Axonal transport and sorting of herpes simplex virus components in a mature mouse visual system

Abstract

The time course for delivery and transport of two major proteins of herpes simplex virus (HSV) has been determined for mature mouse retinal ganglion cell axons in vivo. Twenty-four hours after intravitreal injection of HSV, valacyclovir was introduced into the drinking water of the mice to inhibit subsequent viral replication. Without treatment, viral spread and replication in periaxonal glial cells confound study of axonal transport. At 2 to 5 days after infection, the animals were sacrificed and contiguous segments of the optic pathway were removed. Immunofluorescence microscopy indicated that the number of infected astrocytes was reduced in the proximal optic nerve and eliminated in the optic tract. Western blots of the retina with antibodies for envelope and capsid components, glycoprotein D (gD) and VP5, respectively, revealed that both components were expressed in retinal homogenates by 2 days. Results of reverse transcription-PCR indicated that there was no gD mRNA present in the treated optic tract 5 days after infection. Therefore, we conclude that gD is transcribed from viral mRNA in the retinal ganglion cell bodies. The gD accumulated in the proximal ganglion cell axon by 2 days and reached the most distal segment after 3 days. The VP5 first appeared in the proximal axons at 4 days, about 48 h after the appearance of gD. Thus, gD entered the axon earlier and independent of VP5. These finding confirm the subassembly model of viral transport in neurons and suggest that there is a 4- to 5-day window for initiation of effective antiviral treatment with valacyclovir.

FIG. 1.

Diagram of the path of retinal ganglion cell axons. A single retinal ganglion cell has an axon (indicated in white) about 15 mm long extending from the neuron cell body in the retina through the optic nerve head (ONH), optic nerve (ON1 [green] and ON2 [yellow]), optic chiasm (OC [orange]), and optic tract (OT [red]) toward the thalamus, where it synapses with neurons in the lateral geniculate nucleus (LG).

FIG. 2.

(A) EM immunostained section of retinal ganglion cell in the ganglion cell layer (GCL) 24 h after infection. The cell contains many immunoreactive viral capsids and virions (arrows). The inner limiting membrane (asterisk) underlies the ganglion cell bodies. Bar, 0.45 μm. Inset, light micrograph of the region of the GCL that was sampled. The thin section has not been stained with heavy metals. INL, inner nuclear layer; ONL, outer nuclear layer. (B) Western blots of retinas at 2, 3, 4, and 5 days after infection with equivalent titers of the F strain of HSV. The F strain expresses both gD and VP5 at each time point. Lane (−), retinal homogenates from an uninfected animal; lane (+), viral stock aliquots. Molecular markers for the VP5 gels are 176 and 113 kDa, and those for the gD gels are 64 and 49 kDa.

FIG. 3.

Five days after intraocular infection, we found HSV immunostained astroglial cells (asterisks) in the optic nerve in the ON1 segment of the animal that was not treated with valacyclovir (− Val) after infection (A and B). In contrast, there were relatively few HSV-positive glial cells (asterisk) in sections from the animal treated with the antiviral drug (+ Val) (D and E). Rare HSV-positive astroglial cells were identified in sections of the optic tracts from the animals that received no valacyclovir (C). In contrast, no HSV-positive glial cells were found in the optic tracts of animals that received valacyclovir 24 h after infection (F). For panels A, C, D, and F, sections were immunostained with a polyclonal antiserum to HSV. For panels B and E, sections were immunostained with a marker for astrocytes, GFAP. Magnification, ×400.

FIG. 4.

(A) EM of infected astrocytes in ON1 3 days after intraocular infection. Without valacyclovir treatment, there is significant secondary infection of glial cells. Unenveloped capsids are found in the nucleus (lower inset), and enveloped virions (upper inset) are found in the cytoplasm of astrocytes in the ON1 segment. a, axon; n, nucleus. Bar, 2 μm. (B) Western blots of immunoprecipitated VP5 in the segments of the optic pathways of untreated (− Val) and valacyclovir-treated (+ Val) animals infected intraocularly with HSV 5 days before sacrifice. OC, optic chiasm; OT, optic tract. (C) Densitometric scans of the gels in panel B. The percentage of the total density is expressed per lane. There is significantly more capsid protein in the ON1 and ON2 segments of the pathway in the untreated animals. By contrast, there are more similar amounts of VP5 in all segments of the treated animals.

FIG. 5.

(A) The viral envelope glycoprotein is delivered to the axon 2 days before the capsid protein is recognized in the axon. Western blots of optic pathways of animals at 2, 3, 4, and 5 days after intravitreal infection with HSV followed 24 h later with valacyclovir treatment are shown. The gD (arrows) reached the ON1 and ON2 segments in the first 48 h after infection. By 24 h later, it was recognizable in all four segments. The VP5 (arrows) was first identified at 4 days after infection and had reached the optic tract (OT) by 5 days. Lanes (−), optic pathway homogenates from an uninfected animal; lanes (+), viral stock aliquots. The markers are 50 kDa for gD gels and 182 and 114 kDa for VP5 gels. OC, optic chiasm. (B) At 5 days after intraocular injection of HSV, the mRNA for gD is present in infected retinas and optic tracts of animals not treated with valacyclovir (HSV+, Val−). No PCR products for gD were seen in the tissues taken from infected animals that received valacyclovir (HSV+, Val+). Retinas and optic tracts of animals with no infection were also negative (HSV−, Val−). The internal control, 15S RNA, was detected in all samples (15S primers). No bands were seen in control RT-PCRs for 15S PCR product when reverse transcriptase was omitted (RT control). HSV, infected animals; Val, animals treated with valacyclovir. Re, retina; OT, optic tract. On the left are markers for 250 bp (gD) and 360 bp (15S).