STRUCTURAL AND FUNCTIONAL STUDIES OF MEMBRANE-SPANNING DOMAIN OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 ENVELOPE GLYCOPROTEIN IN VIRAL REPLICATION Pubblico
Shang, Liang (2009)
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
About this Dissertation
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