scholarly journals Incorporation of Three Endocytosed Varicella-Zoster Virus Glycoproteins, gE, gH, and gB, into the Virion Envelope

2005 ◽  
Vol 79 (2) ◽  
pp. 997-1007 ◽  
Author(s):  
Lucie Maresova ◽  
Tracy Jo Pasieka ◽  
Elizabeth Homan ◽  
Erick Gerday ◽  
Charles Grose
Keyword(s):  
Varicella Zoster Virus ◽  
Virion Assembly ◽  
Varicella Zoster ◽  
Virus Particles ◽  
Transmission Electron ◽  
Cleavage Assay ◽  
Viral Glycoproteins ◽  
Cytoplasmic Tails ◽  
Virion Envelope ◽  
Infected Cells

ABSTRACT The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection. Using surface biotinylation of VZV-infected cells followed by a glutathione cleavage assay, we showed that endocytosis was independent of antibody binding to gE, gH, and gB. Subsequently, with this assay, we demonstrated that biotinylated gE, gH, and gB retrieved from the cell surface were incorporated into nascent virus particles isolated after density gradient sedimentation. To confirm and extend this finding, we repeated the above sedimentation step and specifically detected envelopes decorated with Streptavidin-conjugated gold beads on a majority of complete virions through examination by transmission electron microscopy. In addition, a gE-gI complex and a gE-gH complex were found on the virions. Therefore, the above studies established that VZV subsumed a postendocytosis trafficking pathway as one mechanism by which to deliver viral glycoproteins to the site of virion assembly in the cytoplasm. Furthermore, since a recombinant VZV genome lacking only endocytosis-competent gE cannot replicate, these results supported the conclusion that the endocytosis-envelopment pathway is an essential component of the VZV life cycle.

scholarly journals A Functional YNKI Motif in the Short Cytoplasmic Tail of Varicella-Zoster Virus Glycoprotein gH Mediates Clathrin-Dependent and Antibody-Independent Endocytosis

2003 ◽  
Vol 77 (7) ◽  
pp. 4191-4204 ◽  
Author(s):  
Tracy Jo Pasieka ◽  
Lucie Maresova ◽  
Charles Grose
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Cytoplasmic Tail ◽  
Dependent Manner ◽  
Independent Manner ◽  
Varicella Zoster ◽  
Cytoplasmic Tails ◽  
Infected Cells ◽  
Simplex Virus ◽  
Alignment Analysis

ABSTRACT The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern. This observation was unexpected because all herpesvirus gH homologues have short endodomains not known to contain trafficking motifs. Further investigation demonstrated that VZV gH, when expressed alone with its chaperone gL, was capable of endocytosis in a clathrin-dependent manner, independent of gE, gI, or gB. Upon inspection of the short gH cytoplasmic tail, we discovered a putative tyrosine-based endocytosis motif (YNKI). When the tyrosine was replaced with an alanine, endocytosis of gH was blocked. Utilizing an endocytosis assay dependent on biotin labeling, we further documented that endocytosis of VZV gH was antibody independent. In control experiments, we showed that gE, gI, and gB also internalized in an antibody-independent manner. Alignment analysis of the VZV gH cytoplasmic tail to other herpesvirus gH homologues revealed two important findings: (i) herpes simplex virus type 1 and 2 homologues lacked an endocytosis motif, while all other alphaherpesvirus gH homologues contained a potential motif, and (ii) the VZV gH and simian varicella virus gH cytoplasmic tails were likely longer in length (18 amino acids) than predicted in the original sequence analyses (12 and 16 amino acids, respectively). The longer tails provided the proper context for a functional endocytosis motif.

scholarly journals A Novel Human Skin Tissue Model To Study Varicella-Zoster Virus and Human Cytomegalovirus

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Megan G. Lloyd ◽  
Nicholas A. Smith ◽  
Michael Tighe ◽  
Kelsey L. Travis ◽  
Dongmei Liu ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Human Skin ◽  
Mouse Models ◽  
Human Cytomegalovirus ◽  
Fetal Tissue ◽  
Target Cells ◽  
Varicella Zoster ◽  
Adult Human ◽  
Infected Cells ◽  
Adult Human Skin

ABSTRACT The herpesviruses varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) are endemic to humans. VZV causes varicella (chicken pox) and herpes zoster (shingles), while HCMV causes serious disease in immunocompromised patients and neonates. More effective, less toxic antivirals are needed, necessitating better models to study these viruses and evaluate antivirals. Previously, VZV and HCMV models used fetal tissue; here, we developed an adult human skin model to study VZV and HCMV in culture and in vivo. While VZV is known to grow in skin, it was unknown whether skin could support an HCMV infection. We used TB40/E HCMV and POka VZV strains to evaluate virus tropism in skin organ culture (SOC) and skin xenograft mouse models. Adult human skin from reduction mammoplasties was prepared for culture on NetWells or mouse implantation. In SOC, VZV infected the epidermis and HCMV infected the dermis. Specifically, HCMV infected fibroblasts, endothelial cells, and hematopoietic cells, with some infected cells able to transfer infection. VZV and HCMV mouse models were developed by subcutaneous transplantation of skin into SCID/beige or athymic nude mice at 2 independent sites. Viruses were inoculated directly into one xenograft, and widespread infection was observed for VZV and HCMV. Notably, we detected VZV- and HCMV-infected cells in the contralateral, uninoculated xenografts, suggesting dissemination from infected xenografts occurred. For the first time, we showed HCMV successfully grows in adult human skin, as does VZV. Thus, this novel system may provide a much-needed preclinical small-animal model for HCMV and VZV and, potentially, other human-restricted viruses. IMPORTANCE Varicella-zoster virus and human cytomegalovirus infect a majority of the global population. While they often cause mild disease, serious illness and complications can arise. Unfortunately, there are few effective drugs to treat these viruses, and many are toxic. To complicate this, these viruses are restricted to replication in human cells and tissues, making them difficult to study in traditional animal models. Current models rely heavily on fetal tissues, can be prohibitively expensive, and are often complicated to generate. While fetal tissue models provide helpful insights, it is necessary to study human viruses in human tissue systems to fully understand these viruses and adequately evaluate novel antivirals. Adult human skin is an appropriate model for these viruses because many target cells are present, including basal keratinocytes, fibroblasts, dendritic cells, and lymphocytes. Skin models, in culture and xenografts in immunodeficient mice, have potential for research on viral pathogenesis, tissue tropism, dissemination, and therapy.

