Neuron-to-cell spread of pseudorabies virus in a compartmented neuronal culture system

Abstract

Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.

FIG. 1.

Trichamber neuron culture system. The trichamber system consists of a Teflon ring outlined with a thin strip of silicone grease and seated inside a 35-mm tissue culture dish (a). The solid arrowheads indicate the central barriers where the axons have to penetrate underneath. The S (Soma)-compartment contains neuron cell bodies which have been cultured for 2 weeks (b) while the M (Methocel)- and N (Neurite)-compartments have an extensive network of axons (c and d). The solid arrows indicate the parallel grooves etched on the surface of the tissue culture dish (c). Note that the neurites grow between the grooves and not in the grooves.

FIG. 2.

Neurons infected with PRV Becker do not retract their neurites early during infection. Cultured neurons in the S-compartment were mock infected (a) and infected with PRV Becker (b) at very high MOI for 10 h. At each hour, phase contrast images were taken using a Nikon Eclipse TE epifluorescence microscope to chart the growth of several randomly selected axons from the N-compartment. Images were processed and axons traced using Scion Image software.

FIG. 3.

Infectious particles are not released from axon terminals late during infection. Cultured neurons in the S-compartment were infected with PRV Becker at high MOI. The medium in the N-compartment as well as the total content of the S-compartment were harvested at various time points postinfection (0, 12, 24, and 36 hours postinfection) and titered on PK15 cells. Five chambers were used for each time point. The standard deviations are: S-compartment (12 h, ±2.0 × 10⁵; 24 h, ±2.2 × 10⁵; 36 h, ±1.5 10⁵); N-compartment (12 h, ±1.6 × 10³). The hollow circle beside each data set represents the average value for that particular set of data.

FIG. 4.

Neuron-to-cell spread of infection is not cell type specific. PK15 cells, Madin-Darby bovine kidney cells, pig embryonic fibroblasts, and rat embryonic fibroblasts were plated in the N-compartment. Neurons in the S-compartment were then infected with PRV Becker at high MOI for 48 h. Infected cells were then harvested from both the S- and N-compartments and titered on PK15 cells. Six chambers were used for each type of infection. The standard deviations are: N-compartment (PK15 cells, ±1.6 × 10⁸, MDBK cells, ±6.6 × 10⁵, pig embryonic fibroblasts, ±2.8 × 10⁶, rat embryonic fibroblasts, ±2.4 × 10⁶); S-compartment (PK15 cells, ±8.3 × 10⁵, MDBK cells, ±8.2 × 10⁵, pig embryonic fibroblasts, ±2.7 × 10⁵, rat embryonic fibroblasts, ±7.8 × 10⁵). The open circle beside each data set represents the average value for that particular set of data.

FIG. 5.

Neuron-to-cell spread of infection requires axons. A: Neurons were infected with PRV 180 and either left untreated as a control or axotomized to remove all axons in the M-compartment. Infected cells were harvested at 24 h postinfection from S- and N-compartments and titered on PK15 cells. Five chambers were used for each type of infection and treatment. The open circle beside each data set represents the average value for that particular set of data. The standard deviations are: N-compartment (not axotomized, ±4.1 × 10⁷); S-compartment (not axotomized, ±3.8 × 10⁴, axotomized, ±2.6 × 10⁴). B: Immunofluorescence experiment on PRV Becker-infected neurons in the trichamber system. Culture samples in the S- (a to c), M- (d to f), and N-compartments (g to i) were labeled with antibodies against VP5 (a, d, and g), DiI (b, e, and h) and the nuclear dye Hoechst shown in the merged image (c, f, and i). Scale bar: 20 μm.

FIG. 6.

Neuron-to-cell transmission of infection does not require gD. A: Neurons in the S-compartment were infected at high MOI with either PRV Becker or GS442, a complemented gD null virus that expresses GFP. At 24 h postinfection, infected cells in the S- and N-compartments were harvested and titered in PK15 cells. Five chambers were used in each type of infection. The open circle beside each data set represents the average value for that particular set of data. The standard deviations are: N-compartment (Becker, ±1.0 × 10⁷); S-compartment (Becker, ±1.6 × 10⁵; GS442, ±5.9 × 10¹). B: Immunofluorescence experiment on PRV Becker- and GS442-infected neurons in the trichamber system. Culture samples in the S- (a to c), M- (d to f), and N-comparments (g to i) were labeled with antibodies against GFP (a, d, and g), DiI (b, e, and h), and the nuclear dye Hoechst shown in the merged image (c, f, and i). Scale bar: 20 μm.

FIG. 7.

