Wallen Jackson

Varicella-zoster virus infection induces autophagy in both cultured cells and human skin vesicles.

ABSTRACT When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.

Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response

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.

Complete DNA Sequence Analyses of the First Two Varicella-Zoster Virus Glycoprotein E (D150N) Mutant Viruses Found in North America: Evolution of Genotypes with an Accelerated Cell Spread Phenotype

ABSTRACT Varicella-zoster virus (VZV) is considered to be one of the most genetically stable of all the herpesviruses. Yet two VZV strains with a D150N missense mutation within the gE glycoprotein were isolated in North America in 1998 and 2002. The mutant strains have an accelerated cell spread phenotype, which distinguishes them from all wild-type and laboratory viruses. Since the VZV genome contains 70 additional open reading frames (ORFs), the possibility existed that the phenotypic change was actually due to an as-yet-undiscovered mutation or deletion elsewhere in the genome. To exclude this hypothesis, the entire genomes of the two mutant viruses were sequenced and found to contain 124,883 (VZV-MSP) and 125,459 (VZV-BC) nucleotides. Coding single-nucleotide polymorphisms (SNPs) were identified in 14 ORFs. One missense mutation was discovered in gH, but none was found in gB, gI, gL, or gK. There were no coding SNPs in the major regulatory protein ORF 62. One polymorphism was discovered which could never have been anticipated based on current knowledge of herpesvirus genomics, namely, the origins of replication differed from those in the prototype strain but not in a manner expected to affect cell spread. When the two complete mutant VZV sequences were surveyed in their entirety, the most reasonable conclusion was that the increased cell spread phenotype was dependent substantially or solely on the single D150N polymorphism in glycoprotein gE. The genomic results also expanded the evolutionary database by identifying which VZV ORFs were more likely to mutate over time.

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 Varicella-Zoster Virus (VZV) ORF9 Protein Interacts with the IE62 Major VZV Transactivator

ABSTRACT The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.

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.

Egress of Light Particles among Filopodia on the Surface of Varicella-Zoster Virus-Infected Cells

ABSTRACT Varicella-zoster virus (VZV) is renowned for its very low titer when grown in cultured cells. There remains no single explanation for the low infectivity. In this study, viral particles on the surfaces of infected cells were examined by several imaging technologies. Few surface particles were detected at 48 h postinfection (hpi), but numerous particles were observed at 72 and 96 hpi. At 72 hpi, 75% of the particles resembled light (L) particles, i.e., envelopes without capsids. By 96 hpi, 85% of all particles resembled L particles. Subsequently, the envelopes of complete virions and L particles were investigated to determine their glycoprotein constituents. Glycoproteins gE, gI, and gB were detected in the envelopes of both types of particles in similar numbers; i.e., there appeared to be no difference in the glycoprotein content of the L particles. The viral particles emerged onto the cell surface amid actin-based filopodia, which were present in abundance within viral highways. Viral particles were easily detected at the base of and along the exterior surfaces of the filopodia. VZV particles were not detected within filopodia. In short, these results demonstrate that VZV infection of cultured cells produces a larger proportion of aberrant coreless particles than has been seen with any other previously examined alphaherpesvirus. Further, these results suggested a major disassociation between capsid formation and envelopment as an explanation for the invariably low VZV titer in cultured cells.

The Attenuated Genotype of Varicella-Zoster Virus Includes an ORF0 Transitional Stop Codon Mutation

ABSTRACT 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.

Delayed Biosynthesis of Varicella-Zoster Virus Glycoprotein C: Upregulation by Hexamethylene Bisacetamide and Retinoic Acid Treatment of Infected Cells

ABSTRACT In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.

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.