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Coronavirus utilization of trafficki...

Pu, Yinghui.

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Coronavirus utilization of trafficking pathways for cell entry.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Coronavirus utilization of trafficking pathways for cell entry./
作者:	Pu, Yinghui.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2008,
面頁冊數:	177 p.
附註:	Source: Dissertations Abstracts International, Volume: 70-02, Section: B.
Contained By:	Dissertations Abstracts International70-02B.
標題:	Microbiology. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3308076
ISBN:	9780549542568

Coronavirus utilization of trafficking pathways for cell entry.
Pu, Yinghui.

Coronavirus utilization of trafficking pathways for cell entry. - Ann Arbor : ProQuest Dissertations & Theses, 2008 - 177 p.

Source: Dissertations Abstracts International, Volume: 70-02, Section: B.

Thesis (Ph.D.)--University of Arkansas for Medical Sciences, 2008.

This item must not be sold to any third party vendors.

Murine coronavirus mouse hepatitis virus (MHV) is an enveloped RNA virus. Although the receptor for MHV has been identified in the early 90's, the molecular mechanism underlying MHV entry remains largely unknown. In this dissertation, I employed chemical, physiological and molecular approaches to systematically analyze the events that lead to cell entry by two closely related but apparently distinct MHV strains (MHV-A59 and MHV-2). I began with comparative analyses on the entry pathways utilized by these two MHV strains. When DBT cell, a murine astrocytoma cell line, was treated with lysosomotropic agents such as chloroquine and bafilomycin A1, which raise the intracellular pH, we found a significant reduction in virus titer and viral gene expression for MHV-2 but not for MHV-A59, indicating that the two strains have distinct dependence on low pH for infection. Further experiments with inhibitors of the clathrin-mediated pathway such as chlorpromazine, hypertonic sucrose medium and siRNA specific to the clathrin heavy chain, demonstrated that MHV-2 utilizes the clathrin-mediated endocytic pathway for entry while the entry by MHV-A59 is independent of the clathrin-pathway. In addition, using recombinant mutant MHVs that have a single amino acid mutation in the spike protein, which has altered the cleavability of the spike and hence the fusogenicity of the virus, we established that the spike protein and more specifically the cleavability of the spike protein is the determinant for the differential pH requirement and selection of the entry pathways. Interestingly, when the cells were transiently over-expressed with a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, MHV-2 gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection that are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis, suggesting that MHV-2 infection may provide a useful model for identifying novel components of the clathrin-mediated endocytic pathway that is independent of Eps15. We then determined whether lipid rafts/caveolae-mediated endocytosis is involved in MHV-A59 entry. Our results showed that MHV-A59 gene expression and infectivity were significantly inhibited when cells were depleted of cholesterol by treatment with methyl-β-cyclodextrin, indicating that MHV-A59 infection requires the integrity of lipid composition. However, over-expression of a dominant-negative mutant of caveolin-1 did not have any effect on MHV-A59 infection while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. Complete knockdown of endogenous caveolin-1 expression by an siRNA specific to caveolin-1 did not inhibit MHV-A59 infection, nor did the treatments with cytoskeleton disrupting agents (nacodazole and cytochalasin D). These results demonstrate that MHV-A59 utilizes a non-clathrin- and non-caveolin-mediated strategy for entry.

ISBN: 9780549542568Subjects--Topical Terms:

536250
Microbiology.
Subjects--Index Terms:

Clathrin

Coronavirus utilization of trafficking pathways for cell entry.
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520 $a Murine coronavirus mouse hepatitis virus (MHV) is an enveloped RNA virus. Although the receptor for MHV has been identified in the early 90's, the molecular mechanism underlying MHV entry remains largely unknown. In this dissertation, I employed chemical, physiological and molecular approaches to systematically analyze the events that lead to cell entry by two closely related but apparently distinct MHV strains (MHV-A59 and MHV-2). I began with comparative analyses on the entry pathways utilized by these two MHV strains. When DBT cell, a murine astrocytoma cell line, was treated with lysosomotropic agents such as chloroquine and bafilomycin A1, which raise the intracellular pH, we found a significant reduction in virus titer and viral gene expression for MHV-2 but not for MHV-A59, indicating that the two strains have distinct dependence on low pH for infection. Further experiments with inhibitors of the clathrin-mediated pathway such as chlorpromazine, hypertonic sucrose medium and siRNA specific to the clathrin heavy chain, demonstrated that MHV-2 utilizes the clathrin-mediated endocytic pathway for entry while the entry by MHV-A59 is independent of the clathrin-pathway. In addition, using recombinant mutant MHVs that have a single amino acid mutation in the spike protein, which has altered the cleavability of the spike and hence the fusogenicity of the virus, we established that the spike protein and more specifically the cleavability of the spike protein is the determinant for the differential pH requirement and selection of the entry pathways. Interestingly, when the cells were transiently over-expressed with a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, MHV-2 gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection that are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis, suggesting that MHV-2 infection may provide a useful model for identifying novel components of the clathrin-mediated endocytic pathway that is independent of Eps15. We then determined whether lipid rafts/caveolae-mediated endocytosis is involved in MHV-A59 entry. Our results showed that MHV-A59 gene expression and infectivity were significantly inhibited when cells were depleted of cholesterol by treatment with methyl-β-cyclodextrin, indicating that MHV-A59 infection requires the integrity of lipid composition. However, over-expression of a dominant-negative mutant of caveolin-1 did not have any effect on MHV-A59 infection while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. Complete knockdown of endogenous caveolin-1 expression by an siRNA specific to caveolin-1 did not inhibit MHV-A59 infection, nor did the treatments with cytoskeleton disrupting agents (nacodazole and cytochalasin D). These results demonstrate that MHV-A59 utilizes a non-clathrin- and non-caveolin-mediated strategy for entry.
590 $a School code: 0365.
650 4 $a Microbiology. $3 536250
650 4 $a COVID-19. $3 3554449
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