Characterization of hsv glycoprotein-glycosaminoglycan interactions
The two herpes simplex virus (HSV) serotypes, HSV-1 and HSV-2, demonstrate different tissue tropisms in the human host. There is increasing evidence that the HSV serotypes may enter host cells differently, which may account, in part, for the different behaviours of these closely related viruses....
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2009
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The two herpes simplex virus (HSV) serotypes, HSV-1 and HSV-2, demonstrate
different tissue tropisms in the human host. There is increasing evidence that the HSV
serotypes may enter host cells differently, which may account, in part, for the different
behaviours of these closely related viruses. Initial adsorption of HSV to the host cell is
mediated by the interaction of viral glycoproteins with cell surface glycosaminoglycans
such as heparan sulfate (HS) and chondroitin sulfate (CS). To investigate the
contribution of various cell surface components in the infection pathway, we isolated a
mutant cell line, sog9, which is unable to synthesize glycosaminoglycans (Banfield et al.,
1995a). Although HSV-1 and HSV-2 infection of sog9 cells is diminished, the cells are
still infected at about 0.5% efficiency, which suggests that these cells normally express at
least one nonglycosaminoglycan receptor. Sog9 cells were used to test whether
glycosaminoglycan analogs, such as dextran sulfate (DS), could functionally substitute
for cellular glycosaminoglycans to initiate HSV infection. High-molecular-weight DS
added either prior to or during inoculation stimulated HSV-1 but not HSV-2 infection by
up to 35-fold. By contrast, DS added after viral adsorption had no effect on infection
efficiency. Moreover, DS stimulated HSV-1 infection at 4°C, indicating that this
compound impinged on an early, energy-independent step in infection. Using
radiolabeled virus, it was demonstrated that HSV-1 is more efficient than HSV-2 in
adsorbing to DS immobilized on microtiter wells. This raised the possibility that only
HSV-1 could engage additional receptors to initiate infection in the presence of DS. To
determine which viral component(s) facilitated DS stimulation, a panel of intertypic
recombinants and deletion mutant viruses was investigated. These assays showed that
DS stimulation of infection is mediated primarily by glycoprotein B (gB-1) and that this
interaction was mediated by a domain other than the heparin-binding region in gB-1.
Taken together, these results provide direct evidence that a principle role for cell surface
glycosaminoglycans in HSV infection is to provide an efficient matrix for virus
adsorption.
To investigate further the interactions of HSV with cell surface
glycosaminoglycans, a novel cell line, sog9-EXT1, was used in this study. The
expression of EXT1, an enzyme in the heparan sulfate synthesis pathway, restores HS
synthesis in sog9 cells (McCormick et al., 1998). Moreover, sog9-EXT1 cells are fully
susceptible to HSV-1 infection. Heparin and DS competition assays demonstrated that
HSV-1 attachment to sog9-EXT1 cells is mediated by an interaction with heparan sulfate
moieties on the cell surface. To determine which structural features of heparan sulfate
were important for mediating attachment to sog9-EXT1 cells and control cell lines
expressing HS, HSV-1 infection in the presence of chemically modified heparin
compounds was examined. It was found that 2-O-, 6-O- and N-sulfate groups of heparan
sulfate are involved in viral attachment. Using HSV-1 mutants deleted for the heparin-binding
domains of gB and glycoprotein C (gC), it was shown that HSV-1 attachment to
glycosaminoglycans on sog9-EXT1 cells is primarily mediated by gC.
