Anterograde spread of herpes simplex virus type 1 requires glycoprotein E and glycoprotein I but not Us9

Abstract

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.

FIG. 1.

Model systems to study HSV-1 neuronal spread. (A) Mouse flank model. Virus was scratch inoculated onto the skin, where it replicates, spreads to innervating neurons, and travels in a retrograde direction to the neuron cell body in the DRG. After replicating in the DRG, the virus travels in an anterograde direction back to the skin and into the dorsal horn of the spinal cord. Motor neurons also innervate the skin, allowing virus to reach the ventral horn of the spinal cord by retrograde transport. (B) Mouse retina model. Virus is injected into the vitreous body, from which it infects the retina as well as other structures of the eye, including the ciliary body, iris, and skeletal muscles of the orbit. From the retina, the virus is transported into the optic nerve and optic tract (OT) (anterograde direction) and then to the brain along visual pathways. Anterograde spread is detected in the lateral geniculate nucleus (LGN) and superior colliculus (SC). From the infected ciliary body, iris, and skeletal muscle, the virus spreads in a retrograde direction along motor and parasympathetic neurons and is detected in the oculomotor and Edinger-Westphal nuclei (OMN/EWN). Only first-order sites of spread to the brain are indicated. (Brain images were modified and reproduced from reference with permission from of the publisher. Copyright Elsevier 1992.) (C) Campenot chamber system. Campenot chambers consist of a Teflon ring that divides the culture into three separate compartments. Neurons are seeded into the S chamber and extend their axons into the M and N chambers. Vero cells are seeded into the N chamber 1 day before infection. Virus is added to the S chamber and detected in the N chamber, a measure of anterograde spread.

FIG. 2.

Characterization of mutant viruses. (A) Vero cells were infected with HSV-1 NS or NS-Us9null at an MOI of 5. The titers of cells and media were determined together on Vero cells. Results from one experiment are shown. (B) Dissociated SCG neurons were infected with 2.5 × 10⁵ PFU HSV-1 NS or NS-Us9null. Results shown are the means ± standard errors of two experiments. (C) Dissociated SCG neurons were infected with 2.5 × 10⁵ PFU HSV-1 gInull-R or gInull. Results shown are the means ± standard errors of four experiments. (D) Vero cells were infected with HSV-1, and extracts were separated by SDS-PAGE and then probed by Western blotting with antibodies against VP5 (αVP5), gE (αgE), gI (αgI), Us9 (αUs9), or actin (αactin). (E) Extracts from Vero cells infected with NS, NS-Us9null, Us9-, or Us9R were immunoprecipitated with anti-gI MAb Fd69. The extracts (Ext) and immunoprecipitates (IP) were probed by Western blotting with the indicated antibodies.

FIG. 3.

Anterograde spread in Campenot chambers. A total of 1 × 10⁵ PFU WT, mutant, or rescue strain HSV-1 was added to the S chamber. The titers of the contents of the S (A and B) and N (C and D) chambers were determined on Vero cells 24 (A and C) or 48 (B and D) hpi. Results shown are means ± standard errors of six chambers per virus strain at each time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 4.

Anterograde transport into the optic nerve. Mice were infected in the vitreous body with WT, mutant, or rescue strain HSV-1, and tissues were harvested 5 dpi. Sections were stained for immunofluorescence with anti-HSV-1 (red), the neuronal marker anti-Thy1 (green), and DAPI (blue). (A) Ganglion cell neurons form the innermost layer of the retina, which is shown in green at the top of each image. Magnification, ×200. (B) The retina is shown on the left side of each image, while the optic nerve is shown on the right, at the site of exit from the retina. Magnification, ×100.

FIG. 5.

Spread to the brain. Mice were infected in the vitreous body with WT, mutant, or rescue strain HSV-1, and tissues were harvested 5 dpi. (A) Anterograde transport to the optic tract (black arrows) and multisynaptic retrograde spread to the suprachiasmatic nucleus (white arrows). (B) Anterograde transport to the optic tract (black arrows) and anterograde spread to the lateral geniculate nucleus (black arrowheads). (C) Anterograde spread to the superior colliculus (black arrows) and retrograde spread to the oculomotor and Edinger-Westphal nuclei (white arrows). Magnification, ×20.

FIG. 6.

Glycoprotein, tegument, and capsid antigens are transported into the optic tract. Mice were infected in the vitreous body with 4 × 10⁴ PFU HSV-1 NS, NS-Us9null, Us9-, or Us9R, and tissues were harvested 5 dpi. Optic tracts ipsilateral and contralateral to the injected eye are shown at ×200 magnification. Antigen staining in the contralateral optic tract is indicated with black arrows. Brains were stained with the following antibodies against glycoprotein, tegument, or capsid: R122 anti-gD (NS, NS-Us9null) or R118 anti-gC (Us9-, Us9R) (A); PSU74 anti-VP22 (B); and NC-L anti-capsid (C). The antibodies against VP22 and light capsid produced higher background staining levels than did the antibodies against gD and gC.

FIG. 7.

Zosteriform disease in the mouse flank model. Mice were scratch inoculated with 5 × 10⁵ PFU NS, NS-Us9null, or Us9-. (A) The percentage of mice surviving was recorded for 15 dpi. For comparisons of NS to NS-Us9null or Us9-, P < 0.01; for comparisons of Us9- to NS-Us9null, P > 0.05. (B) The severity of zosteriform disease was measured 3 to 11 dpi. For comparisons of NS to NS-Us9null or Us9-, P < 0.001; for comparisons of Us9- to NS-Us9null, P > 0.05. For NS and NS-Us9null, n = 13; for Us9-, n = 4. (C) Zosteriform disease was photographed 7 dpi. (D) Zosteriform lesions from NS- or NS-Us9null-infected mice were stained with anti-HSV-1 and counterstained with hematoxylin. Viral antigen staining is indicated by the arrows. (E) Viral titers in zosteriform lesions 6 dpi. **, P < 0.01 (n = 9). (F) Viral titers in DRG 5 dpi. ***, P < 0.001 (n = 5).

FIG. 8.

Viral spread to the spinal cord. Mice were flank inoculated with 4 × 10⁵ PFU NS or NS-Us9null. Spinal cords were dissected 5 (A) or 3 (B) dpi. Sections were stained with anti-HSV-1 and counterstained with cresyl violet. The boxed areas at ×40 magnification are shown at ×200 magnification below. Black arrows indicate viral antigen staining at 3 dpi.