Autophagy Quantification and STAT3 Expression in a Human Skin Organ Culture Model for Innate Immunity to Herpes Zoster.

The goal of this project was to document the autophagy response in human neonatal skin organ culture (SOC) after infection with varicella-zoster virus (VZV). The VZV-infected SOC model has attributes of herpes zoster, in that an injection of virus into the skin is analogous to exit of virus from the sensory nerve termini into skin during herpes zoster. Cultures were maintained for 28 days and periodically examined for an autophagy response by quantitation of autophagosomes with Imaris software. Expression of the STAT3 protein was plentiful in the VZV-infected SOC. Abundant autophagy was observed in VZV-infected SOC between 14 and 28 days after infection, while autophagy in mock-infected SOC was minimal (p = 0.0003). The autophagic response after infection of SOC with a recombinant VZV genome containing the herpes simplex virus ICP34.5 neurovirulence gene was similar to wild-type VZV (p = 0.3). These results suggested that the VZV-infected SOC system resembled biopsy data from herpes zoster infection of skin. An enhanced autophagy response has now been reported after infection with two additional alpha herpesviruses besides VZV, namely, pseudorabies virus and duck enteritis herpes virus; both lack the ICP34.5 protein.

Cellular Stress Response to Varicella-Zoster Virus Infection of Human Skin Includes Highly Elevated Interleukin-6 Expression.

BACKGROUND: The infectious cycle of varicella-zoster virus (VZV) after reactivation from the dorsal root ganglia includes replication and assembly of complete enveloped virions in the human skin to cause the characteristic herpes zoster (shingles). METHODS: To pursue studies of innate immunity to VZV infection, we have adapted a fetal skin organ culture model to a human neonatal foreskin explant model. RESULTS: Abundant expression of VZV IE62, gE, and gC was visualized by confocal microscopy while numerous enveloped virions were observed by electron microscopy in infected skin organ cultures. Microarray experiments demonstrated that the patterns of upregulated transcripts differed between VZV-infected cells and VZV-infected skin explants. One result stood out, namely a >30-fold elevated interleukin (IL)-6 level in the infected skin explant that was not present in the infected monolayer culture. The IL-6 results in the polyermase chain reaction (PCR) assay were reproduced by quantitative PCR testing with newly designed primers. To determine if increased transcription was accompanied by increased IL-6 expression, we quantitated the levels of IL-6 protein in the explant media at increasing intervals after infection. We found a statistically significant increase in IL-6 protein levels secreted into the media from VZV-infected skin explants as compared with mock-infected explants. CONCLUSIONS: The cellular stress response to VZV infection in neonatal skin explants included highly elevated levels of IL-6 transcription and expression. This skin organ model could be adapted to other viruses with a skin tropism, such as herpes simplex virus.

Identification of Herpes Zoster-Associated Temporal Arteritis Among Cases of Giant Cell Arteritis.

PURPOSE: To examine whether herpes zoster antigen (also called varicella-zoster virus antigen) was detectable in temporal artery biopsies taken from individuals with giant cell arteritis (GCA). DESIGN: Retrospective comparative case series. METHODS: Sections of formalin-fixed paraffin-embedded temporal arteries were examined first by hematoxylin-eosin (H&E) staining to establish the diagnosis of GCA. Adjacent sections of the same biopsy were then examined by immunohistochemistry, using 2 different monoclonal antibodies against a major antigen of varicella-zoster virus called gE. Pathologic specimens were obtained from patients cared for at the University of Iowa and Washington University in St. Louis ophthalmology clinics. RESULTS: The study included biopsies from 25 patients with symptoms of GCA as well as positive H&E pathology and 25 patients with symptoms compatible with GCA but negative H&E pathology. Among the GCA-positive group, 3 patients had positive staining for herpes zoster antigen. Among the GCA-negative group, herpes zoster antigen was not detected in any biopsy. In both groups of patients, false-positive staining for herpes zoster antigen was detected in the presence of calcifications in the arteries. False-positive staining was also detected on some extra-arterial skeletal muscle and erythrocytes. CONCLUSION: Herpes zoster antigen was detected in 3 of 25 temporal arteries from patients with biopsy-proven GCA. One of the 3 positive cases was noteworthy because the patient had had herpes zoster ophthalmicus diagnosed 3 weeks before the onset of GCA symptoms. False-positive staining for herpes zoster antigen was detected on several temporal artery biopsies.

Varicella-Zoster Virus Infectious Cycle: ER Stress, Autophagic Flux, and Amphisome-Mediated Trafficking.