PRV Bartha is defective in neuron-to-cell spread of infection. A: Neurons in the S-compartment were infected at high MOI with either PRV Becker or PRV Bartha. At 24 h postinfection, infected cells in the S- and N-compartments were harvested and titered in PK15 cells. Five chambers were used in each type of infection. The open circle beside each data set represents the average value for that particular set of data. The standard deviations are: N-compartment (Becker, ±3.4 × 10⁸); S-compartment (Becker, ±1.5 × 10⁵, Bartha, ±5.9 × 10⁴). B: Immunofluorescence experiment on PRV Becker and PRV Bartha infected neurons in the trichamber system. PK15 cells and axons in the N-compartment of PRV Becker-infected cells (a to d) and PRV Bartha-infected cells (e to h) were labeled with antibodies against VP5 (b and f) and the lipid dye DiI (a and e). The cells were stained with the nuclear dye Hoechst and are shown in the merged image (c and g). Images at the focal plane of PK15 cells clearly show clusters of PRV Becker infected cells (d to h). Scale bar: 20 μm.

FIG. 8.

gE, gI and Us9 are kinetically delayed during neuron-to-cell transmission of infection. A: Neurons in the S-compartment were infected at high MOI with PRV Becker, PRV 758, PRV 98 and PRV 160. At 24 h postinfection, infected cells in the S- and N-compartments were harvested and titered in PK15 cells. Seven chambers in two separate experiments were used in each type of infection. The standard deviations are: N-compartment (Becker, ±3.5 × 10⁷, 758, ±2.1 × 10⁵, 160, ±9.0 × 10³, 98, ±1.2 × 10⁶); S-compartment (Becker, ±2.3 × 10⁷, 758, ±2.7 × 10⁵, 160, ±1.2 × 10⁴, 98, ±1.3 × 10⁶). B: Neurons in the S-compartment were infected at high MOI with PRV Becker and PRV 758. At 0, 12, 24, and 36 h postinfection, infected cells in the N-compartment were harvested and titered in PK15 cells. Three chambers were used for each time point. The standard deviations are: N-compartment; Becker (12 h, ±5.0 × 10⁴, 24 h, ±9.1 × 10⁶, 36 h, ±9.3 × 10⁷,); 758 (24 h, ±3.2 × 10⁴, 36 h, ±7.1 × 10⁶). C: Neurons in the S-compartment were infected at high MOI with PRV Becker, PRV Bartha, PRV BaBe, or PRV 158. At 24 h postinfection, infected cells in the S- and N-compartments were harvested and titered in PK15 cells. Six chambers were used in each type of infection. The standard deviations are: N-compartment (Becker, ±1.4 × 10⁷; 158, ±9.5 × 10⁶); S-compartment (Becker, ±5.6 × 10⁵, Bartha, ±5.8 × 10⁵; 158, ±2.4 × 10⁵; BaBe, ±8.0 × 10⁵). The open circles beside each data set represent the average value for that particular set of data.

FIG.9.

Electron micrographs of distal axons show that viral capsids are enclosed in vesicles. Electron micrographs were obtained from infected cell bodies in the S-compartment (a, b, c, and d) and axons in the M-compartment (e, f, g, and h). The areas enclosed by the dotted boxes (a, c, e, and g) are enlarged and shown either to the right of the original micrograph (b and d) or as an inset (f and h). The micrographs taken from the S-compartment were either from the cell bodies (a and b) or from the proximal segment of the axons (c and d). Scale bars shown are either 500 nm (a and c) or 100 nm (b, d, e, f, g, and h).

FIG. 10.

PRV Bartha but not gE, gI, or Us9 null single mutants have a slight defect in axon-mediated infection of neurons. A: High-titer viral inoculum from PRV Becker and PRV Bartha were incubated in the N-compartment for 1 hour before being replaced with regular medium. At 24 h postinfection, infected neuron cell bodies in the S-compartment were harvested, lysed, and titered on PK15 cells. A total of five chambers were used for each type of infection. The standard deviations are: S-compartment (Becker, ±1.2 × 10⁵, Bartha, ±2.1 × 10³). B: Immunofluorescence experiment on axon mediated infection of neurons by PRV Becker. Confocal microscopy images show the S- (a to c), M- (d to f), and N-compartments (g to i) being labeled with either antibodies against VP5 (a, d, and g), or DiI (b, e, and h). Merged images are shown in panel B (c, f, and i). Scale bar: 20 μm. C: High-titer viral inocula from PRV Becker, PRV 758, PRV 98, and PRV 160 were incubated in the N-compartment for 1 hour before being replaced with regular medium. At 24 h postinfection, infected neuron cell bodies in the S-compartment were harvested, lysed, and titered on PK15 cells. A total of five chambers were used for each type of infection. The standard deviations are: S-compartment (Becker, ±5.9 × 10⁵; 758, ±9.1 × 10⁵; 160, ±5.7 × 10⁵; 98, ±6.7 × 10⁵). The open circle beside each data set shown in the scatter plots represents the average value for that particular set of data.