Varicella vaccine meningitis is an uncommon delayed adverse event of vaccination. Varicella vaccine meningitis has been diagnosed in 12 children, of whom 3 were immunocompromised. We now report two additional cases of vaccine meningitis in twice-immunized immunocompetent children and we perform further testing on a prior third case. We used three methods to diagnose or investigate cases of varicella vaccine meningitis, none of which have been used previously on this disease. These include metagenomic next-generation sequencing and cytokine multiplex profiling of cerebrospinal fluid and immunology exome analysis of white blood cells. In one new case, the diagnosis was confirmed by metagenomic next-generation sequencing of cerebrospinal fluid. Both varicella vaccine virus and human herpesvirus 7 DNA were detected. We performed cytokine multiplex profiling on the cerebrospinal fluid of two cases and found ten elevated biomarkers: interferon gamma, interleukins IL-1RA, IL-6, IL-8, IL-10, IL-17F, chemokines CXCL-9, CXCL-10, CCL-2, and G-CSF. In a second new case, we performed immunology exome sequencing on a panel of 356 genes, but no errors were found. After a review of all 14 cases, we concluded that (i) there is no common explanation for this adverse event, but (ii) ingestion of an oral corticosteroid burst 3–4 weeks before onset of vaccine meningitis may be a risk factor in some cases.

The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.IMPORTANCE The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV's usage being closer to HSV1's usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.

Varicella-zoster virus vaccination is recommended for virtually all young children in the United States, Canada, and several other countries. Varicella vaccine is a live attenuated virus that retains some of its neurotropic properties. Herpes zoster caused by vaccine virus still occurs in immunized children, although the rate is much lower than in children who had wild-type varicella. It was commonly thought that 2 varicella vaccinations would protect children against the most serious complication of meningitis following herpes zoster; however, 2 meningitis cases have already been published. We now report a third case of varicella vaccine meningitis and define risk factors shared by all 3 immunized adolescents. The diagnosis in cerebrospinal fluid in this third case was verified by amplifying and sequencing portions of the viral genome, to document fixed alleles found only in the vaccine strain. Viral antibody was also detected in the cerebrospinal fluid by confocal microscopy. When compared with the other 2 cases, remarkably all 3 were 14 years old when meningitis occurred. All 3 were treated with intravenous acyclovir, with complete recovery. The adolescent in our case report also had recurrent asthma, which was treated with both prednisone tablets and beclomethasone inhaler before onset of meningitis. When the 3 cases were considered together, they suggested that immunity to varicella-zoster virus may be waning sufficiently in some twice-immunized adolescents to make them vulnerable to varicella vaccine virus reactivation and subsequent meningitis. This complication rarely happens in children after wild-type varicella.

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.

ABSTRACT 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.

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.

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.

Objective:Varicella-zoster virus (VZV) infection may trigger the inflammatory cascade that characterizes giant cell arteritis (GCA).Methods:Formalin-fixed, paraffin-embedded GCA-positive temporal artery (TA) biopsies (50 sections/TA) including adjacent skeletal muscle and normal TAs obtained postmortem from subjects >50 years of age were examined by immunohistochemistry for presence and distribution of VZV antigen and by ultrastructural examination for virions. Adjacent regions were examined by hematoxylin & eosin staining. VZV antigen-positive slides were analyzed by PCR for VZV DNA.Results:VZV antigen was found in 61/82 (74%) GCA-positive TAs compared with 1/13 (8%) normal TAs (p < 0.0001, relative risk 9.67, 95% confidence interval 1.46, 63.69). Most GCA-positive TAs contained viral antigen in skip areas. VZV antigen was present mostly in adventitia, followed by media and intima. VZV antigen was found in 12/32 (38%) skeletal muscles adjacent to VZV antigen-positive TAs. Despite formalin fixation, VZV DNA was detected in 18/45 (40%) GCA-positive VZV antigen-positive TAs, in 6/10 (60%) VZV antigen-positive skeletal muscles, and in one VZV antigen-positive normal TA. Varicella-zoster virions were found in a GCA-positive TA. In sections adjacent to those containing VZV, GCA pathology was seen in 89% of GCA-positive TAs but in none of 18 adjacent sections from normal TAs.Conclusions:Most GCA-positive TAs contained VZV in skip areas that correlated with adjacent GCA pathology, supporting the hypothesis that VZV triggers GCA immunopathology. Antiviral treatment may confer additional benefit to patients with GCA treated with corticosteroids, although the optimal antiviral regimen remains to be determined.

