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Title: Intracellular transport and stability of varicella-zoster virus glycoprotein K

Abstract

VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.

Authors:
 [1];  [1];  [2]
  1. Division of Infectious Diseases and Immunology, Saint Louis University School of Medicine, Saint Louis, Missouri 63110-0250 (United States)
  2. Division of Infectious Diseases and Immunology, Saint Louis University School of Medicine, Saint Louis, Missouri 63110-0250 (United States). E-mail: heinemtc@slu.edu
Publication Date:
OSTI Identifier:
20975213
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 358; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2006.08.021; PII: S0042-6822(06)00579-4; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; CELL MEMBRANES; GLYCOPROTEINS; HALF-LIFE; LABELLING; MORPHOGENESIS; VIRUSES

Citation Formats

Hall, Susan L., Govero, Jennifer L., and Heineman, Thomas C. Intracellular transport and stability of varicella-zoster virus glycoprotein K. United States: N. p., 2007. Web. doi:10.1016/j.virol.2006.08.021.
Hall, Susan L., Govero, Jennifer L., & Heineman, Thomas C. Intracellular transport and stability of varicella-zoster virus glycoprotein K. United States. doi:10.1016/j.virol.2006.08.021.
Hall, Susan L., Govero, Jennifer L., and Heineman, Thomas C. Tue . "Intracellular transport and stability of varicella-zoster virus glycoprotein K". United States. doi:10.1016/j.virol.2006.08.021.
@article{osti_20975213,
title = {Intracellular transport and stability of varicella-zoster virus glycoprotein K},
author = {Hall, Susan L. and Govero, Jennifer L. and Heineman, Thomas C.},
abstractNote = {VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.},
doi = {10.1016/j.virol.2006.08.021},
journal = {Virology},
number = 2,
volume = 358,
place = {United States},
year = {Tue Feb 20 00:00:00 EST 2007},
month = {Tue Feb 20 00:00:00 EST 2007}
}
  • Varicella-zoster virus (VZV) encodes two multiply inserted membrane proteins, open reading frame (ORF) 39 protein (ORF39p) and glycoprotein K (gK). The HSV-1 homologs of these proteins are believed to act in conjunction with each other during viral egress and cell-cell fusion, and they directly influence each other's intracellular trafficking. However, ORF39p and VZV gK have received very limited study largely due to difficulties in producing antibodies to these highly hydrophobic proteins. To overcome this obstacle, we introduced epitope tags into both ORF39p and gK and examined their intracellular distributions in transfected and infected cells. Our data demonstrate that both ORF39pmore » and gK accumulate predominately in the ER of cultured cells when expressed in the absence of other VZV proteins or when coexpressed in isolation from other VZV proteins. Therefore, the transport of VZV ORF39p and gK does not exhibit the functional interdependence seen in their HSV-1 homologs. However, during infection, the primary distributions of ORF39p and gK shift from the ER to the Golgi, and they are also found in the plasma membrane indicating that their intracellular trafficking during infection depends on other VZV-encoded proteins. During infection, ORF39p and gK tightly colocalize with VZV envelope glycoproteins B, E and H; however, the coexpression of ORF39p or gK with other individual viral glycoproteins is insufficient to alter the transport of either ORF39p or gK.« less
  • Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an inmore » vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.« less
  • A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX;more » gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.« less
  • The major features that distinguish recurrent herpes simplex virus infections from zoster are illustrated in this article by two case histories. The clinical and epidemiologic features that characterize recurrent herpes simplex virus and varicella-zoster virus infections are reviewed. It is noted that herpesvirus infections are more common and severe in patients with cellular immune deficiency. Each virus evokes both humoral and cellular immune response in the course of primary infection. DNA hybridization studies with RNA probes labelled with sulfur-35 indicate that herpes simplex viruses persist within neurons, and that varicella-zoster virus is found in the satellite cells that encircle themore » neurons.« less
  • Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with (/sup 3/H)glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gpmore » 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.« less