Expression of Varicella-Zoster Virus Immediate-Early Regulatory Protein IE63 in Neurons of Latently Infected Human Sensory Ganglia

Abstract

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediateearly 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.Varicella-zoster virus (VZV), the human alphaherpesvirus that causes varicella during primary infection, establishes a lifelong latency in neurons of the sensory ganglia along the cerebrospinal axis (7). Sensory ganglia are composed of heterogeneous populations of neurons surrounded by satellite cells and other nonneuronal cells; axons extend from these neurons to innervate the skin and mucous membranes (50). Herpes zoster (shingles) results from reactivation of latent virus within ganglion cells and transfer of newly synthesized infectious particles to the skin via axonal transport (7).The number and type of neural cells that harbor latent virus are fundamental to the question of VZV latency. Studies to address this question have used methods to detect viral DNA, RNA, and proteins in cadaver ganglia. Although RNA is especially vulnerable to RNA-degrading enzymes during the postmortem interval between death and fixation of histological specimens at autopsy, DNA and proteins are reasonably stable (16). Nevertheless, reports using either VZV DNA or protein detection to assess the numbers of cells that contain VZV in human cadaver ganglia have yielded estimates that are extremely variable. By in situ hybridization and PCR methods, VZV DNA has been reported in as few as 1.5% of neurons exclusively (none in satellite cells) to as many as 30% of ganglion cells (neurons as well as satellite cells) (24, 26). Most recently,...

Varicella-Zoster Virus-Derived Major Histocompatibility Complex Class I-Restricted Peptide Affinity Is a Determining Factor in the HLA Risk Profile for the Development of Postherpetic Neuralgia

“…tested and in VZV IE63 for HLA-A*02. These proteins are expressed during the early stage of viral reactivation, and there is evidence to support that these proteins may also be expressed during latency (35,41). One possible hypothesis is that there is strong evolutionary pressure to introduce mutations in these proteins that remove potential high-affinity peptides.…”

“…Similar results were found for a larger selection of HLA alleles, other affinity cutoffs, and other affinity prediction methods (see Tables S2 to S5 in the supplemental material). Interestingly, VZV IE63, another VZV immediate early protein that is found in ganglia and is implicated in VZV reactivation (35,36), also shows depletion for several HLA alleles, including the abundant HLA-A*02 allele. Indeed, together with VZV IE62, VZV IE63 has the lowest affinity for HLA-A*02.…”

A comparison of herpes simplex virus type 1 and varicella-zoster virus latency and reactivation

“…VZV IE63-encoded protein has been detected in ganglionic neurons removed at autopsy, although this is not a common event (Mahalingam et al, 1996;Kennedy et al, 2000;Zerboni et al, 2010). Whether IE63 and other VZV proteins are expressed in human ganglia is an active area of investigation.…”

Effect of Time Delay after Necropsy on Analysis of Simian Varicella-Zoster Virus Expression in Latently Infected Ganglia of Rhesus Macaques

“…To test the possibility that our detection of SVV ORF 63 in latently infected monkey ganglia was due to neuromelanin, which can confound immunohistochemical detection of VZV proteins in human ganglia (9), we stained sections of lumbar ganglia from monkey GI39 (which contained SVV ORF 63 protein) with Nile blue solution as described previously (9). Nile blue is known to bind to neuromelanin in tissue sections, resulting in a dark green stain that is resistant to acetone extraction, whereas binding to cell nuclei and lipofuscin produces blue staining.…”