ABSTRACT 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.

BioTechniquesVol. 57, No. 5 BioFeedback / Letter to the EditorOpen AccessNuclear LC3-positive puncta in stressed cells do not represent autophagosomesErin M. Buckingham, John E. Carpenter, Wallen Jackson & Charles GroseErin M. BuckinghamVirology Laboratory, University of Iowa Children's Hospital, Iowa City, IASearch for more papers by this author, John E. CarpenterVirology Laboratory, University of Iowa Children's Hospital, Iowa City, IASearch for more papers by this author, Wallen JacksonVirology Laboratory, University of Iowa Children's Hospital, Iowa City, IASearch for more papers by this author & Charles Grose*Address correspondence to Charles Grose, Virology Laboratory, University of Iowa Children's Hospital, Iowa City, IA. E-mail: E-mail Address: charles-grose@uiowa.eduVirology Laboratory, University of Iowa Children's Hospital, Iowa City, IASearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114226AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail This letter points out an important potential artifact when using immunolabeling techniques with confocal microscopy to identify autophagosomes. Our laboratory has been investigating autophagy induced by varicella-zoster virus (VZV), a human herpesvirus. Although closely related to herpes simplex virus type 1 (HSV-1), VZV lacks the inhibitors of autophagy harbored within the HSV genome (1–3). Therefore, autophagy is abundant after VZV infection (4–6). Many of our studies have relied on autophagosome quantitation to gauge the level of autophagy under varying conditions of infection (7). For these assays, we have enumerated autophagosomes (puncta) in the cytoplasm after immunolabeling with commercial LC3 antibody reagents (7). Depending on the conditions, we occasionally noticed what appeared to be puncta in the nuclei of the infected cells. In two recent autophagy articles (8, 9), other investigators had identified LC3-positive puncta in the nuclei of their stressed cells. They have implied that these LC3-positive puncta may be related to autophagy. Based on our extensive observations investigating VZV-induced autophagy, we postulate that these nuclear puncta are not related to autophagosome production but instead are related to the antibody reagent and other experimental conditions under which the microscopy experiment is carried out.Based on prior autophagy experiments in our laboratory, we first postulated that different anti-LC3 antibodies led to different levels of nuclear LC3 staining. The panels in Figure 1 illustrate the main differences between using a rabbit monoclonal antibody (#2057–1; Epitomics, Cambridge, MA) versus a rabbit polyclonal antibody (#sc-28266; Santa Cruz, Biotechnology, Inc., Dallas, TX) in different cell lines. In immortalized human keratinocytes (TERT-HFK), both uninfected and VZV-infected cells were labeled with anti-LC3 antibodies, but there was a much greater amount of nuclear LC3 staining with the rabbit polyclonal anti-LC3 reagent (panels A and B). Panel D demonstrates that immunoreactivity (red) to VZV IE62, an abundant viral protein, was present in panel B.Figure 1. Effects of antibody choice and permeabilization on nuclear LC3 immunoreactivity.(A–D) TERT-HFK cells, uninfected (A and C) or virus infected (B and D), were permeabilized with 0.05% Triton X-100 and labeled with Epitomics rabbit monoclonal anti-LC3 (Epi LC3; panel A) or Santa Cruz rabbit polyclonal anti-LC3 (SC LC3; panel B) (both green), MAb 5C6 against VZV IE62 protein (red) (panel D), and the blue fluorescent H33342 DNA stain (Invitrogen) (panels A–D). Infections and immunolabeling were performed as described (6). Briefly, cells were grown on glass coverslips in 6-well dishes. After infection, monolayers were fixed, permeabilized, and blocked in 5% nonfat milk with 2.5% normal goat serum in PBS for 2 h. Cells were then immunolabeled (primary antibody overnight at 4°C; secondary antibody for 2 h at RT). Coverslips were mounted on glass slides and viewed on a Zeiss 710 Laser Scanning Confocal Microscope (Zeiss, Pleasanton, CA). Images were analyzed using Zen 2009 (Zeiss) software. LC3 puncta were seen in the cytoplasm of TERT-HFK cells when labeled with a rabbit monoclonal antibody against LC3 (A), but a rabbit polyclonal antibody against LC3 labeled mostly puncta in the nucleus, with some cytoplasmic staining (B). (E and F) Infected melanoma cells were permeabilized with 0.05% Triton X-100 and labeled with DNA stain (blue) and either Epitomics rabbit monoclonal anti-LC3 (Epi LC3; panel E) or Santa Cruz rabbit polyclonal anti-LC3 (SC LC3; panel F). In these cells, the monoclonal antibody labeled mainly cytoplasmic LC3 (E), but the polyclonal antibody stained primarily nuclear LC3 (F). (G–I) Three infected melanoma monolayers were