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Experimental Determination of the Topology of the HIV-1 gp41 C-Terminal Tail

Steckbeck, Jonathan (2012) Experimental Determination of the Topology of the HIV-1 gp41 C-Terminal Tail. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    The C-terminal tail (CTT) of HIV gp41 has been traditionally viewed as a cytoplasmic domain. Genetic studies demonstrating functional interactions between the CTT and various intracellular partners have implicitly reinforced this view. However, antibody neutralization data and biochemical studies have suggested that the CTT is, or can be, externally localized under certain condition. Additionally, other studies have demonstrated that the CTT is dispensable for in vitro virus replication. After nearly three decades of HIV research, the function and structure of the CTT remain elusive. Our goals, then, were twofold: (i) to determine the overall conservation of the CTT in an attempt to provide an understanding of the functional and structural relevance of the CTT; and, (ii) to provide an experimental topological map of the CTT in an attempt to understand and align observed CTT topology(ies) with the functional necessity of a cytoplasmic CTT. We believe that we made significant contributions to the understanding of CTT topology and its relationship to current published functional studies. The initial studies demonstrated that the CTT sequence is conserved at a level that is intermediate between the highly variable gp120 region and the relatively conserved gp41 ectodomain. Additionally, physicochemical and structural properties of CTT sequences were found to be conserved in spite of the relatively high sequencevariability. These studies demonstrated for the first time that the CTT sequence, while highly variable, contains highly conserved structural and chemical properties that suggest a functional requirement for the CTT. Topology studies of the CTT indicated that the topology of the CTT can be distinct between the surface of Env-expressing cells and viral particles. Additionally, dynamic rearrangement of the CTT was observed as a function of antibody neutralization. These findings prompted a theoretical study of gp41 CTT predicted topology and the proposal of a topological model that we believe is consistent with all published studies regarding the localization of the CTT.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmailORCID
    Thesis AdvisorMontelaro, Ronald C.rmont@pitt.edu
    Committee MemberCascio, Michaelcasciom@duq.edu
    Committee MemberDay, Billybday@pitt.edu
    Committee MemberKlein-Seetharaman, Judithjudithks@cs.cmu.edu
    Committee MemberSmithgall, Thomastsmithga@pitt.edu
    Title: Experimental Determination of the Topology of the HIV-1 gp41 C-Terminal Tail
    Status: Published
    Abstract: The C-terminal tail (CTT) of HIV gp41 has been traditionally viewed as a cytoplasmic domain. Genetic studies demonstrating functional interactions between the CTT and various intracellular partners have implicitly reinforced this view. However, antibody neutralization data and biochemical studies have suggested that the CTT is, or can be, externally localized under certain condition. Additionally, other studies have demonstrated that the CTT is dispensable for in vitro virus replication. After nearly three decades of HIV research, the function and structure of the CTT remain elusive. Our goals, then, were twofold: (i) to determine the overall conservation of the CTT in an attempt to provide an understanding of the functional and structural relevance of the CTT; and, (ii) to provide an experimental topological map of the CTT in an attempt to understand and align observed CTT topology(ies) with the functional necessity of a cytoplasmic CTT. We believe that we made significant contributions to the understanding of CTT topology and its relationship to current published functional studies. The initial studies demonstrated that the CTT sequence is conserved at a level that is intermediate between the highly variable gp120 region and the relatively conserved gp41 ectodomain. Additionally, physicochemical and structural properties of CTT sequences were found to be conserved in spite of the relatively high sequencevariability. These studies demonstrated for the first time that the CTT sequence, while highly variable, contains highly conserved structural and chemical properties that suggest a functional requirement for the CTT. Topology studies of the CTT indicated that the topology of the CTT can be distinct between the surface of Env-expressing cells and viral particles. Additionally, dynamic rearrangement of the CTT was observed as a function of antibody neutralization. These findings prompted a theoretical study of gp41 CTT predicted topology and the proposal of a topological model that we believe is consistent with all published studies regarding the localization of the CTT.
    Date: 08 February 2012
    Date Type: Publication
    Defense Date: 15 December 2011
    Approval Date: 08 February 2012
    Submission Date: 17 January 2012
    Release Date: 08 February 2012
    Access Restriction: No restriction; The work is available for access worldwide immediately.
    Patent pending: No
    Number of Pages: 215
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    Uncontrolled Keywords: Membrane protein topology, HIV, protein biochemistry
    Schools and Programs: School of Medicine > Biochemistry and Molecular Genetics
    Date Deposited: 08 Feb 2012 12:34
    Last Modified: 16 Jul 2014 17:02

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