Ph.D., Faculty of Health Sciences, University of the Witwatersrand, 2011 / The
HIV-‐1
pandemic
continues
unabated.
Although
treatments
exist
that
can
substantially
alleviate
the
morbidity
and
mortality
associated
with
HIV,
there
is
still
a
need
for
improved
anti-‐HIV
treatments
that
reduce
toxicities
and
administration
frequency
and
mediate
sustained
inhibition
of
viral
replication.
Given
the
high
mutability
and
variability
of
the
virus,
a
strategy
that
is
garnering
increasing
focus
is
the
targeting
of
host
factors
that
the
virus
requires
to
replicate,
so-‐called
HIV-‐dependency
factors
(HDFs).
It
is
hoped
this
will
reduce
the
emergence
of
viral
drug
resistance.
A
number
of
genome-‐wide
screens
have
been
performed
to
identify
HDFs,
although
many
remain
to
be
validated,
particularly
in
relevant
cells
lines.
An
objective
of
this
thesis
was
to
validate
three
host
factors
as
HDFs,
in
both
TZM-‐bl
reporter
and
T
cell-‐derived
cell
lines,
and
to
examine
their
potential
as
anti-‐HIV-‐1
therapeutic
targets
through
exploitation
of
the
cellular
gene
silencing
pathway,
RNA
interference
(RNAi).
These
were
HIV-‐1
Tat
specific
factor
1
(HTATSF1),
DEAD
(Asp-‐Glu-‐Ala-‐Asp)
box
polypeptide
3,
X-‐
linked
(DDX3X)
and
SWI/SNF
related,
matrix
associated,
actin
dependent
regulator
of
chromatin,
subfamily
b,
member
1
(SMARCB1),
selected
because
they
had
been
previously
implicated
in
HIV-‐
1
pathogenesis.
The
well-‐characterised
HDF,
PC4
and
SFRS1
interacting
protein
1
(PSIP1)/lens
epithelium-‐derived
growth
factor
(LEDGF)/p75,
was
included
in
the
study
as
a
positive
control.
Cassettes
expressing
short
hairpin
RNAs
(shRNAs)
targeting
the
four
host
proteins
were
generated,
although
shRNAs
did
not
suppress
endogenous
ddx3x
mRNA
levels.
The
ability
of
shRNAs
to
inhibit
HIV-‐1
replication
in
the
reporter
cell
line,
TZM-‐bl,
was
examined.
These
HeLa-‐
derived
cells
are
permissive
for
R5-‐tropic
HIV-‐1
infection
and
contain
an
integrated
luciferase
gene
driven
by
the
viral
promoter.
shRNAs
mediated
a
dose-‐dependent
inhibition
of
luciferase
activity
in
cells
infected
with
a
HIV-‐1
subtype
B
molecular
clone
and,
although
production
of
the
viral
protein
p24
was
unaltered,
infectious
particle
production
was
decreased
in
cells
treated
with
a
shRNA
suppressing
HTATSF1.
Little
effect
was
observed
with
a
shRNA
targeting
SMARCB1,
suggesting
that
this
may
not
function
as
an
HDF
under
these
conditions.
No
effect
on
infectious
particle
production
was
seen
with
the
shRNA
targeting
PSIP1,
which
was
a
result
of
the
long
half-‐
life
of
this
protein,
highlighting
a
limitation
of
using
such
reporter
systems
for
HDF
validation.
Importantly,
shRNAs
were
not
associated
with
any
cytotoxic
effects
in
TZM-‐bl
cells.
Whether
HTATSF1
is
a
potential
therapeutic
target
was
interrogated
further
in
the
more
relevant
T
cell-‐derived
SupT1
cell
line.
Lentiviruses
were
used
to
generate
populations
where
>90%
had
one
copy
of
the
integrated
shRNA
expression
cassette.
Replication
of
the
subtype
B
molecular
clone
p81A-‐4
was
significantly
inhibited
in
the
shH1-‐expressing
SupT1
cell
line,
which
targets
HTATSF1,
for
over
14
days
post-‐infection,
although
inhibition
was
not
as
pronounced
asthat
observed
in
the
shP1-‐expressing
SupT1
cell
line,
which
targets
PSIP1.