fixed and permeabilized with increasing amounts of Triton X-100 ' 0.02% (G), 0.05% (H), or 0.1% (I) ' and then labeled with a rabbit polyclonal antibody to LC3 (green) and the DNA stain (blue). Although occasional nuclei showed some LC3 staining at the 0.02% Triton X-100 concentration, nuclear LC3 staining increased greatly from 0.05% to 0.1%. All images are shown are at a final magnification of 630×, and all scale bars represent 20 µm.In Figure 1, panels E and F show similar results in melanoma cells (4). Panel E shows syncytia cytopathology induced by VZV infection. Interestingly, nuclei (blue color) within the syncytia did not show LC3 staining after immunolabeling with the rabbit monoclonal antibody (panel E), although cytoplasmic puncta were easily seen. In contrast, immunolabeling with the rabbit polyclonal antibody caused prominent nuclear LC3 staining, as noted by the green puncta within the blue nuclei (panel F). Similar nuclear LC3 patterns were also seen in infected MRC-5 fibroblasts after immunolabeling with a polyclonal antibody (not shown). In short, nuclear puncta were more easily seen with polyclonal anti-LC3 antibodies, regardless of the infected cell substrate.We next postulated that the conditions for permeabilization of the cells before immunolabeling were very important in determining the level of nuclear LC3 staining. All of the studies cited above used Triton X-100 for permeabilization. In previous experiments, we have used Triton concentrations ranging from 0.02% to 0.1% for 1 h at room temperature (RT), to permeabilize cells before immunolabeling with anti-LC3 antibody. In the experiment shown in Figure 1, panels G–I, infected cells were fixed with 2% paraformaldehyde and permeabilized with 0.02% (G), 0.05% (H), or 0.1% (H) Triton X-100 and then immunolabeled with the rabbit polyclonal antibody against LC3. As the amount of Triton X-100 was increased, more nuclei contained LC3 staining. Note in particular that almost every blue nucleus in panel I contained green puncta. This effect has been observed in many different experiments in this laboratory using different cell types and conditions of infection. We also observed that some nuclear puncta were larger than typically seen in the cytoplasm of VZV-infected cells; whereas true cytoplasmic puncta are 590 nm ± 240 nm (10), some nuclear puncta were greater than 1000 nm in diameter.After observing these differences between cytoplasmic and nuclear puncta, we postulated that the nuclear puncta were not typical double-membraned autophagosomes. Because our laboratory has used transmission electron microscopy (TEM) to examine virus-infected cells for many years, we have a large archive of micrographs. We have already documented cytoplasmic autophagosomes by TEM in an earlier paper (5). For this report, we reexamined more than 70 micrographs to look for any structures that resembled autophagosomes within the nuclei of uninfected or infected cells (Figure 2). In virus-infected monolayers during later time points (11), many viral capsids were seen in the nuclei; capsids measure 75–100 nm in diameter (5). Because autophagosomes typically are 4-to 6-fold larger, we should be able to easily identify these structures, if they were present in the nuclei of these cells (Figure 2, E–H). Note the diameter of a virion in a cytoplasmic vacuole as another size control (Figure 2, circled in panel H). However, we found no double-membraned structures within nuclei (Figure 2). We also examined nuclear preparations from uninfected MRC-5 fibroblasts for the presence of structures that resembled autophagosomes. As seen in representative TEM images in Figure 2, A–D, no double-membraned structures were found in uninfected nuclei. Therefore, punctate LC3 staining within nuclei of cells was not attributable to the LC3-II embedded in the membranes of autophagosomes or organelles resembling autophagosomes.Figure 2. Absence of double-membraned autophagosomal structures in electron micrographs of nuclei of uninfected and virus-infected cells.(A–D) Nuclei isolated from uninfected cells showed no double-membraned structures resembling autophagosomes when examined by transmission electron microscopy (TEM). (B) Magnification of the region within the black box in panel A. (E–H) Melanoma cells infected with VZV showed no autophagosomal-like structures in the nucleus. Cells were infected for 24 h (E) or 72 h (F–H) and imaged by TEM. Black arrows within nuclei point to viral capsids approximately 75– 100 nm in diameter. The circle in (H) encloses virions within a cytoplasmic vacuole. Cy = cytoplasm; Nu = nucleus. Scale bars are included in each panel.We were intrigued when we read two recent autophagy papers in which the authors had observed what were called nuclear puncta identified by various anti-LC3 antibody reagents (8, 