virus particles. (A) Enveloped virus particles were detected in the distal-axon of Becker-infected explants but were not present in explants infected with PRV 160 (B).

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 neurons per virus were examined for the presence of capsid puncta moving in the anterograde direction. In order to quantify the number of puncta undergoing axonal transport in neurons infected with PRV GS443, eight different axon shafts were imaged where capsids could be visualized moving >50 μm without dropping in and out of the plane of focus. An equal number of axons were examined for PRV 368 for the same time period. (A) Neurons were infected with PRV GS443 and imaged with the Leica SP5 confocal microscope between 13 and 14 h postinfection. Blue, red, and yellow arrowheads track the anterograde movement of individual fluorescent puncta through the field of view during a 1-min interval (see Movie S1 in the supplemental material). (B) A neuron infected for 13.5 h with PRV 368 (green capsid, Us9 null) shows robust green fluorescence in the soma (black arrowhead) but no presence of capsids in the axon (enlarged image, see Movie S2 in the supplemental material).

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 are merged overlays of differential interference contrast, GFP, and RFP. Blue, red, and yellow arrowheads track the anterograde movement of individual fluorescent puncta through the field of view for 1.5 min (see Movie S3 in the supplemental material). Red and blue arrowheads highlight individual yellow puncta, whose composition is a mix of GFP-VP26 and RFP-VP26. The yellow arrowhead marks a predominantly red punctum and a predominantly green punctum transported together that eventually separate. (asterisks)