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|Title:||Glycoprotein K of herpes simplex virus (HSV): Role in viral egress and HSV-induced cell-cell fusion|
|Authors:||Hutchinson, Lloyd M.|
|Advisor:||Johnson, David C.|
|Keywords:||Medical Sciences;Medical Sciences|
|Abstract:||<p>The fusion of cellular and viral membranes induced by herpes simplex virus type 1 (HSV-l) is essential for many stages of the replication cycle including virus penetration into host cells, nucleocapsid envelopment, virus egress and transmission of virus from infected to uninfected cells. Wild-type infections also induce low levels of cell-cell fusion, and syncytial mutants of HSV cause cells to fuse extensively. Previous studies have demonstrated that a large fraction of syncytial viruses contain mutations in the HSV-l UL53 gene, indicating that UL53 likely plays a central role in HSV -induced membrane fusion. Consequently, the objectives of this thesis are as follows: 1) Identify and characterize the UL53 gene product produced by wild-type and syncytial strains of HSV -I, and 2) Investigate the role of this protein in HSV -induced membrane fusion and in HSV-l replication. A single 40-kDa protein containing unprocessed, high-mannose N-linked oligosaccharides was detected in cells infected with wild-type and syncytial strains of HSV -I. This protein was designated gK, the ninth HSV -l glycoprotein to be described. gK is unique among HSV -1 glycoproteins since all other HSV glycoproteins examined to date exist as two proteins species; an immature protein with unprocessed oligosaccharides and a mature fonn containing complex N-linked oligosaccharides and O-linked oligosaccharides acquired during post-translational processing in the Golgi apparatus. The gK protein also exhibited signs of heat induced aggregation, an attribute which is commonly observed in proteins spanning the membrane multiple times. Low levels of gK were expressed in HSV -infected cells relative to HSV glycoproteins with a direct role in membrane fusion. which is more consistent with a regulatory role for gK in the membrane fusion process. Wild-type gK. but not other HSV glycoproteins. inhibited cell-cell fusion induced by HSV -1 strains encoding a mutant form of gK (gKˢʸⁿ). As such. the recessive phenotype displayed by gKˢʸⁿ may be symptomatic of a loss-of-function mutation, which disrupts a regulatory function governing membrane fusion. Glycoprotein K was not detected in HSV-l virions and immunofluorescence microscopy demonstrated that gK is not transported to the surfaces of cells infected with either wild-type or syncytial HSV. Instead, gK accumulates in the perinuclear and nuclear membranes of cells, and is unlikely to reach the Goigi apparatus because gK oligosaccharides remained sensitive to endoglycosidase H, These findings are in contrast to the behaviour of all other HSV glycoproteins described to date, which are expressed on the cell surface and incorporated into the virion envelope. Therefore gK must influence membrane fusion indirectly. since the protein is absent from the virion and does not reach the plasma membrane. Furthermore, these results imply that syn mutations in gK induce fusion between the surface membranes of HSV -infected cells by deregulating some aspect of virus replication. Resident proteins are maintained within intracellular compartments (eg. Goigi membranes) by one of three mechanisms: retrieval signals. retention through oligomerization, or retention through membrane thickness. Increasing gK protein synthesis by 10-20 fold did not overcome the block in cell surface transport, and this property is typical of membrane proteins maintained within intracellular compartments by retention signals. Furthermore, gK expressed in the absence of other HSV proteins by using a recombinant adenovirus vector, exhibited the traits and subcellular distribution of gK expressed in HSV -infected cells. These observations indicate that gK contains the targeting signals responsible for gK retention in the endoplasmic reticulum (ER) and nuclear envelope. Although the process controlling gK retention is still uncertain, gK oligomeric structures were observed in cells overexpressing the gK protein, suggesting that the fonnation of oligomeric assemblies may contribute to gK retention in the ER. In addition, sequence analysis identified a tyrosine-based motif (YTK[FILM]) conserved by the gK homologs of eight different alphaherpesviruses, which may have the potential to act as an ER retrieval signal. To further investigate the role of gK in membrane fusion and HSV replication, a gK-negative mutant (F-gKβ) was constructed. Since gK was found to be essential for virus replication, F-gKβ was propagated on complementing cells which can express gK. F-gKβ produced normal plaques characterized by rounded cells when plated on complementing cells, and F-gKβ produced syncytia on cells expressing low levels of gK. In contrast, F-gKβ formed microscopic plaques (consisting of 3-6 infected cells) on gK-negative cell lines and these plaques were reduced by 10² to 10⁶ in number. In the absence of gK, large quantities of unenveloped caps ids accumulated in the cytoplasm of HSV-infected cells and virus panicles did not reach the cell surface. The few enveloped panicles that were assembled displayed a reduced capacity to enter cells and initiate infection. Overexpression of gK also caused defects in virus egress, although virions accumulated in the perinuclear space, and large multilamellar membranous structures juxtaposed with the nuclear envelope were observed. Together these results demonstrate that gK regulates or facilitates HSV egress from cells. In addition, these findings raise the possibility that defects in gK may influence cell-cell fusion by altering the cell surface transport of viral particles, or other viral andlor cellular components which govern the fusion process.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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