Latent herpes simplex virus infection of sensory neurons alters neuronal gene expression

Abstract

The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. (33)P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities (n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.

FIG. 1.

Scatterplots comparing ³³P signal intensities in microarrays after background subtraction and normalization. Each point represents the log₁₀ phosphorimager intensity value normalized to the median intensity of the microarray for each filter. (A and B) Results from duplicate array hybridizations. The x and y axes each represent one of the two duplicate arrays. HSV, one representative probe generated from pooled ganglia 30 days after infection. Mock, one representative probe generated from pooled ganglia 30 days after mock infection, and from the same experiment as the HSV probe shown. (C) Results for HSV-infected versus mock-infected ganglia. y axis, one of the two duplicate HSV arrays; x axis, one of the duplicate Mock arrays.

FIG. 2.

Neuronal expression of host genes confirmed by in situ hybridization. Mouse trigeminal ganglia latently infected with HSV at 30 days p.i. hybridized with radiolabeled DNA probes for cellular genes. Neurons were identified morphologically by their large size and staining pattern. (A) Muscarinic acetylcholine receptor gene (Chrm1). Grains indicate nuclear and cytoplasmic distribution of the mRNA in neurons (magnification, ×200). (B) Protein tyrosine phosphatase sigma gene (Ptprs). Grains cluster over neurons (magnification, ×400).

FIG. 3.

Quantitative real-time RNA PCR. This example illustrates that the level of Stat1 RNA normalized to Gapdh RNA is higher in latently infected ganglia (HSV) than in mock-infected ganglia (Mock). ΔRn, fluorescence difference from background determined by using Sequence Detection System software (Applied Biosystems). Each assay was performed in triplicate. The relative level of Stat1 was determined by the difference in average cycle number at a constant threshold ΔRn. Gapdh, mouse Gapdh mRNA; Stat1, mouse Stat1 mRNA. View is expanded around exponential phase.

FIG. 4.

Kinetic expression profiles of selected genes whose up-regulation at day 30 p.i. correlated significantly with HSV latency. HSV/Mock, ratios of mean normalized signal intensities in HSV-infected samples to those in mock-infected samples.. Each bar for day 3 or 10 p.i. represents an average ratio from one experiment, and each bar for day 30 p.i. represents average ratios from all three experiments.