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. 2005 Jul;79(14):9019-25.
doi: 10.1128/JVI.79.14.9019-9025.2005.

Herpes simplex virus type 1 latency-associated transcript expression protects trigeminal ganglion neurons from apoptosis

Francisco J Branco  1 , Nigel W Fraser
Affiliations

Herpes simplex virus type 1 latency-associated transcript expression protects trigeminal ganglion neurons from apoptosis

Francisco J Branco et al. J Virol. 2005 Jul.

Abstract

Upon infection of murine trigeminal ganglia with herpes simplex virus type 1 (HSV-1), an immune response is initiated resulting in significant infiltration of CD8+ T cells. Previous investigators have observed a lack of apoptosis in HSV-1 trigeminal ganglia even in the presence of cytotoxic immune cells. To determine the role of the latency-associated transcript (LAT) in inhibiting apoptosis, we examined mice during acute and latent infection with HSV-1 (strain 17 or a LAT-negative deletion mutant strain 17 N/H) by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and fluorescence-activated cell sorting (FACS). FACS analysis revealed CD8+ T cells in the trigeminal ganglia by day 7, with more being present in 17- than 17 N/H-infected trigeminal ganglia (6.22% versus 3.5%) and a decrease in number through day 30 (2.7% to 1.2%). To detect apoptotic CD8+ T cells, sections were assayed by TUNEL and stained for CD8+ T cells. By day 7, approximately 10% of CD8+ T cells in both 17- and 17 N/H-infected trigeminal ganglia had undergone apoptosis. By day 30, 58% and 74% of all T cells had undergone apoptosis in 17- and 17 N/H-infected trigeminal ganglia, respectively. Furthermore, no HSV strain 17-infected trigeminal ganglion neurons were apoptotic, but 0.087% of 17deltaSty and 0.98% of 17 N/H-infected neurons were apoptotic. We conclude that the antiapoptotic effect of LAT appears to require the LAT promoter, with most of the antiapoptotic effect mapping within the StyI (+447) to the HpaI (+1667) region and a minor contribution from the upstream StyI (+76) to StyI (+447) region.

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Figures

FIG. 1.
FIG. 1.
Map of HSV-1 genome and LAT deletion mutants. (A) Linear map of the HSV-1 genome with its unique long (UL) and unique short (US) regions flanked by inverted repeat (IR) elements. (B) LAT region of the HSV-1 genome. The LAT region is enlarged to show the latency-active promoter (LAP1) and the 2-kb LAT intron. (C) LAT deletion region in 17 N/H. (D) Exon 1 deletion region in 17ΔSty. (E) LAT deletion region in 17Δ A/H.
FIG. 2.
FIG. 2.
Detection of LAT expression and apoptosis in 17+- and 17 N/H-infected trigeminal ganglia. BALB/c mice were infected with 17+ (A, B) and 17 N/H (C, D). At 30 days postinfection, mice were sacrificed, trigeminal ganglia were extracted and fixed, and sections were cut and processed for in situ hybridization for LAT message (A, C) and apoptosis (B, D) using the fluorescein in situ cell death detection kit (Roche). Sections were visualized by light microscopy and fluorescent microscopy using a fluorescein filter.
FIG. 3.
FIG. 3.
Detection of apoptotic neurons in 17 N/H-infected trigeminal ganglia. Trigeminal ganglia were explanted 30 days postinfection from 17 N/H-infected BALB/c mice. Neuronal apoptosis was detected using the fluorescein in situ cell death detection kit (Roche). Sections were visualized by light microscopy (A) and fluorescent microscopy (B).
FIG. 4.
FIG. 4.
Percentage of neurons in 17+-, 17ΔSty-, 17 N/H-, and mock-infected trigeminal ganglia that are apoptotic. 17+-, 17ΔSty-, 17 N/H-, and mock-infected BALB/c mice were sacrificed 30 days postinfection, and trigeminal ganglia were explanted and sectioned. Neurons were identified by morphology, and apoptosis was determined by the presence of fluorescent nuclei using the fluorescein in situ cell death detection kit (Roche). The asterisk indicates statistically significant differences (P < 0.05) between 17 N/H- and 17ΔSty-infected trigeminal ganglia and between mock- and 17+-infected trigeminal ganglia.
FIG. 5.
FIG. 5.
Trigeminal ganglion sections stained with hematoxylin and eosin. Trigeminal ganglia were explanted at day 15 postinfection from mock (A)-, 17+ (B)-, and 17 N/H (C)-infected BALB/c mice.
FIG. 6.
FIG. 6.
Detection of CD8+ T cells in trigeminal ganglia. Trigeminal ganglia of mice infected with 17+ (A, B, C) or 17 N/H (D, E, F) or mock infected (G, H, I) are shown at day 7 (A, D, G), 15 (B, E, H), and 30 (C, F, I). Trigeminal ganglia were sectioned and stained with a monoclonal antibody against the antigen CD8a followed by a fluorescein-conjugated secondary antibody. Sections were visualized by fluorescence microscopy.
FIG. 7.
FIG. 7.
FACS analysis of infiltrating CD8+ T cells. 17+ (A, B, C)- and 17 N/H (D, E, F)-infected BALB/c mice were sacrificed on day 7 (A, D), 15 (B, E), and 30 (C, F) postinfection. Trigeminal ganglia were explanted, dissociated by collagenase, and stained with a PE-conjugated anti-CD8a monoclonal antibody. Cells were analyzed by flow cytometry.
FIG. 8.
FIG. 8.
CD8+ T-cell infiltration into 17+- and 17 N/H-infected trigeminal ganglia. Trigeminal ganglia from 17+ (white bars)- and 17 N/H (black bars)-infected mice were explanted at 7, 15, and 30 days and analyzed by flow cytometry (Fig. 7). Values for the number of CD8+ T cells detected in trigeminal ganglia relative to the total number of cells counted were graphed.
FIG. 9.
FIG. 9.
Localization of CD8+ T cells and apoptosis in 17 N/H-infected trigeminal ganglia. BALB/c mice were infected by corneal scarification with 2.5 × 104 PFU of 17 N/H virus in each eye. At 30 days postinfection, mice were sacrificed and trigeminal ganglia were extracted and sectioned for slides. Slides were stained for CD8a antigen and apoptosis using TUNEL. Sections were viewed by light microscopy (A), UV microscopy using a rhodamine filter to view CD8a staining (B), and UV microscopy using a fluorescein filter to view TUNEL staining (C). (D) Fields B and C were merged for colocalization of CD8 cells and apoptosis.
FIG. 10.
FIG. 10.
FACS analysis of CD8+ T cells and apoptosis. 17+ (A, B, C)- and 17 N/H (D, E, F,)-infected BALB/c mice were sacrificed on day 7 (A, D), 15 (B, E), and 30 (C, F) postinfection. Trigeminal ganglia were explanted, dissociated by collagenase, and stained for CD8+ T cells with a PE-conjugated anti-CD8a monoclonal antibody and TUNEL using fluorescein-conjugated nucleotides. Cells were analyzed by flow cytometry.
FIG. 11.
FIG. 11.
Apoptosis of CD8+ T cells infiltrating trigeminal ganglia. BALB/c mice were infected with 17+ (white bars) and 17 N/H (black bars) by corneal scarification and sacrificed at 30 days postinfection. Explanted trigeminal ganglia were digested with collagenase, and dissociated cells were stained for CD8+ T cells and apoptosis using TUNEL. Cells were analyzed by FACS, and values were graphed.
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