scholarly journals Particle Assembly and Ultrastructural Features Associated with Replication of the Lytic Archaeal Virus Sulfolobus Turreted Icosahedral Virus

2009 ◽  
Vol 83 (12) ◽  
pp. 5964-5970 ◽  
Author(s):  
Susan K. Brumfield ◽  
Alice C. Ortmann ◽  
Vincent Ruigrok ◽  
Peter Suci ◽  
Trevor Douglas ◽  
...  
Keyword(s):  
Time Course ◽  
Plaque Assay ◽  
Ultrastructural Changes ◽  
Progeny Virus ◽  
Particle Assembly ◽  
Virus Particles ◽  
Transmission Electron ◽  
Archaeal Viruses ◽  
Infected Cells ◽  
Sulfolobus Turreted Icosahedral Virus

ABSTRACT Little is known about the replication cycle of archaeal viruses. We have investigated the ultrastructural changes of Sulfolobus solfataricus P2 associated with infection by Sulfolobus turreted icosahedral virus (STIV). A time course of a near synchronous STIV infection was analyzed using both scanning and transmission electron microscopy. Assembly of STIV particles, including particles lacking DNA, was observed within cells, and fully assembled STIV particles were visible by 30 h postinfection (hpi). STIV was determined to be a lytic virus, causing cell disruption beginning at 30 hpi. Prior to cell lysis, virus infection resulted in the formation of pyramid-like projections from the cell surface. These projections, which have not been documented in any other host-virus system, appeared to be caused by the protrusion of the cell membrane beyond the bordering S-layer. These structures are thought to be sites at which progeny virus particles are released from infected cells. Based on these observations of lysis, a plaque assay was developed for STIV. From these studies we propose an overall assembly model for STIV.

scholarly journals Replication of Varicella-Zoster Virus in Human Skin Organ Culture

2005 ◽  
Vol 79 (17) ◽  
pp. 11501-11506 ◽  
Author(s):  
Shannon L. Taylor ◽  
Jennifer F. Moffat
Keyword(s):  
Organ Culture ◽  
Varicella Zoster Virus ◽  
Ex Vivo ◽  
Plaque Assay ◽  
Rodent Models ◽  
Fetal Skin ◽  
Varicella Zoster ◽  
Transmission Electron ◽  
Models Of Disease ◽  
Skin Organ Culture

ABSTRACT Varicella-zoster virus (VZV) infection is restricted to humans, which hinders studies of its pathogenesis in rodent models of disease. To facilitate the study of VZV skin tropism, we developed an ex vivo system using human fetal skin organ culture (SOC). VZV replication was analyzed by plaque assay, transmission electron microscopy, and histology. The yield of infectious VZV from SOC increased ∼100-fold over 6 days, virions were abundant, and lesions developed that contained VZV antigens and resembled varicella and zoster lesions. The SOC system for VZV replication has applications for testing virus mutants and antiviral drugs.

scholarly journals A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL

2015 ◽  
Vol 112 (19) ◽  
pp. 6056-6061 ◽  
Author(s):  
Yi Xing ◽  
Stefan L. Oliver ◽  
TuongVi Nguyen ◽  
Claudio Ciferri ◽  
Avishek Nandi ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Neutralizing Antibodies ◽  
Mutational Analysis ◽  
Natural Immunity ◽  
Varicella Zoster ◽  
Glycoprotein Complex ◽  
Negative Stain ◽  
Virion Envelope ◽  
Negative Stain Electron Microscopy ◽  
A Site

Varicella-zoster virus (VZV), of the familyAlphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

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

2016 ◽  
Vol 90 (19) ◽  
pp. 8673-8685 ◽  
Author(s):  
Erin M. Buckingham ◽  
Keith W. Jarosinski ◽  
Wallen Jackson ◽  
John E. Carpenter ◽  
Charles Grose
Keyword(s):  
Varicella Zoster Virus ◽  
Endocytic Pathway ◽  
Autophagic Flux ◽  
Varicella Zoster ◽  
Viral Particles ◽  
Cytoplasmic Vesicles ◽  
Infected Cells ◽  
Autophagy Pathway ◽  
Simplex Virus ◽  
2D And 3D

ABSTRACTVaricella-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.IMPORTANCEVZV 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.

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

2006 ◽  
Vol 81 (2) ◽  
pp. 761-774 ◽  
Author(s):  
Cristian Cilloniz ◽  
Wallen Jackson ◽  
Charles Grose ◽  
Donna Czechowski ◽  
John Hay ◽  
...  
Keyword(s):  
Amino Acids ◽  
Confocal Microscopy ◽  
Varicella Zoster Virus ◽  
Transcriptional Activation ◽  
Protein Function ◽  
Transfected Cells ◽  
Varicella Zoster ◽  
Transcriptional Activation Domain ◽  
Infected Cells ◽  
Orf9 Protein

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.

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