Varicella-zoster virus (VZV) induces abundant autophagy. Of the nine human herpesviruses, the VZV genome is the smallest (~124 kbp), lacking any known inhibitors of autophagy, such as the herpes simplex virus ICP34.5 neurovirulence gene. Therefore, this review assesses the evidence for VZV-induced cellular stress, endoplasmic-reticulum-associated degradation (ERAD), and autophagic flux during the VZV infectious cycle. Even though VZV is difficult to propagate in cell culture, the biosynthesis of the both N- and O-linked viral glycoproteins was found to be abundant. In turn, this biosynthesis provided evidence of endoplasmic reticulum (ER) stress, including a greatly enlarged ER and a greatly diminished production of cellular glycoproteins. Other signs of ER stress following VZV infection included detection of the alternatively spliced higher-molecular-weight form of XBP1 as well as CHOP. VZV infection in cultured cells leads to abundant autophagosome production, as was visualized by the detection of the microtubule-associated protein 1 light chain 3-II (LC3-II). The degree of autophagy induced by VZV infection is comparable to that induced in uninfected cells by serum starvation. The inhibition of autophagic flux by chemicals such as 3-methyladenine or ATG5 siRNA, followed by diminished virus spread and titers, has been observed. Since the latter observation pointed to the virus assembly/trafficking compartments, we purified VZ virions by ultracentrifugation and examined the virion fraction for components of the autophagy pathway. We detected LC3-II protein (an autophagy marker) as well as Rab11 protein, a component of the endosomal pathway. We also observed that the virion-containing vesicles were single-walled; thus, they are not autophagosomes. These results suggested that some VZ virions after secondary envelopment were transported to the outer cell membrane in a vesicle derived from both the autophagy and endosomal pathways, such as an amphisome. Thus, these results demonstrate that herpesvirus trafficking pathways can converge with the autophagy pathway.

Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.

UNLABELLED: Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE: VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

Defensive Perimeter in the Central Nervous System: Predominance of Astrocytes and Astrogliosis during Recovery from Varicella-Zoster Virus Encephalitis.

UNLABELLED: Varicella-zoster virus (VZV) is a highly neurotropic virus that can cause infections in both the peripheral nervous system and the central nervous system. Several studies of VZV reactivation in the peripheral nervous system (herpes zoster) have been published, while exceedingly few investigations have been carried out in a human brain. Notably, there is no animal model for VZV infection of the central nervous system. In this report, we characterized the cellular environment in the temporal lobe of a human subject who recovered from focal VZV encephalitis. The approach included not only VZV DNA/RNA analyses but also a delineation of infected cell types (neurons, microglia, oligodendrocytes, and astrocytes). The average VZV genome copy number per cell was 5. Several VZV regulatory and structural gene transcripts and products were detected. When colocalization studies were performed to determine which cell types harbored the viral proteins, the majority of infected cells were astrocytes, including aggregates of astrocytes. Evidence of syncytium formation within the aggregates included the continuity of cytoplasm positive for the VZV glycoprotein H (gH) fusion-complex protein within a cellular profile with as many as 80 distinct nuclei. As with other causes of brain injury, these results suggested that astrocytes likely formed a defensive perimeter around foci of VZV infection (astrogliosis). Because of the rarity of brain samples from living humans with VZV encephalitis, we compared our VZV results with those found in a rat encephalitis model following infection with the closely related pseudorabies virus and observed similar perimeters of gliosis. IMPORTANCE: Investigations of VZV-infected human brain from living immunocompetent human subjects are exceedingly rare. Therefore, much of our knowledge of VZV neuropathogenesis is gained from studies of VZV-infected brains obtained at autopsy from immunocompromised patients. These are not optimal samples with which to investigate a response by a human host to VZV infection. In this report, we examined both flash-frozen and paraffin-embedded formalin-fixed brain tissue of an otherwise healthy young male with focal VZV encephalitis, most likely acquired from VZV reactivation in the trigeminal ganglion. Of note, the cellular response to VZV infection mimicked the response to other causes of trauma to the brain, namely, an ingress of astrocytes and astrogliosis around an infectious focus. Many of the astrocytes themselves were infected; astrocytes aggregated in clusters. We postulate that astrogliosis represents a successful defense mechanism by an immunocompetent human host to eliminate VZV reactivation within neurons.

The pros and cons of autophagic flux among herpesviruses.

