STRUCTURAL AND FUNCTIONAL STUDIES OF MEMBRANE-SPANNING DOMAIN OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 ENVELOPE GLYCOPROTEIN IN VIRAL REPLICATION Open Access

Shang, Liang (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/70795789x?locale=en
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Abstract

STRUCTURAL AND FUNCTIONAL STUDIES OF THE MEMBRANE-SPANNING
DOMAIN OF HUMAN IMMUNODEFICIENCY VIRUS TYPE I ENVELOPE
GLYCOPROTEIN IN VIRAL REPLICATION

LIANG SHANG

The membrane-spanning domain (MSD) of the human immunodeficiency viruses type I
(HIV-1) envelope glycoprotein (Env) is critical for its biological activities. In this
dissertation, genetic approaches are used to study the structural and functional roles of
the MSD in viral replication. By examining a series of C-terminal truncation mutants of
HIV-1 gp41, we show that the entire MSD is required for normal Env incorporation and
viral infection. In contrast, a region of 17 residues (K681 to A698) is sufficient to stably
anchor the protein in the membrane, allow efficient transport to the plasma membrane,
and mediate WT levels of cell-cell fusion. Truncation to the residues that are N-terminal
to F697 resulted in an Env complex that was secreted from the cells. Based on the
analysis of these mutants, a "snorkeling" model is proposed for the structure of HIV-1
MSD. In this model, the 12 residues (L682-L693) form an intramembrane helical "core"
and the flanking residues K681 and R694 are buried in the lipid while their side chains
interact with polar head groups. In order to understand roles of the highly conserved
hydrophobic residues and glycine motifs in the MSD "core", we constructed recovery-of-
function mutants by initially replacing the "core" with 12 leucine residues and then
reintroducing the specific residues. We show here that conservation of the MSD core
sequence is not required for normal expression, processing, intracellular transport, and
viral incorporation of HIV-1 Env. However, specific residues in the MSD do play a critical
role in the Env-mediated virus-cell fusion and subsequent viral infection by modulating
conformational integrity of the Env ectodomain. Substitutions in the MSD "core" result in
several minor and localized conformational changes in gp120 and the gp41 ectodomain.
These conformational changes impair initiation of the early events that are required for
formation of the 6-helix bundle and subsequent fusion pores; while they does not
influence the specific virus-cell attachment, nor the recognition of HIV-1 Env to the CD4
receptor and CXCR4 coreceptor.

Table of Contents

TABLE OF CONTENTS

INTRODUCTION………………………………………………………………………………..1
Enveloped Viruses and Envelope Glycoproteins…………………………………...1
Membrane Fusion and Fusion Mediated by Viral Envelope Glycoproteins……...7
Human Immunodeficiency Virus Type I…………………………………………….15
HIV-1 Envelope Glycoprotein………………………………………………………..28
The Membrane-spanning Domain of HIV-1 Env…………………………………..45

TRUNCATION OF THE MEMBRANE-SPANNING DOMAIN OF HUMAN
IMMUNODEFICIENCY VIRUS TYPE I ENVELOPE GLYCOPROTEIN DEFINES
ELEMENTS REQUIRED FOR FUSION, INCORPORATION AND INFECTIVITY……..49

ROLE OF THE MEMBRANE-SPANNING DOMAIN OF HUMAN IMMUNODEFICIENCY
VIRUS TYPE I ENVELOPE GLYCOPROTEIN IN CELL-CELL FUSION AND VIRUS
INFECTION…………………………………………………………………………………….84

RESIDUES IN THE MEMBRANE-SPANNING DOMAIN CORE CONTRIBUTE TO
CONFORMATIONAL INTEGRITY AND FUSOGENICITY OF THE HUMAN
IMMUNODEFICIENCY VIRUS TYPE I ENVELOPE GLYCOPROTEIN……………….122

DISCUSSION…………………………………………………………………………………154

GENERAL REFERENCE LIST……………………………………………………………..163

LIST OF ABBREVIATIONS…………………………………………………………………194

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