Molecular Aspects of Varicella-Zoster Virus Latency.

Primary varicella-zoster virus (VZV) infection causes varicella (chickenpox) and the establishment of a lifelong latent infection in ganglionic neurons. VZV reactivates in about one-third of infected individuals to cause herpes zoster, often accompanied by neurological complications. The restricted host range of VZV and, until recently, a lack of suitable in vitro models have seriously hampered molecular studies of VZV latency. Nevertheless, recent technological advances facilitated a series of exciting studies that resulted in the discovery of a VZV latency-associated transcript (VLT) and provide novel insights into our understanding of VZV latency and factors that may initiate reactivation. Deducing the function(s) of VLT and the molecular mechanisms involved should now be considered a priority to improve our understanding of factors that govern VZV latency and reactivation. In this review, we summarize the implications of recent discoveries in the VZV latency field from both a virus and host perspective and provide a roadmap for future studies.

Deletion of a Predicted ß-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons.

UNLABELLED: We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31-68 mutation spans a well-defined ß-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31-68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. IMPORTANCE: Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted ß-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.

Roles of conserved residues within the pre-NH2-terminal domain of herpes simplex virus 1 DNA polymerase in replication and latency in mice.

The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) is essential for viral DNA synthesis and production of infectious virus in cell culture. While mutations that affect 5'-3' polymerase activity have been evaluated in animal models of HSV-1 infection, mutations that affect other functions of HSV-1 Pol have not. In a previous report, we utilized bacterial artificial chromosome technology to generate defined HSV-1 pol mutants with lesions in the previously uncharacterized pre-NH2-terminal domain. We found that the extreme N-terminal 42 residues (deletion mutant polΔN43) were dispensable for replication in cell culture, while residues 44-49 (alanine-substitution mutant polA6) were required for efficient viral DNA synthesis and production of infectious virus. In this study, we sought to address the importance of these conserved elements in viral replication in a mouse corneal infection model. Mutant virus polΔN43 exhibited no meaningful defect in acute or latent infection despite strong conservation of residues 1-42 with HSV-2 Pol. The polA6 mutation caused a modest defect in replication at the site of inoculation, and was severely impaired for ganglionic replication, even at high inocula that permitted efficient corneal replication. Additionally, the polA6 mutation resulted in reduced latency establishment and subsequent reactivation. Moreover, we found that the polA6 replication defect in cultured cells was exacerbated in resting cells as compared to dividing cells. These results reveal an important role for the conserved motif at residues 44-49 of HSV-1 Pol for ganglionic viral replication.

Bovine herpesvirus 1 regulatory proteins bICP0 and VP16 are readily detected in trigeminal ganglionic neurons expressing the glucocorticoid receptor during the early stages of reactivation from latency.

Bovine herpesvirus 1 (BHV-1) establishes a lifelong latent infection in sensory neurons following acute infection. Increased corticosteroid levels, due to stress, increases the incidence of reactivation from latency. Within minutes, corticosteroids activate the glucocorticoid receptor and transcription of promoters containing a glucocorticoid receptor element. A single intravenous injection of the synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. Lytic cycle viral gene expression is detected within 6 h after dexamethasone treatment of calves latently infected with BHV-1. Cellular transcription factors are induced by dexamethasone in trigeminal ganglionic neurons within 1.5 h after dexamethasone treatment, suggesting they promote viral gene expression during the early phases of reactivation from latency, which we operationally defined as the escape from latency. In this study, immunohistochemistry was utilized to examine viral protein expression during the escape from latency. Within 1.5 h after dexamethasone treatment, bICP0 and a late protein (VP16) were consistently detected in a subset of trigeminal ganglionic neurons. Most neurons expressing bICP0 also expressed VP16. Additional studies revealed that neurons expressing the glucocorticoid receptor also expressed bICP0 or VP16 at 1.5 h after dexamethasone treatment. Two other late proteins, glycoprotein C and D, were not detected until 6 h after dexamethasone treatment and were detected in only a few neurons. These studies provide evidence that VP16 and the promiscuous viral trans-activator (bICP0) are expressed during the escape from latency, suggesting they promote the production of infectious virus in a small subset of latently infected neurons.

Role of oxidative stress in rabies virus infection.

Recent studies in an experimental model of rabies indicated that there are major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult mouse dorsal root ganglion (DRG) neurons are a good in vitro model for studying the mechanisms involved in rabies virus-induced degeneration of neurites (axons) because, unlike other neuronal cell types, these neurons are fairly permissive to rabies virus infection. DRG neurons infected with the challenge virus standard-11 (CVS) strain of rabies virus show axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress, and also reduced axonal growth in comparison with mock-infected DRG neurons. Treatment with the antioxidant N-acetyl cysteine prevented the reduction in axonal outgrowth that occurred with CVS infection. The axonal swellings with 4-HNE-labeled puncta were found to be associated with aggregations of actively respiring mitochondria. We postulate that rabies virus infection likely induces mitochondrial dysfunction resulting in oxidative stress and degenerative changes involving neuronal processes. This mitochondrial dysfunction may be the result of either direct or indirect effects of the virus on the mitochondrial electron-transport chain or it may occur through other mechanisms. Further investigations are needed to gain a better understanding of the basic mechanisms involved in the oxidative damage associated with rabies virus infection. This information may prove helpful in the design of future therapeutic effects for this dreaded ancient disease.

A locus on mouse chromosome 6 that determines resistance to herpes simplex virus also influences reactivation, while an unlinked locus augments resistance of female mice.

During studies to determine a role for tumor necrosis factor (TNF) in herpes simplex virus type 1 (HSV-1) infection using TNF receptor null mutant mice, we discovered a genetic locus, closely linked to the TNF p55 receptor (Tnfrsf1a) gene on mouse chromosome 6 (c6), that determines resistance or susceptibility to HSV-1. We named this locus the herpes resistance locus, Hrl, and showed that it also mediates resistance to HSV-2. Hrl has at least two alleles, Hrl(r), expressed by resistant strains like C57BL/6 (B6), and Hrl(s), expressed by susceptible strains like 129S6 (129) and BALB/c. Although Hrl is inherited as an autosomal dominant gene, resistance to HSV-1 is strongly sex biased such that female mice are significantly more resistant than male mice. Analysis of backcrosses between resistant B6 and susceptible 129 mice revealed that a second locus, tentatively named the sex modifier locus, Sml, functions to augment resistance of female mice. Besides determining resistance, Hrl is one of several genes involved in the control of HSV-1 replication in the eye and ganglion. Remarkably, Hrl also affects reactivation of HSV-1, possibly by interaction with some unknown gene(s). We showed that Hrl is distinct from Cmv1, the gene that determines resistance to murine cytomegalovirus, which is encoded in the major NK cell complex just distal of p55 on c6. Hrl has been mapped to a roughly 5-centimorgan interval on c6, and current efforts are focused on obtaining a high-resolution map for Hrl.