The investigation of HSV-glycosaminoglycan interactions, and certainly of other
aspects of the viral life cycle, is facilitated by the generation of HSV mutants. The
construction of HSV recombinants has traditionally involved time-consuming
purification procedures. To overcome this, attempts were made to develop i) an HSV-2
cosmid set, which contains overlapping cosmids representing the entire HSV-2 genome
and ii) an HSV-2 bacterial artificial chromosome (BAC), which can be manipulated by
bacterial genetics to generate the desired recombinant. This work demonstrates the
potential advantages and disadvantages of using these different systems to construct HSV
recombinants. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate |
author |
Dyer, Angela Patricia |
spellingShingle |
Dyer, Angela Patricia Characterization of hsv glycoprotein-glycosaminoglycan interactions |
author_facet |
Dyer, Angela Patricia |
author_sort |
Dyer, Angela Patricia |
title |
Characterization of hsv glycoprotein-glycosaminoglycan interactions |
title_short |
Characterization of hsv glycoprotein-glycosaminoglycan interactions |
title_full |
Characterization of hsv glycoprotein-glycosaminoglycan interactions |
title_fullStr |
Characterization of hsv glycoprotein-glycosaminoglycan interactions |
title_full_unstemmed |
Characterization of hsv glycoprotein-glycosaminoglycan interactions |
title_sort |
characterization of hsv glycoprotein-glycosaminoglycan interactions |
publishDate |
2009 |
url |
http://hdl.handle.net/2429/11004 |
work_keys_str_mv |
AT dyerangelapatricia characterizationofhsvglycoproteinglycosaminoglycaninteractions |
_version_ |
1718588727073177600 |
spelling |
ndltd-UBC-oai-circle.library.ubc.ca-2429-110042018-01-05T17:35:36Z Characterization of hsv glycoprotein-glycosaminoglycan interactions Dyer, Angela Patricia The two herpes simplex virus (HSV) serotypes, HSV-1 and HSV-2, demonstrate different tissue tropisms in the human host. There is increasing evidence that the HSV serotypes may enter host cells differently, which may account, in part, for the different behaviours of these closely related viruses. Initial adsorption of HSV to the host cell is mediated by the interaction of viral glycoproteins with cell surface glycosaminoglycans such as heparan sulfate (HS) and chondroitin sulfate (CS). To investigate the contribution of various cell surface components in the infection pathway, we isolated a mutant cell line, sog9, which is unable to synthesize glycosaminoglycans (Banfield et al., 1995a). Although HSV-1 and HSV-2 infection of sog9 cells is diminished, the cells are still infected at about 0.5% efficiency, which suggests that these cells normally express at least one nonglycosaminoglycan receptor. Sog9 cells were used to test whether glycosaminoglycan analogs, such as dextran sulfate (DS), could functionally substitute for cellular glycosaminoglycans to initiate HSV infection. High-molecular-weight DS added either prior to or during inoculation stimulated HSV-1 but not HSV-2 infection by up to 35-fold. By contrast, DS added after viral adsorption had no effect on infection efficiency. Moreover, DS stimulated HSV-1 infection at 4°C, indicating that this compound impinged on an early, energy-independent step in infection. Using radiolabeled virus, it was demonstrated that HSV-1 is more efficient than HSV-2 in adsorbing to DS immobilized on microtiter wells. This raised the possibility that only HSV-1 could engage additional receptors to initiate infection in the presence of DS. To determine which viral component(s) facilitated DS stimulation, a panel of intertypic recombinants and deletion mutant viruses was investigated. These assays showed that DS stimulation of infection is mediated primarily by glycoprotein B (gB-1) and that this interaction was mediated by a domain other than the heparin-binding region in gB-1. Taken together, these results provide direct evidence that a principle role for cell surface glycosaminoglycans in HSV infection is to provide an efficient matrix for virus adsorption. To investigate further the interactions of HSV with cell surface glycosaminoglycans, a novel cell line, sog9-EXT1, was used in this study. The expression of EXT1, an enzyme in the heparan sulfate synthesis pathway, restores HS synthesis in sog9 cells (McCormick et al., 1998). Moreover, sog9-EXT1 cells are fully susceptible to HSV-1 infection. Heparin and DS competition assays demonstrated that HSV-1 attachment to sog9-EXT1 cells is mediated by an interaction with heparan sulfate moieties on the cell surface. To determine which structural features of heparan sulfate were important for mediating attachment to sog9-EXT1 cells and control cell lines expressing HS, HSV-1 infection in the presence of chemically modified heparin compounds was examined. It was found that 2-O-, 6-O- and N-sulfate groups of heparan sulfate are involved in viral attachment. Using HSV-1 mutants deleted for the heparin-binding domains of gB and glycoprotein C (gC), it was shown that HSV-1 attachment to glycosaminoglycans on sog9-EXT1 cells is primarily mediated by gC. The investigation of HSV-glycosaminoglycan interactions, and certainly of other aspects of the viral life cycle, is facilitated by the generation of HSV mutants. The construction of HSV recombinants has traditionally involved time-consuming purification procedures. To overcome this, attempts were made to develop i) an HSV-2 cosmid set, which contains overlapping cosmids representing the entire HSV-2 genome and ii) an HSV-2 bacterial artificial chromosome (BAC), which can be manipulated by bacterial genetics to generate the desired recombinant. This work demonstrates the potential advantages and disadvantages of using these different systems to construct HSV recombinants. Science, Faculty of Microbiology and Immunology, Department of Graduate 2009-07-20T20:22:13Z 2009-07-20T20:22:13Z 1999 2000-05 Text Thesis/Dissertation http://hdl.handle.net/2429/11004 eng For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. 9990897 bytes application/pdf |