In
contrast
to
a
previous
report,
no
change
in
the
ratio
of
unspliced
to
singly-‐
or
multiply-‐spliced
HIV-‐1
transcripts
were
detected
in
shH1-‐expressing
SupT1
cells,
suggesting
that
HTATSF1
does
not
function
as
a
splicing
cofactor
in
this
system.
A
slight
rebound
in
p24
levels
at
14
days
post-‐infection
was
accompanied
by
increased
HTATSF1
expression
and
a
decrease
in
the
percentage
of
cells
with
transgene
expression
in
the
population.
In
addition,
there
was
a
slight
decrease
in
shH1-‐derived
guide
strand
expression,
but
no
change
in
transcription
rates
of
the
htatsf1
gene,
suggesting
that
cells
within
the
population
with
shH1
expression
and
HTATSF1
suppression
may
have
a
growth
disadvantage.
Thus,
although
this
work
demonstrates
for
the
first
time
that
HTATSF1
functions
as
an
HDF
in
T
cell-‐derived
SupT1
cells,
it
may
not
constitute
a
viable
therapeutic
target.
A
second
objective
of
this
thesis
was
to
examine
the
feasibility
of
transcriptional
gene
silencing
(TGS)
of
HDFs
as
an
anti-‐HIV
strategy.
TGS
is
a
small
RNA-‐induced
gene
silencing
pathway
that
operates
through
chromatin
remodelling
with
the
potential
to
mediate
long-‐term
silencing
of
gene
expression.
Thus,
its
application
may
reduce
the
frequency
of
drug
administration
and
associated
toxicities.
Short
interfering
RNAs
(siRNAs)
targeting
the
htatsf1
promoter
were
able
to
reduce
target
mRNA
expression,
which
was
accompanied
by
decreased
htatsf1
transcription
rates
in
HEK293T
cells,
suggesting
silencing
via
a
TGS
mechanism.
The
htatsf1
silencing
inhibited
infectious
HIV-‐1
particle
production
from
TZM-‐bl
cells.
This
work
provides
proof
of
principle
that
TGS
induction
at
a
HDF
may
inhibit
HIV-‐1
replication.
siRNAs
targeting
the
ddx3x
promoter
did
not
induce
TGS.
To
examine
whether
gene
susceptibility
to
TGS
may
be
influenced
by
promoter
architectures,
49
promoter
features
were
examined
for
enrichment
in
genes
at
which
small
RNA-‐induced
TGS
has
been
reported.
Initially,
the
TGS
group
was
compared
to
a
random
set
of
2,000
promoters
and
then
all
other
promoters
in
the
genome.
To
control
for
gene
activation,
two
further
analyses
were
performed
comparing
the
TGS
group
features
to
those
from
promoters
active
in
the
THP-‐1
cell
line
and
housekeeping
genes.
Whilst
difficult
to
ascribe
differences
between
the
TGS
group
and
the
control
groups
to
anything
beyond
a
variation
in
the
proportion
of
active
genes
within
each
group,
there
was
enrichment
for
certain
promoter
features
that
are
independent
of
activity;
the
TGS
group
was
characterised
by
broad
transcription
start
regions,
high
CpG
content
and
a
single
expression
profile.
Moreover,
the
fraction
of
promoters
with
reported
non-‐coding
RNA
overlap
was
greater
in
the
TGS
group
than
the
control
groups.
Thus,
there
is
some
evidence
that
a
number
of
promoter
features
are
associated
with
TGS
susceptibility.
It
is
hoped
this
novel
analysis
will
facilitate
selection
of
future
TGS
targets,
including
HDFs.
In
summary,
the
work
presented
in
this
thesis
paves
the
way
for
development
of
improved
anti-‐HIV
therapies
involving
HDF-‐targeted
TGS-‐based
gene
therapies
that
mediate
sustained
inhibition
of
the
virus.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/11306 |
| Date | 14 February 2012 |
| Creators | Green, Victoria Andress |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf |
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