Saturday, June 26, 2010

Seunghee Hong Dissertation on XMRV

This is a PhD dissertation by Seunghee Hong on XMRV in prostate
cancer, with Ms. Hong's advisor being Robert Silverman, co-discoverer
of XMRV. As you can see by looking at the table of contents, the whole
thing is a hundred and sixty-something pages long and is basically a
book on XMRV, complete with overviews of the IFN-2-5A/RNase L
pathways, XPR1 cellular receptors, XMRV gene expression, etc. For
anyone up to the challenge, it looks to be a pretty good resource for
some good old fashioned book learnin'.

http://etd.ohiolink.edu/send-pdf.cgi/Hong%20Seunghee.pdf?case1251739728

-----------------------------------------------


IMPLICATIONS FOR XMRV INFECTIONS IN PROSTATE CANCER
by SEUNGHEE HONG
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Thesis Advisor: Dr. Robert H. Silverman
Department of Molecular Biology and Microbiology- Molecular Virology Program
CASE WESTERN RESERVE UNIVERSITY
January, 2010

http://etd.ohiolink.edu/send-pdf.cgi/Hong%20Seunghee.pdf?case1251739728


TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................4
LIST OF FIGURES
....................................................................................................5
ACKNOWLEDGEMENTS
..........................................................................................7
LIST OF ABBREVIATIONS
........................................................................................9
ABSTRACT ............................................................................................................
12

CHAPTER 1. INTRODUCTION...................................................................................
14
IFN system in innate immunity
.................................................................................
14
2-5A/RNase L pathway in the interferon anti-viral system
............................................ 15
Role of RNase L in the biology of prostate cancer
...................................................... 18
Identification of xenotropic gammaretrovirus in prostate tumor tissues
.......................... 19
Replication-competent XMRV genome has been constructed
...................................... 30
XPR1 as a receptor for xenotropic and polytropic murine leukemia
viruses .................... 31
SEVI dramtically boosts HIV-1 infection
....................................................................
34
Retrovirus Restriction Factors
...................................................................................
35
Summary of Introduction
..........................................................................................
39

CHAPTER 2. MATERIALS AND METHODS
.............................................................. 41
Cell Culture ............................................................................................................
41
XPR1 Expression and XMRV Infection of MEFs and CHO Cells
................................... 41
Nested RT-PCR Assays for XPR1
............................................................................
42
XMRV preparations and infectivity assays
................................................................. 43
Effect of SEVI on viral infectivity
...............................................................................
43
Immunofluorescence assay
.....................................................................................
44
Effects of semen on viral infectivity
...........................................................................
44
Preparation of PSA, PSMA, PAP
............................................................................
45
Plasmids and experiments with recombinant viruses
................................................. 45
Isolation of RNA in EPS
..........................................................................................
46
Depletion of XPR1 mRNA using siRNA
.....................................................................
47
Quantitive RT-PCR
.................................................................................................
47
Nested RT-PCR .....................................................................................................
49
Illumina cDNA microarray for gene expression profiles
............................................... 50
Preparation of in vitro co-cultures
.............................................................................
51

CHAPTER 3. XPR1 as a host cellular receptor for XMRV
........................................... 52
Summary ..............................................................................................................
52
Introduction ...........................................................................................................
52
Results .................................................................................................................
55
TCID50 of infectious XMRV supernatant was determined.
........................................... 55
Expression of human XPR1 in mouse cell does not render susceptible to
XMRV infection.
...............................................................................................................
56
XPR1 expression renders hamster cells susceptible to XMRV Infection.
...................... 57
Downregulation of XPR1 in prostate human cells reduces XMRV
infectivity. ................. 62
Discussion.............................................................................................................
68

