Varicella-zoster virus (VZV), the highly contagious agent of chickenpox (varicella) and shingles (zoster), is spread by infectious virions that are abundant in the vesicular fluid of cutaneous lesions and which are shed from the skin. Virions secreted from cells grown in vitro, however, are not infectious. Electron microscopic (EM) observations have led to the postulate that in cultured cells nucleocapsids of VZV are enveloped while passing through the inner nuclear membrane and travel within the cisternal compartment to the trans Golgi network (TGN), where they are diverted from the secretory pathway to prelysosomes, in which the virions are degraded. Since VZV envelope glycoproteins (gps), like those of newly synthesized lysosomal enzymes, contain phosphorylated oligosaccharides, it is proposed that binding of VZV to Man 6-P receptors (MPRs) in the TGN is responsible for directing newly synthesized VZV to prelysosomes. Because MPRs also cycle to the cell surface, binding of VZV to MPRs of the plasma membrane might facilitate infection of target cells if VZV in vivo were to escape diversion to prelysosomes. Preliminary data indicate that: (i) the phosphorylated mannose residues of viral envelope gps are present on complex oligosaccharides; the enzymes responsible for phosphorylating these residues are different from those which phosphorylate acid hydrolases; (ii) purified cation independent 125I-MPR (MPRci) binds to immobilized VZV gp I; (iii) Man 6-P and other phosphorylated monosaccharides protect cells from infection by cell-free VZV in vitro with a rank order of efficacy that is comparable to that for affinity for MPRs; Man 6-P protection declines within 30 min of application of VZV, a time course compatible with an action directed against viral entry (iv) dephosphorylation of virions by exposure to alkaline phosphatase destroys viral infectivity; (v) LM and EM immunocytochemistry reveal that within infected cells in culture VZV is found in the TGN and co-distributes in intracellular vacuoles with the immunoreactivity of the MPRci. We now propose to determine where in infected cells VZV gps are assembled into the viral envelope, the identity and relative internal acidity of the organelles through which enveloped VZV virions pass during their maturation, and the identity of the intracellular compartments in which VZ virions bind to MPRci. We will also ascertain the structures of VZV-associated oligosaccharides, how they are synthesized in infected cells, and how comparable they are to oligosaccharides of gps of other viruses. In addition, we will characterize the binding of MPRci to VZV-associated Man 6-P bearing oligosaccharides, using as reagents iodinated or colloidal gold-labeled affinity purified ectodomain of the MPRci. The role of the MPRci in viral entry will be tested with human epidermoid KB cells (HEKB), which lack MPRci. If HEKB cells cannot be infected with VZV, we will determine whether transfection with cDNA encoding the MPRci confers infectability on them. Also to be studied are whether unoccupied MPRci at cell surfaces are necessary for infection of HELF by VZV and whether VZV infection disrupts recycling of MPRci to the plasma membrane. Finally, the mechanism by which infectious cell-free VZV particles escape intracellular degradation and reach vesicle fluid in vivo will be investigated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI027187-05
Application #
2063739
Study Section
Virology Study Section (VR)
Project Start
1989-12-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Pediatrics
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Gershon, Anne A; Gershon, Michael D (2010) Perspectives on vaccines against varicella-zoster virus infections. Curr Top Microbiol Immunol 342:359-72
Gershon, Michael D; Gershon, Anne A (2010) VZV infection of keratinocytes: production of cell-free infectious virions in vivo. Curr Top Microbiol Immunol 342:173-88