Erin M. Buckingham

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

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.

The role of telomeres in the ageing of human skin.

Abstract:  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.

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

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.

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

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. 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.

The p53/p21WAF/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.

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.

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.

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

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.

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.

Nuclear LC3-positive puncta in stressed cells do not represent autophagosomes.

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), (2), (3). Therefore, autophagy is abundant after VZV infection (4), (5), (6). Many of our studies have relied upon 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 of Figure 1 illustrate the main differences between using a rabbit monoclonal antibody (Epitomics #2057-1) versus a rabbit polyclonal antibody (Santa Cruz #sc-28266) 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 (compare panels A and B). Panel D demonstrates that VZV IE62 immunoreactivity (red), 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&C) or virus-infected (B&D), were permeabilized with 0.05% Triton X-100 and labeled with either Epitomics rabbit ... Panels E and F of Figure 1 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 (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 (F). Similar nuclear LC3 patterns were also seen in infected MRC-5 fibroblasts after immunolabeling with a polyclonal reagent (not shown). In short, nuclear puncta were more easily seen with polyclonal anti-LC3 reagents, regardless of the infected cell substrate. We next postulated that the conditions for permeabilization of the cells before immunolabeling were very important to the level of nuclear LC3 staining. All articles cited above have used Triton X-100 for permeabilization. In previous experiments, we have used Triton concentrations ranging from 0.02% to 0.1% for one hour at room temperature (RT), to permeabilize cells before immunolabeling with LC3 antibody. In the experiment shown in Figure 1 (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, 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 had 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, namely, true cytoplasmic puncta are 590 nm± 240 nm (10) whereas some nuclear puncta were >1000 nm in diameter. After observing the above differences between cytoplasmic and nuclear puncta, we postulated that the nuclear puncta were not typical double-membraned autophagosomes. Since 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 re-examined over 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 timepoints (11), many viral capsids were seen in the nuclei; capsids measure 75–100 nm in diameter (5). Since autophagosomes typically are 4–6 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 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 due to the LC3-II embedded in the membrane of autophagosomes or an organelle resembling an autophagosome. 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 ... 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). Further, 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 pointing out the optimal experimental conditions for their use in the detection of cytoplasmic puncta by confocal microscopy techniques.