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The role of cell surface architecture in the HSV-1 lifecycle and in HA binding ability of CD44

University of British Columbia

HSV-1 resistant cell lines derived from murine fibroblast L cells were isolated and found to have various cell surface alterations resulting from abnormal processing of glycosaminoglycans and oligosaccharides. These cell lines are relatively resistant to infection because heparan sulfate, a ubiquitous glycosaminoglycan, is absent in these mutant cells, and HSV uses heparan sulfate as an initial attachment receptor to initiate infection. One such mutant, sog8, harbors defects in both N-glycosylation and glycosaminoglycan synthesis. Surprisingly, sog8 cells were more susceptible to HSV infection than sog9 cells, which are only defective in heparan sulfate biosynthesis. This suggests that the display of cell surface N-linked glycans plays a role in viral infection. Sog8 cells were also completely unable to support HSV egress. Taken together, these results are consistent with the hypothesis that defects in sog8 cells may alter fusion between viral and cellular membranes at multiple stages of the virus life cycle. These mutant cell lines were also used to investigate the ligand binding requirements for an important cell surface adhesion molecule, CD44, which binds hyaluronan. On the basis of the known properties of several cell lines with defects in glycosylation and glycosaminoglycan synthesis, I determined that chondroitin sulfate biosynthesis is required for inducible hyaluronan binding by CD44. Moreover, in the absence of fully processed oligosaccharides, chondroitin sulfate is not essential for hyaluronan binding, indicating that the effect of chondroitin sulfate depends upon the glycosylation state of the cell. I also investigated the expression of a putative tumour suppressor gene, EXT-1 in sog9 cells. Our laboratory determined that EXT-1 encodes a heparan sulfate polymerase enzyme that restores expression of cell surface heparan sulfate in sog9 cells. As expected, EXT-1 expression also rescues HSV infection of sog9 cells. I characterized the initial interaction of HSV with these cells in an attempt to identify the specific heparan sulfate proteoglycans with which HSV interacts. I identified at least three polypeptides that were recognized by the virus. Further characterization of these proteins will be necessary to determine their potential function.

Thesis/Dissertation

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2009-06-29

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http://hdl.handle.net/2429/9861

Microbiology and Immunology

University of British Columbia

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