Autophagy has been intensively studied in herpes simplex virus type 1 (HSV-1), a human alphaherpesvirus. The HSV-1 genome encodes a well-known neurovirulence protein called ICP34.5. When the gene encoding this protein is deleted from the genome, the virus is markedly less virulent when injected into the brains of animal models. Subsequent characterization of ICP34.5 established that the neurovirulence protein interacts with BECN1, thereby inhibiting autophagy and facilitating viral replication in the brain. However, an ortholog of the ICP34.5 gene is lacking in the genomes of other closely related alphaherpesviruses, such as varicella-zoster virus (VZV). Further, autophagosomes are easily identified in the exanthem (rash) that is the hallmark of both VZV diseases-varicella and herpes zoster. Inhibition of autophagy leads to diminished VZV titers. Finally, no block is detected in studies of autophagic flux following VZV infection. Thus autophagy appears to be proviral during VZV infection while antiviral during HSV-1 infection. Because divergence to this degree is extremely unusual for 2 closely related herpesviruses, we postulate that VZV has accommodated its infectious cycle to benefit from autophagic flux, whereas HSV-1 has captured cellular immunomodulatory genes to inhibit autophagy.

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection.

Autophagy is a process by which misfolded and damaged proteins are sequestered into autophagosomes, before degradation in and recycling from lysosomes. We have extensively studied the role of autophagy in varicella-zoster virus (VZV) infection, and have observed that vesicular cells are filled with >100 autophagosomes that are easily detectable after immunolabeling for the LC3 protein. To confirm our hypothesis that increased autophagosome formation was not secondary to a block, we examined all conditions of VZV infection as well as carrying out two assessments of autophagic flux. We first investigated autophagy in human skin xenografts in the severe combined immunodeficiency (SCID) mouse model of VZV pathogenesis, and observed that autophagosomes were abundant in infected human skin tissues. We next investigated autophagy following infection with sonically prepared cell-free virus in cultured cells. Under these conditions, autophagy was detected in a majority of infected cells, but was much less than that seen after an infected-cell inoculum. In other words, inoculation with lower-titered cell-free virus did not reflect the level of stress to the VZV-infected cell that was seen after inoculation of human skin in the SCID mouse model or monolayers with higher-titered infected cells. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.

Focal encephalitis following varicella-zoster virus reactivation without rash in a healthy immunized young adult.

Herein we describe an episode of focal varicella-zoster virus (VZV) encephalitis in a healthy young man with neither rash nor radicular pain. The symptoms began with headaches and seizures, after which magnetic resonance imaging detected a single hyperintense lesion in the left temporal lobe. Because of the provisional diagnosis of a brain tumor, the lesion was excised and submitted for pathological examination. No tumor was found. But the tissue immunostained positively for VZV antigens, and wild-type VZV sequences were detected. In short, this case represents VZV reactivation, most likely in the trigeminal ganglion, in the absence of clinical herpes zoster.

Autophagy and the effects of its inhibition on varicella-zoster virus glycoprotein biosynthesis and infectivity.

Autophagy and the effects of its inhibition or induction were investigated during the entire infectious cycle of varicella-zoster virus (VZV), a human herpesvirus. As a baseline, we first enumerated the number of autophagosomes per cell after VZV infection compared with the number after induction of autophagy following serum starvation or treatment with tunicamycin or trehalose. Punctum induction by VZV was similar in degree to punctum induction by trehalose in uninfected cells. Treatment of infected cells with the autophagy inhibitor 3-methyladenine (3-MA) markedly reduced the viral titer, as determined by assays measuring both cell-free virus and infectious foci (P < 0.0001). We next examined a virion-enriched band purified by density gradient sedimentation and observed that treatment with 3-MA decreased the amount of VZV gE, while treatment with trehalose increased the amount of gE in the same band. Because VZV gE is the most abundant glycoprotein, we selected gE as a representative viral glycoprotein. To further investigate the role of autophagy in VZV glycoprotein biosynthesis as well as confirm the results obtained with 3-MA inhibition, we transfected cells with ATG5 small interfering RNA to block autophagosome formation. VZV-induced syncytium formation was markedly reduced by ATG5 knockdown (P < 0.0001). Further, we found that both expression and glycan processing of VZV gE were decreased after ATG5 knockdown, while expression of the nonglycosylated IE62 tegument protein was unchanged. Taken together, our cumulative results not only documented abundant autophagy within VZV-infected cells throughout the infectious cycle but also demonstrated that VZV-induced autophagy facilitated VZV glycoprotein biosynthesis and processing.

Dyskeratosis Congenita Dermal Fibroblasts are Defective in Supporting the Clonogenic Growth of Epidermal Keratinocytes.