9). The authors were uncertain as to their function but speculated that the nuclear puncta may be related to autophagy. We know of no reason why our data about VZV-induced autophagy should not be applicable broadly. In particular, viral capsids in the nucleus provide a valuable marker for any structures >100 nm. Yet we see no structures compatible in size with autophagosomes in nuclei. Based on data acquired in our VZV-induced autophagy system, therefore, we postulate that these nuclear puncta are not related to autophagosome production.Instead, we conclude that a likely explanation for nuclear puncta is the formation of LC3 aggregates within the nuclei and their detection by the primary anti-LC3 antibody reagent (12). LC3-II protein is easily detectable in the nucleus (13). Earlier studies of direct precipitation of antigen by antibody by this laboratory and others demonstrate that aggregates are readily bound by polyclonal antisera (14). Furthermore, aggregate detection is more pronounced with polyclonal as opposed to monoclonal antibody reagents. In addition, the nonionic detergent Triton X-100 does not hinder the detection of antigen—antibody aggregates at the concentrations used for permeabilization (15).Finally, we note that we are not advising that polyclonal anti-LC3 antibody reagents be avoided in autophagy studies. We are instead pointing out the optimal experimental conditions for their use in the detection of cytoplasmic puncta by confocal microscopy techniques.Author contributionsE.M.B. designed experiments, obtained and analyzed the data, and prepared the manuscript. J.E.C. and W.J. contributed to the study design, obtained and analyzed the data, and contributed to manuscript preparation. C.G. supervised the study and prepared the manuscript.AcknowledgmentsWe thank A. Klingelhutz (University of Iowa) for his gift of the TERT-HFK cells. This autophagy research was supported by National Institutes of Health (NIH) grant AI89716 (C.G.). The University of Iowa Central Microscopy Research Facility is supported by NIH grant 1S10RR025439. This paper is subject to the NIH Public Access Policy.Competing interestsThe authors declare no competing interests.References1. Gobeil, P.A. and D.A. Leib. 2012. Herpes simplex virus gamma 34.5 interferes with autophagosome maturation and antigen presentation in dendritic cells. MBio 3:e00267–12.Crossref, Medline, CAS, Google Scholar2. Davison, A.J. and J.E. Scott. 1986. The complete DNA sequence of varicella-zoster virus. J. Gen. Virol. 67:1759–1816.Crossref, Medline, CAS, Google Scholar3. Orvedahl, A., D. Alexander, Z. Talloczy, Q. Sun, Y. Wei, W. Zhang, D. Burns, D.A. Leib, and B. Levine. 2007. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe. 1:23–35.Crossref, Medline, CAS, Google Scholar4. Carpenter, J.E., W. Jackson, L. 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Immunol. 119:1645–1651.Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByAutophagy modulating agents as chemosensitizers for cisplatin therapy in cancer7 November 2020 | Investigational New Drugs, Vol. 39, No. 2The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress16 September 2019 | Autophagy, Vol. 16, No. 7Histone Deacetylase HDAC6 Inhibitor CAY10603 Blocks G1/S of the Cell Cycle and Promotes Senescence of Murine Fibroblasts Transformed with E1A and cHa-ras Oncogenes26 August 2019 | Cell and Tissue Biology, Vol. 13, No. 4Standard Immunohistochemical Assays to Assess Autophagy in Mammalian Tissue30 June 2017 | Cells, Vol. 6, No. 3Development of LC 3/ GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy20 March 2017 | The EMBO Journal, Vol. 36, No. 8Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy PathwaysJournal of Virology, Vol. 90, No. 19Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus18 February 2016 | Traffic, Vol. 17, No. 4Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction2 June 2015 | PLOS ONE, Vol. 10, No. 6 Vol. 57, No. 5 STAY CONNECTED Metrics History Received 20 August 2014 Accepted 29 September 2014 Published online 3 April 2018 Published in print November 2014 Information© 2014 Author(s)Author contributionsE.M.B. designed experiments, obtained and analyzed the data, and prepared the manuscript. J.E.C. and W.J. contributed to the study design, obtained and analyzed the data, and contributed to manuscript preparation. C.G. supervised the study and prepared the manuscript.AcknowledgmentsWe thank A. Klingelhutz (University of Iowa) for his gift of the TERT-HFK cells. This autophagy research was supported by National Institutes of Health (NIH) grant AI89716 (C.G.). The University of Iowa Central Microscopy Research Facility is supported by NIH grant 1S10RR025439. This paper is subject to the NIH Public Access Policy.Competing interestsThe authors declare no competing interests.PDF download

Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.

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.

Significance Varicella-zoster virus (VZV) is an important pathogen, which causes varicella and herpes zoster in humans. In general, there are similarities in virus-host interactions between the alphaherpesviruses. One notable exception is the response to autophagy. VZV infection induces autophagy. This is in contrast to herpes simplex virus (HSV), which has two genes that inhibit autophagy, ICP34.5 and US11; neither is present in the smaller VZV genome. In this study, we found that VZV-induced autophagic flux was not blocked. These results reinforce prior observations showing a proviral effect of autophagy on VZV infectivity and spread. These VZV findings also exhibit similarities with recent data about a requirement for early phase autophagy during Epstein–Barr virus infection, a phylogenetically distant gammaherpesvirus.

ABSTRACT 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.

ABSTRACT Highly pure (>95%) terminally differentiated neurons derived from pluripotent stem cells appear healthy at 2 weeks after infection with varicella-zoster virus (VZV), and the cell culture medium contains no infectious virus. Analysis of the healthy-appearing neurons revealed VZV DNA, transcripts, and proteins corresponding to the VZV immediate early, early, and late kinetic phases of replication. Herein, we further characterized virus in these neuronal cells, focusing on (i) transcription and expression of late VZV glycoprotein C (gC) open reading frame 14 (ORF14) and (ii) ultrastructural features of virus particles in neurons. The analysis showed that gC was not expressed in most infected neurons and gC expression was markedly reduced in a minority of VZV-infected neurons. In contrast, expression of the early-late VZV gE glycoprotein (ORF68) was abundant. Transcript analysis also showed decreased gC transcription compared with gE. Examination of viral structure by high-resolution transmission electron microscopy revealed fewer viral particles than typically observed in cells productively infected with VZV. Furthermore, viral particles were more aberrant, in that most capsids in the nuclei lacked a dense core and most enveloped particles in the cytoplasm were light particles (envelopes without capsids). Together, these results suggest a considerable deficiency in late-phase replication and viral assembly during VZV infection of neurons in culture.