CHAPTER 4. SEVI boosts infections by XMRV, a Human Retrovirus
Associated with Prostate Cancer
..................................................................................................................
71
Summary ..............................................................................................................
71
Introduction ...........................................................................................................
71
Results .................................................................................................................
73
SEVI promotes XMRV infection of human prostatic and non-prostatic cell
types. ......... 73
SEVI potently increases the infectious titer of XMRV.
............................................... 80
SEVI and semen enhance infectivity at the level of virion attachment
and fusion. ......... 80
XMRV RNA isolated and sequenced from expressed prostatic secretions.
.................. 88
Discussion.............................................................................................................
98
Potent enhancement of XMRV infectivity mediated by SEVI or semen.
........................ 98
Host restriction of XMRV replication by RNase L.
...................................................... 99
The SEVI effect and presence of XMRV in
EPS........................................................ 100

CHAPTER 5. Gene expression profiles regulated by XMRV infection in
human primary prostate cell cultures
...............................................................................................................
102
Summary .............................................................................................................
102
Introduction ..........................................................................................................
103
Results ................................................................................................................
105
Discussion............................................................................................................
139

CHAPTER 6. Stromal-epithelial cell interaction and apoptosis affected
by XMRV infection in prostate cancer progression
..........................................................................................................
144
Summary .............................................................................................................
144
Introduction ..........................................................................................................
144
Results ................................................................................................................
146
Discussion............................................................................................................
157

CHAPTER 7. DISCUSSION AND PERSPECTIVE
.................................................... 160
Summary .............................................................................................................
160
XPR1 as an XMRV receptor
...................................................................................
160
Host restriction factors
..........................................................................................
161
XMRV and prostate carcinogenesis
........................................................................
163
XMRV as a sexually transmitted infection
............................................................... 164
Possible involvement of IL-8 in IFN systems
............................................................ 166
Apoptosis induced by Camptothecin upon XMRV
infection........................................ 168
Antiviral effects of camptothecin
.............................................................................
168
BIBLIOGRAPHY ...................................................................................................
169

----------------------------------

Implications for XMRV Infections in Prostate Cancer
Abstract
By SEUNGHEE HONG

The xenotropic murine leukemia virus - related virus (XMRV) has
recently been identified in prostate cancer tissues and may contribute
to prostate tumorigenesis. XMRV is the first murine leukemia virus
(MLV) -related virus found in humans and a full-length,
replication-competent XMRV genome was recently constructed from
prostate tissue RNA. Currently, little is known about how this virus
infects and/or is transmitted in the human population or how this
virus promotes prostate tumorigenesis.

To determine how XMRV recognizes the host and initiates infection, we
sought to identify the XMRV receptor. (Chapter 3) Expression of human
xenotropic and polytropic retrovirus receptor 1 (XPR1) in resistant
hamster cells rendered them susceptible to infection by XMRV.
Moreover, down-regulation of XPR1 in susceptible human cell lines
reduced XMRV infectivity, thus implicating XPR1 as a cognate receptor
for XMRV. (Chapter 4) In addition, amyloidogenic fibrils known as
semen-derived enhancer of virus infection (SEVI) derived from prostate
acid phosphatase dramatically enhanced XMRV infections of various
human cells including prostate epithelial and stromal cells. The
enhancing effect of SEVI occurred at the level of viral attachment and
entry but did not bypass the requirement for XPR1. Furthermore, XMRV
RNA was detected in prostate secretions of some prostate cancer
patients, implying that XMRV infection is found in an environment that
secrets a natural enhancer of viral infection, SEVI. (Chapter 5) To
discover global effects of XMRV infection in primary prostate cells,
gene expression profiles upon XMRV infection were determined. Our
results revealed that XMRV induced changes in expression of a
relatively small number of genes. Among those, IL-8, a chemokine
previously shown to be involved in prostate carcinogenesis and
metastasis, was induced by XMRV infection. (Chapter 6) To study
dynamic interactions between prostate stromal and epithelial cells
upon XMRV infection, co-culture experiments were performed but were
inconclusive. However, XMRV was observed to inhibit the ability of the
camptothecin, an inhibitor of topoisomerase I, to induce apoptosis in
primary prostate stromal cells. (Chapter 7) These investigations
provide insight into how XMRV infects and spreads in the human
population with implications for prostate carcinogenesis.
 

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