Anterograde transport of herpes simplex virus proteins in axons of peripheral human fetal neurons: an immunoelectron microscopy study

Abstract

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.

FIG. 1

Diagram of the experimental DRG-EC model. Axons usually reach the ECs by 7 to 10 days of culture, at which time virus is selectively inoculated into the inner chamber. Reprinted from reference with permission from the Royal Microscopical Society.

FIG. 2

SIEM of virus particles present on the surface of MRC5 cells and on neurons in the positive control of the model. The inset shows a low-magnification view of immunolabelled virions on the surface of MRC-5 cells 36 h postinfection Bar, 100 nm. The 30-nm gold particles (arrows) label gC, and the 10-nm particles (arrowheads) label VP5. Similarly immunolabelled HSV on the surface of axons in positive controls of the DRG-EC model fixed 1 h after addition of HSV is shown in the main panel. No similar virus particles were seen on the surface of axons in the OC of the infected experimental model.

FIG. 3

TIEM of infected MRC-5 cells showing the labelling pattern for VP5, VP16, and glycoprotein, 24 h postinfection. (A) Examples of virus particles singly labeled for VP5. (B) Single labelling for VP16. (C) Single labelling for gB (10-nm particles). Note the more diffuse reticular labelling for glycoprotein over vesicular membranes (arrows) as well as virus particles. Bar, 100 nm. (D) Double labelling for VP5 (10 nm) and VP16 (5 nm). Bar, 100 nm. (Inset) High-magnification view of VP5 and VP16 double labelling of a virus with clear definition of ultrastructural morphology. Bar, 100 nm.

FIG. 4

TEM of axons in the infected (D to F) or noninfected (A to C) model at 24 h postinfection. The morphology of virus in tangential sections and membranous vesicles can overlap, making distinction difficult. (A to C) Membranous vesicles in a noninfected axon (arrows). (D) Both a membranous vesicle (arrow) and a nucleocapsid (arrowhead). (E and F) Viral nucleocapsids in the infected model (arrowheads). Note the close association of capsid with microtubules in panel F. Bar, 100 nm.

FIG. 5

SIEM of the mid-region of axons in the OC of the model, fixed 24 h after virus was inoculated into the IC. (A) Colocalization of 10-nm immunogold labelling VP5 and 20-nm immunogold labelling VP16. The 10- and 20-nm labels are intermingled within 150 nm of each other. Bar, 100 nm. (B) Dually labelled axons for VP5 and gC, but showing only the 30-nm immunolabel for gC since there was no colocalization with the 10-nm immunogold label for VP5. In fact, immunolabels for VP5 and glycoproteins could not usually be observed in the same high-power field. Note the small diameter of some axon fascicles. Bar, 100 nm.

FIG. 6

TEM of axons. Longitudinal (A) and transverse (B) sections of axons in OC are shown. Microtubules are easily visible in the longitudinal sections and in some axons in the transverse sections. Bar, 200 nm.

FIG. 7

TIEM of axons in the outside chamber of the model fixed 24 h after HSV infection of the inner chamber. Bars, 100 nm. (A) Colocalization of gold particles labelling VP5 (10 nm) and VP16 (5 nm) in a cluster. (B and C) Spatial separation of collections of immunolabel for VP5 (10 nm; arrowheads) and glycoprotein C (5 nm; arrows) in a cross section (B) and longitudinal section (C) of axons. (D) Similar pattern to panels B and C with double labelling for VP5 (10 nm) and glycoprotein B (5 nm); the immunolabel for VP5 was arranged around a dense structure, and that for gB was arranged around microvesicles. Identical findings were obtained with immunolabelling for gD. (E) Immunolabel for gB 5-nm gold particles was arrayed around the walls of 60- to 200-mm-diameter vesicles. The inset shows a higher-magnification view of glycoprotein immunolabelling of an axonal vesicle. Bar, 100 nm. (F) Distinct example of a nucleocapsid with immunolabel (10 nm) for VP16. Although this section was also immunolabelled for glycoprotein, none is present near this capsid. Ultrathin sections immediately before and after this section showed only indistinct virus structures.