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|Title:||Biosynthesis, Processing and Intracellular Transport of Herpes Simplex Virus Glycoproteins|
|Authors:||Choudhury, Ghosh Nandini|
|Advisor:||Ghosh, Hara P.|
|Abstract:||<p>The work in this thesis is directed towards a better understanding of herpes simplex virus (HSV) glycoprotein biogenesis and structure-function relationship.</p> <p>The synthesis and post-translational maturation of HSV type-2 (HSV-2) glycoproteins were studied using an ionophore, monensin. Monensin, by blocking Golgi functions, affected the post-translational processing of three of the five HSV-2 glycoproteins. The HSV-2 glycoproteins gC (gC-2), gD (gD-2) and gE (gE-2) synthesized in the presence of monensin contained incompletely processed N-linked glycans, indicating that the final processing steps for these glycoproteins involved the Golgi apparatus. The processing of N-linked oligosaccharide chains of gB-2 and gG-2 was however found to proceed normally in the presence of monensin. These two glycoproteins had escaped the inhibitory effect of monensin either because of their individual structures or their processing could have involved a pathway different from the rest of the HSV-2 glycoproteins. The intracellular HSV-2 population formed in the presence of monensin contained fully processed gB-2 and gG-2 along with the immature forms of gC-2, gD-2 and gE-2. On the other hand, the extracellular population of HSV-2 was found to contain only fully processed gG-2 in the presence of monensin. This suggested that gG-2 might be transported to the plasma membrane by a yet different pathway and in the presence of monensin, envelopment of HSV-2 can occur at the regions of plasma membrane modified by the insertion of gG-2. Monensin was found to markedly decrease the infectivity of HSV-2 without affecting the yield of the virus particles to the same extent. These findings, together with the processing data indicated differences in the maturation of the glycoproteins of HSV-1 and HSV-2.</p> <p>The herpes virus qlycoproteins are known to be secreted from the infected cells. Studies with HSV-2 infected cells demonstrated that out of five glycoproteins (gB-2, gC-2, gD-2, gE-2 ,and gG~2) specified by HSV-2, gG-2 is the major glycoprotein secreted into the medium. The size of the secreted form of gG-2 was identical to its intracellular fully processed form. Partial peptide analysis for the intracellular and the secreted gG-2 showed similar patterns, indicating the identity of the two proteins. The data weaken the possibility of removal of the membrane anchoring domain of gG-2 as a cause for its secretion. Though gB-2 and gD-2 were not found to be secreted from the HSV-2 infected cells, monoclonal antibodies, immunoprecipitated the faster migrating forms of gC-2 and gE-2 from the medium. Unlike gG-2, the decrease in molecular weight of the secreted forms of gC-2 and gE-2 suggested that these two HSV-2 glycoproteins can be secreted following proteolytic removal of their membrane anchoring domains. Therefore, as with the post-translational processing mechanisms, it is possible that different mechanisms are involved for the secretion of HSV-2 glycoproteins.</p> <p>Genes for some HSV-1 glycoporoteins were well characterized and therefore to study the role of HSV glycoproteins in directing the virus to its site of envelopment (inner nuclear membrane), one of the HSV-1 glycoproteins, gC-1, was expressed in mammalian cells using eukaryotic expressions vectors. The glycoprotein was expressed transiently in COS-1 cells under the control of adenovirus major late promoter (Ad MLP). The glycoprotein was found to be properly processed and glycosylated in the COS-1 cells following transfection. As in the HSV-1 infected cells, the transfected COS-1 cells secreted gC-1 containing both the N- and O-linked oligosaccharide chains. The sizes of the intracellular and secreted gC-1 were found to be identical.</p> <p>Using a different expression plasmid containing mouse wild type dihydrofolate reductase (dhfr) gene as the selectable marker, Chinese hamster ovary (CHO) cell lines constitutively expressing and secreting properly processed gC-1 were established. The glycoprotein was found to be distributed in the nuclear membrane as well as in the cytoplasm of the cells expressing it. As was found in the HSV-1 infected cells, gC-1 in the COS-1 and the CHO cells was found to be present on the cell surface. This indicated that the systems established for expressing gC-1 in the absence of any other HSV-1 proteins synthesize, process and localize gC-1 in a manner similar to the HSV-1 infected cells.</p> <p>Following this, recombinant retroviruses were generated containing gC-1 integrated in the genome. Transformed Ψ-2 cell lines were generated that constitutively produce the gC-1 containing recombinant retroviruses. Upon infecting 3T3 cells with these recombinant retroviruses permanent lines of cells were generated that constitutively express and secrete gC-1. Unlike the gC-1 producing CHO cells, these cells are permissive to HSV infection. The glycosylation and localization of gC-1 in these 3T3 cells were identical to those found in the H5V-1 infected cells.</p> <p>Finally, the above-mentioned system for expressing gC-1 in COS-1 cells was utilized for preliminary identification of domains of gC-1 containing signals for secretion and nuclear localization. The approach involved construction of hybrid glycoprotein genes (a) replacing carboxy terminal region of gC-1 with that of vesicular stomatitis virus (VSV) glycoprotein G and (b) replacing the carboxy terminal amino acids of a transmembrane HSV-1 glycoprotein, gB-1, with that of gC-1. The transient expression of these hybrid glycoproteins in COS-1 cells under the control of Ad MLP led to some preliminary but significant observations. The carboxy terminal sequences of VSV G was found to affect the intracellular localization of the hybrid glycoprotein gC₁G. In contrast to gC-1, which was distributed primarily at the nuclear membrane, gC₁G was found to be localized at the region which could be suggested to correspond to the endoplasmic reticulum (ER) and Golgi apparatus. The difference in the intracellular localization patterns of these two glycoproteins suggested that the nuclear localization signal of gC-1 might be present within its carboxy terminal region that was replaced by VSV G.</p> <p>The other hybrid glycoprotein, gBgC, was found to be localized at the nuclear membrane indicating that the nuclear localization signal thought to be present in the C-terminal part of gB-1 can be replaced by that of gC-1. In addition secretion of this hybrid glycoprotein also suggested that the carboxy terminal amino acid sequences of gC-1 present in this hybrid glycoprotein could contain the information required for its secretion.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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