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Breaking and Entering: Hantavirus Interactions with Host Factors.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Breaking and Entering: Hantavirus Interactions with Host Factors./
Author:	Kleinfelter, Lara Marie.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	198 p.
Notes:	Source: Dissertations Abstracts International, Volume: 80-09, Section: B.
Contained By:	Dissertations Abstracts International80-09B.
Subject:	Microbiology. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13808870
ISBN:	9781392007969

Breaking and Entering: Hantavirus Interactions with Host Factors.
Kleinfelter, Lara Marie.

Breaking and Entering: Hantavirus Interactions with Host Factors. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 198 p.

Source: Dissertations Abstracts International, Volume: 80-09, Section: B.

Thesis (Ph.D.)--Albert Einstein College of Medicine, 2019.

This item is not available from ProQuest Dissertations & Theses.

Hantaviruses are members of the Hantaviridae family within the order Bunyavirales and are classified into Old and New World clades that cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), respectively. The fatality rates of some hantaviruses exceed 50%, yet there are currently no Food & Drug Administration (FDA) approved treatments or vaccines for either disease. Hantavirus entry into cells is mediated by viral glycoproteins, Gn and Gc, which stud the viral envelope. Virions attach to receptors on the cell surface and internalized into endocytic compartments by clathrin-mediated endocytosis, a macropinocytosis-like mechanism, and/or a dynamin-independent pathway. Once inside, virions likely traffic through the endocytic pathway and fuse with the endosomal membrane in response to acidic pH. However, the roles of putative receptors, the entry strategies utilized by different hantaviruses, intracellular trafficking of virus particles, the site of membrane fusion, and the roles of host factors in post-attachment entry steps are poorly understood. This thesis details our investigation into the hantavirus entry pathway and the roles of two critical host factors: cholesterol and Protocadherin-1 (PCDH1). First, we describe our work uncovering cholesterol as a novel host factor required for Old and New World hantavirus entry. We performed a loss-of-function genetic screen on human haploid cells to identify previously unknown host factors required for entry of Andes virus (ANDV), a virulent New World hantavirus. We discovered that genes involved in the sterol response element-binding protein (SREBP) pathway, which regulates cholesterol homeostasis, and cholesterol biosynthesis are critical for both authentic ANDV infection and infection of vesicular stomatitis virus (VSV) particles expressing hantavirus Gn/Gc in place of VSV glycoprotein (VSV-ANDV Gn/Gc). Using in vitro assays, we determined that genetic or pharmacological disruption of the SREBP pathway results in a modest reduction in cell membrane cholesterol that selectively inhibits hantavirus infection. Membrane cholesterol is necessary in cells for maintaining proper membrane rigidity and for forming membrane microdomains, which are important for receptor organization, receptor signaling, and mediating cholesterol-dependent endocytosis. A modest cholesterol reduction does not affect hantavirus Gn/Gc-mediated binding to cell-surface receptors or virus internalization. However, the ability of VSV-ANDV Gn/Gc to undergo fusion with cellular membranes was severely hampered by cholesterol reduction. Furthermore, experiments conducted on protein-free liposomes revealed that hantaviruses have a direct requirement for cholesterol, independent of cellular proteins. We conclude that this exquisite reliance on cholesterol is a unique feature of hantavirus Gn/Gc-mediated fusion, as hantavirus fusion is significantly reduced under conditions where alphavirus fusion, which also relies on cholesterol, is not. Second, we describe our approach to characterize the hantavirus entry pathway and the role of PCDH1 therein. We developed a live cell imaging assay to observe entry of single VSV-ANDV Gn/Gc particles and VSV particles bearing the Gn/Gc of Hantaan virus (HTNV), a virulent Old World hantavirus, in real time to gain a better understanding of virus intracellular trafficking and fusion. We observed that entry of both VSV-ANDV Gn/Gc and VSV-HTNV Gn/Gc occurs through the classical endocytic pathway and that both viruses undergo fusion with kinetics similar to late-fusing viruses. We also identified that most VSV-ANDV Gn/Gc particles fuse from late endosomes while VSV-HTNV Gn/Gc particles fuse from earlier compartments, probably maturing endosomes. PCDH1 is a member of the cadherin superfamily and a type I transmembrane protein composed of seven extracellular cadherin repeats, a transmembrane domain, and a cytoplasmic tail. Previous work characterized PCDH1 as a novel host factor for New World hantaviruses that has relevance to HPS pathogenesis in an animal disease model. However, the mechanistic role of PCDH1 downstream of virus attachment was not explored. Here, we interrogate the role of PCDH1 in virus uptake by truncating the cytoplasmic tail of PCDH1 and by replacing the transmembrane and C-tail portion with three different glycosylphosphatidylinositol (GPI) anchors to reduce PCDH1 endocytosis. We observed that all PCDH1 variants were able to rescue VSV-ANDV Gn/Gc infection in PCDH1-KO cells, suggesting that PCDH1 serves as a virus attachment factor during entry. Further studies on the mechanistic roles of cholesterol, PCDH1, and the hantavirus entry pathway may reveal novel therapeutic targets and treatment strategies for hantavirus diseases.