Varicella-zoster virus (VZV) is the first of the human herpesviruses to be attenuated and subsequently approved as a live vaccine to prevent varicella and herpes zoster. Both the attenuated VZV vaccine, called vaccine Oka or vOka, and the parental strain pOka have been completely sequenced. Yet the specific determinants of attenuation are uncertain. The open reading frame (ORF) with the most single nucleotide polymorphisms (SNPs), ORF62, encodes the regulatory protein IE62, but IE62 studies have failed to define a specific SNP associated with attenuation. We have completed next-generation sequencing of the VZV Ellen genome, a strain known to be highly attenuated by its very limited replication in human skin xenografts in the SCID mouse model of VZV pathogenesis. A comparative analysis of the Ellen sequence with all other complete VZV sequences was extremely informative. In particular, an unexpected finding was a stop codon mutation in Ellen ORF0 (herpes simplex virus UL56 homolog) identical to one found in vOka, combined with the absence of polymorphisms in most Ellen ORFs that were known to be mutated in vOka. The mutated ORF0 protein was also imaged in both two dimensions and three dimensions by confocal microscopy. The probability of two VZV strains not connected by a recent common ancestor having an identical ORF0 SNP by chance would be 1 × 10(-8), in other words, extremely unlikely. Taken together, these bioinformatics analyses strongly suggest that the stop codon ORF0 SNP is one of the determinants of the attenuation genotype of live VZV vaccines.

ABSTRACT Autophagy is a recently recognized component of the life cycle of varicella-zoster virus (VZV). We have documented abundant autophagosome formation in skin vesicles (final site of virion assembly) from randomly selected cases of varicella and zoster. The fact that autophagy was an early event in the VZV replication cycle was documented by finding infected vesicle cells with the VZV IE62 protein confined to the nucleus. Next, we pursued studies in VZV-infected cultured cells to define whether autophagy was preceded by endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). First, we demonstrated that autophagosome formation in infected cells closely resembled that seen after treatment of cells with tunicamycin, a potent initiator of ER stress. Second, we demonstrated a marked expansion of ER size in both VZV-infected cells and cells transfected with the predominant VZV glycoprotein complex gE/gI. An enlarged ER is critical evidence of ER stress, which in turn is relieved by the UPR. To this end, we documented the UPR by detecting the alternatively spliced form of the XBP1 protein as well as CHOP (C/EBP homologous protein), both transcriptional activators of other UPR genes in an ER stress-dependent manner. Because VZV does not encode inhibitors of autophagy, the above results suggested that autophagy was a common event in VZV-infected cells and that it was provoked at least in part by ER stress secondary to overly abundant VZV glycoprotein biosynthesis, which led to UPR activation in an attempt to maintain cellular homeostasis.

The VZV genome is smaller than the HSV genome and only encodes nine glycoproteins. This chapter provides an overview of three VZV glycoproteins: gH (ORF37), gL (ORF60), and gC (ORF14). All three glycoproteins are highly conserved among the alpha herpesviruses. However, VZV gC exhibits unexpected differences from its HSV counterpart gC. In particular, both VZV gC transcription and protein expression are markedly delayed in cultured cells. These delays occur regardless of the virus strain or the cell type, and may account in part for the aberrant assembly of VZV particles. In contrast to VZV gC, the general properties of gH and gL more closely resemble their HSV homologs. VZV gL behaves as a chaperone protein to facilitate the maturation of the gH protein. The mature gH protein in turn is a potent fusogen. Its fusogenic activity can be abrogated when infected cultures are treated with monoclonal anti-gH antibodies.