CHAPTER 2 Identification of amino acid residues important for TLR3 inhibition by the ...

184 Pages·2012·1.86 MB·English

ABSTRACT
MORRISON, CLAYTON RUSSELL. Identification of Amino Acid Residues Critical for
TLR3 Inhibition by the Multifunctional West Nile Virus NS1 Protein. (Under the direction of
Frank Scholle Ph.D.).

The flavivirus West Nile virus (WNV) is maintained in nature through an enzootic
transmission cycle between mosquitoes and birds. However, epizootic transmission to
humans and other animals can occur when an infected mosquito takes a blood meal. While
the levels and duration of viremia in these animals are insufficient to maintain the
transmission cycle, the virus is capable of causing febrile and encephalitic diseases in these
dead end hosts, which can result in death. WNV was first isolated in 1937 in Uganda, Africa,
and has since been found to be endemic to regions of Africa, Europe, and the Middle East. In
1999, WNV was first isolated in New York State in birds and humans and has since spread
throughout the contiguous United States. WNV poses a major public health threat as no
vaccine or antiviral strategies are currently available for humans.
Flaviviruses have evolved numerous mechanisms to both evade detection by the
innate immune system and to actively suppress its activation, thereby promoting virus
dissemination and transmission. The innate immune system is an evolutionarily conserved,
first-line defense against invading microorganisms, and functions by detecting pathogen-
associated molecular patterns (PAMPS) through pattern recognition receptors (PRR). Toll-
like Receptor 3 (TLR3) is a PRR found in the endosomes of many cell types and recognizes
dsRNA of many viruses. Activation of TLR3 triggers a highly coordinated signaling cascade
that results in the production of pro-inflammatory cytokines and type I interferon. These
cytokines synergistically induce an antiviral state in responsive cells and prime the adaptive
immune response.
Recently, work by our laboratory has described a role for the WNV non-structural
protein 1 (NS1) in inhibiting TLR3 signal transduction. TLR3 inhibition occurred in WNV
infected cells, WNV replicon-bearing cells, cells ectopically expressing NS1, andnaïve cells
pretreated with the purified secreted form of NS1. The current study was designed to identify
amino acids of the WNV NS1 protein that are important for TLR3 inhibition. Since the NS1
protein is also absolutely required for virus genome replication, a screening strategy was
developed that would allow for the identification of random mutations in NS1 which relieve
TLR3 inhibition, while still allowing virus genome replication. Fluorescence activated cell
sorting (FACS) was used to isolate cells expressing mutant NS1 proteins of the desired
phenotype. These cells were subsequently clonally expanded, and the sequences of their NS1
genes were determined.
Ultimately, three amino acid residues (G295, P320, and M333) located in the C-
terminal half of NS1 were identified as being important for TLR3 inhibition. Changes at
those positions were further characterized, both ectopically and in recombinant WNV. Each
of the amino acid changes allowed genome replication and TLR3 signaling in cells infected
with recombinant WNV.
Biochemical studies were designed to identify the defects of the NS1 mutants, which
allow TLR3 signaling. The amino acid changes resulted in reduced protein stability,
improper glycosylation, and inefficient cell surface expression and secretion. The cumulative
result of these defects in NS1 processing prevents antagonism of TLR3 signaling by the
mutant proteins.
In summary, this work has confirmed that the WNV NS1 protein does inhibit TLR3
and that specific amino acid residues located within the C-terminal half of NS1 can be
mutated to restore signaling. The identification of these residues may allow for more detailed
studies of the role of TLR3 in sensing WNV infection, the pathological contributions of NS1
antagonism of TLR3, as well as serving as a tool for understanding the mechanism by which
NS1 inhibits TLR3.
© Copyright 2012 by Clayton R Morrison
All Rights Reserved
Identification of Amino Acid Residues Critical for TLR3 Inhibition by the Multifunctional
West Nile Virus NS1 Protein

by
Clayton Russell Morrison

A dissertation submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy

Microbiology

Raleigh, North Carolina
2013

APPROVED BY:

_______________________________ ______________________________
Frank Scholle Ph.D. Scott Laster Ph.D.
Committee Chair

________________________________ ________________________________
Michael Sikes Ph.D. Jeffrey Yoder Ph.D.
DEDICATION
This work is dedicated to my parents for their endless support and encouragement of all my
academic endeavors.
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BIOGRAPHY
Clayton is originally a resident of Atlanta, GA and attended the University of Georgia where
he completed his Bachelor’s degree in Microbiology in 2005. Prior to joining the Department
of Microbiology at North Carolina State University in 2008, he completed Master’s thesis
research in Biology at Georgia College & State University in 2007.
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ACKNOWLEDGMENTS
I am extremely thankful to Dr. Scholle for the opportunity train to under him. Thank you for
your patience and encouragement throughout my time here, and for providing me with
resources and freedom to pursue my research. I am also greatly indebted to the numerous
individuals the NCSU Department of Microbiology, past and present. Thank you all for your
advice, friendship, and for lending an ear when things weren’t going well in the lab.

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TABLE OF CONTENTS
List of Tables ......................................................................................................................... vii
List of Figures ....................................................................................................................... viii

CHAPTER 1: Literature Review ...........................................................................................1
Flavivirus Phylogeny and West Nile Virus Origins ...............................................................1
West Nile Virus Maintenance Cycle and Transmission .........................................................6
West Nile Virus Diseases .......................................................................................................8
West Nile Virus Structure, Genome Organization, and Replication Cycle .........................12
West Nile Virus Structural Proteins .....................................................................................18
West Nile Virus Non-structural Proteins..............................................................................21
Adaptive Immune Responses to West Nile Virus Infection ................................................31
Innate Immune Responses to West Nile Virus Infection .....................................................35
Flavivirus Tactics to Subvert the Innate Immune System ....................................................48
Current Studies .....................................................................................................................56
References ............................................................................................................................59

CHAPTER 2: Identification of amino acid residues important for TLR3 inhibition by
the West Nile virus NS1 protein ...........................................................................................87
Abstract ................................................................................................................................87
Introduction ..........................................................................................................................88
Material and Methods ...........................................................................................................91
Results ................................................................................................................................102
Discussion ..........................................................................................................................114
Tables .................................................................................................................................119
Figures ................................................................................................................................120
References ..........................................................................................................................127

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CHAPTER 3: Improper processing and cellular localization of West Nile virus NS1
mutants allow TLR3 signaling ............................................................................................132
Abstract ..............................................................................................................................132
Introduction ........................................................................................................................133
Material and Methods .........................................................................................................137
Results ................................................................................................................................143
Discussion ..........................................................................................................................152
Figures ................................................................................................................................156
References ..........................................................................................................................163

CHAPTER 4: Summary ......................................................................................................166
Figures ................................................................................................................................171

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