ISBN: 9781392007969Subjects--Topical Terms:

536250
Microbiology.
Subjects--Index Terms:

Hantavirus

Breaking and Entering: Hantavirus Interactions with Host Factors.
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520 $a Hantaviruses are members of the Hantaviridae family within the order Bunyavirales and are classified into Old and New World clades that cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), respectively. The fatality rates of some hantaviruses exceed 50%, yet there are currently no Food & Drug Administration (FDA) approved treatments or vaccines for either disease. Hantavirus entry into cells is mediated by viral glycoproteins, Gn and Gc, which stud the viral envelope. Virions attach to receptors on the cell surface and internalized into endocytic compartments by clathrin-mediated endocytosis, a macropinocytosis-like mechanism, and/or a dynamin-independent pathway. Once inside, virions likely traffic through the endocytic pathway and fuse with the endosomal membrane in response to acidic pH. However, the roles of putative receptors, the entry strategies utilized by different hantaviruses, intracellular trafficking of virus particles, the site of membrane fusion, and the roles of host factors in post-attachment entry steps are poorly understood. This thesis details our investigation into the hantavirus entry pathway and the roles of two critical host factors: cholesterol and Protocadherin-1 (PCDH1). First, we describe our work uncovering cholesterol as a novel host factor required for Old and New World hantavirus entry. We performed a loss-of-function genetic screen on human haploid cells to identify previously unknown host factors required for entry of Andes virus (ANDV), a virulent New World hantavirus. We discovered that genes involved in the sterol response element-binding protein (SREBP) pathway, which regulates cholesterol homeostasis, and cholesterol biosynthesis are critical for both authentic ANDV infection and infection of vesicular stomatitis virus (VSV) particles expressing hantavirus Gn/Gc in place of VSV glycoprotein (VSV-ANDV Gn/Gc). Using in vitro assays, we determined that genetic or pharmacological disruption of the SREBP pathway results in a modest reduction in cell membrane cholesterol that selectively inhibits hantavirus infection. Membrane cholesterol is necessary in cells for maintaining proper membrane rigidity and for forming membrane microdomains, which are important for receptor organization, receptor signaling, and mediating cholesterol-dependent endocytosis. A modest cholesterol reduction does not affect hantavirus Gn/Gc-mediated binding to cell-surface receptors or virus internalization. However, the ability of VSV-ANDV Gn/Gc to undergo fusion with cellular membranes was severely hampered by cholesterol reduction. Furthermore, experiments conducted on protein-free liposomes revealed that hantaviruses have a direct requirement for cholesterol, independent of cellular proteins. We conclude that this exquisite reliance on cholesterol is a unique feature of hantavirus Gn/Gc-mediated fusion, as hantavirus fusion is significantly reduced under conditions where alphavirus fusion, which also relies on cholesterol, is not. Second, we describe our approach to characterize the hantavirus entry pathway and the role of PCDH1 therein. We developed a live cell imaging assay to observe entry of single VSV-ANDV Gn/Gc particles and VSV particles bearing the Gn/Gc of Hantaan virus (HTNV), a virulent Old World hantavirus, in real time to gain a better understanding of virus intracellular trafficking and fusion. We observed that entry of both VSV-ANDV Gn/Gc and VSV-HTNV Gn/Gc occurs through the classical endocytic pathway and that both viruses undergo fusion with kinetics similar to late-fusing viruses. We also identified that most VSV-ANDV Gn/Gc particles fuse from late endosomes while VSV-HTNV Gn/Gc particles fuse from earlier compartments, probably maturing endosomes. PCDH1 is a member of the cadherin superfamily and a type I transmembrane protein composed of seven extracellular cadherin repeats, a transmembrane domain, and a cytoplasmic tail. Previous work characterized PCDH1 as a novel host factor for New World hantaviruses that has relevance to HPS pathogenesis in an animal disease model. However, the mechanistic role of PCDH1 downstream of virus attachment was not explored. Here, we interrogate the role of PCDH1 in virus uptake by truncating the cytoplasmic tail of PCDH1 and by replacing the transmembrane and C-tail portion with three different glycosylphosphatidylinositol (GPI) anchors to reduce PCDH1 endocytosis. We observed that all PCDH1 variants were able to rescue VSV-ANDV Gn/Gc infection in PCDH1-KO cells, suggesting that PCDH1 serves as a virus attachment factor during entry. Further studies on the mechanistic roles of cholesterol, PCDH1, and the hantavirus entry pathway may reveal novel therapeutic targets and treatment strategies for hantavirus diseases.
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