Telomere shortening is associated with cellular senescence and aging. Dyskeratosis congenita (DC) is a premature aging syndrome caused by mutations in genes for telomerase components or telomere proteins. DC patients have very short telomeres and exhibit aging-associated pathologies including epidermal abnormalities and bone marrow failure. Here, we show that DC skin fibroblasts are defective in their ability to support the clonogenic growth of epidermal keratinocytes. Conditioned media transfer experiments demonstrated that this defect was largely due to lack of a factor or factors secreted from the DC fibroblasts. Compared to early passage normal fibroblasts, DC fibroblasts express significantly lower transcript levels of several genes that code for secreted proteins, including Insulin-like Growth Factor 1 (IGF1) and Hepatocyte Growth Factor (HGF). Aged normal fibroblasts with short telomeres also had reduced levels of IGF1 and HGF, similar to early passage DC fibroblasts. Knockdown of IGF1 or HGF in normal fibroblasts caused a reduction in the capacity of conditioned media from these fibroblasts to support keratinocyte clonogenic growth. Surprisingly, reconstitution of telomerase in DC fibroblasts did not significantly increase transcript levels of IGF1 or HGF or substantially increase the ability of the fibroblasts to support keratinocyte growth, indicating that the gene expression defect is not readily reversible. Our results suggest that telomere shortening in dermal fibroblasts leads to reduction in expression of genes such as IGF1 and HGF and that this may cause a defect in supporting normal epidermal proliferation.

Viral cyclins mediate separate phases of infection by integrating functions of distinct mammalian cyclins.

Gammaherpesvirus cyclins have expanded biochemical features relative to mammalian cyclins, and promote infection and pathogenesis including acute lung infection, viral persistence, and reactivation from latency. To define the essential features of the viral cyclin, we generated a panel of knock-in viruses expressing various viral or mammalian cyclins from the murine gammaherpesvirus 68 cyclin locus. Viral cyclins of both gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus supported all cyclin-dependent stages of infection, indicating functional conservation. Although mammalian cyclins could not restore lung replication, they did promote viral persistence and reactivation. Strikingly, distinct and non-overlapping mammalian cyclins complemented persistence (cyclin A, E) or reactivation from latency (cyclin D3). Based on these data, unique biochemical features of viral cyclins (e.g. enhanced kinase activation) are not essential to mediate specific processes during infection. What is essential for, and unique to, the viral cyclins is the integration of the activities of several different mammalian cyclins, which allows viral cyclins to mediate multiple, discrete stages of infection. These studies also demonstrated that closely related stages of infection, that are cyclin-dependent, are in fact genetically distinct, and thus predict that cyclin requirements may be used to tailor potential therapies for virus-associated diseases.

The role of telomeres in the ageing of human skin.

Skin is a self-renewing tissue that is required to go through extensive proliferation throughout the lifespan of an organism. Telomere shortening acts as a mitotic clock that prevents aberrant proliferation such as cancer. A consequence of this protection is cellular senescence and ageing. The telomerase enzyme complex maintains telomere length in germline cells and in cancer cells. Telomerase is also active in certain somatic cells such as those in the epidermis but is almost undetectable in the dermis. Increasing evidence indicates that telomerase plays a significant role in maintenance of skin function and proliferation. Mutations in telomerase component genes in the disease dyskeratosis congenita result in numerous epidermal abnormalities. Studies also indicate that telomerase activity in epidermal stem cells might have roles that go beyond telomere elongation. Telomeres in skin cells may be particularly susceptible to accelerated shortening because of both proliferation and DNA-damaging agents such as reactive oxygen species. Skin might present an accessible tissue for manipulation of telomerase activity and telomere length with the potential of ameliorating skin diseases associated with ageing.

The p53/p21(WAF/CIP) pathway mediates oxidative stress and senescence in dyskeratosis congenita cells with telomerase insufficiency.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal growth arrest, leading to senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in genes for telomere binding proteins or components of telomerase give rise to the premature aging disorder dyskeratosis congenita (DC), which is characterized by extremely short telomeres and an aging phenotype. The current study demonstrates that DC cells signal a DNA damage response through p53 and its downstream mediator, p21(WAF/CIP), which is accompanied by an elevation in steady-state levels of superoxide and percent glutathione disulfide, both indicators of oxidative stress. Poor proliferation of DC cells can be partially overcome by reducing O(2) tension from 21% to 4%. Further, restoring telomerase activity or inhibiting p53 or p21(WAF/CIP) significantly mitigated growth inhibition as well as caused a significant decrease in steady-state levels of superoxide. Our results support a model in which telomerase insufficiency in DC leads to p21(WAF/CIP) signaling, via p53, to cause increased steady-state levels of superoxide, metabolic oxidative stress, and senescence.