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Theses & Dissertations
Boston University Theses & Dissertations
2016
The influence of T cell receptor
signaling on human
immunodeficiency virus infection
Herring, Melissa Beth
http://hdl.handle.net/2144/17014
Boston University
BOSTON UNIVERSITY
SCHOOL OF MEDICINE
Thesis
THE INFLUENCE OF T CELL RECEPTOR SIGNALING ON HUMAN
IMMUNODEFICIENCY VIRUS INFECTION
by
MELISSA B. HERRING
B.S., Louisiana State University, 2009
Ed.M., Boston University, 2014
Submitted in partial fulfillment of the
requirements for the degree of
Master of Science
2016
© 2016 by
MELISSA B. HERRING
All rights reserved
Approved by
First Reader
Andrew J. Henderson, Ph.D.
Assistant Dean Graduate Medical Sciences
Professor of Medicine and Microbiology
Second Reader
Theresa A. Davies, Ph.D.
Assistant Professor of Medical Sciences and Education
Director, M.S. in Oral Health Sciences Program
THE INFLUENCE OF T CELL RECEPTOR SIGNALING ON HUMAN
IMMUNODEFICIENCY VIRUS INFECTION
MELISSA B. HERRING
ABSTRACT
A significant barrier to curing Acquired Immunodeficiency Syndrome
(AIDS) is the presence of latent reservoirs of cells infected with an integrated but
transcriptionally silent provirus that is unaffected by the immune response or by
antiretroviral drugs. Therefore, while antiretroviral therapy is able to suppress
patient viral loads to clinically undetectable levels, upon cessation of treatment,
viral loads rebound rapidly as virus is released from the latent reservoir. An
understanding of the Human Immunodeficiency Virus (HIV) transcriptional
regulatory events that contribute to viral latency is an important step towards
eradicating the latent reservoir. Of particular interest are the mechanisms early in
HIV infection that direct the cell towards productive or latent infection.
The current study aims to determine whether the strength of T cell
receptor (TCR) signaling at the time of HIV infection is correlated with the level of
HIV transcription and to characterize the T cell receptor signaling pathways that
regulate HIV transcription. We hypothesize that the strength of signaling at the
time of infection determines the magnitude of early HIV transcription.
iv
Simultaneous infection and stimulation of Jurkat E6.1 T cells expressing
the C6.5 chimeric antigen receptor shows a 1.5-fold increase in transcription
compared to unstimulated controls while Jurkat E6.1 T cells expressing the B1D2
chimeric antigen receptor shows a nearly 3-fold increase in transcription
compared to unstimulated controls. These results suggest that the strength of
signaling through the TCR at the time of infection determines the magnitude of
HIV transcription early in infection and may contribute to the establishment of
productive or latent infection within the cell.
Simultaneous infection and stimulation of Jurkat E6.1 T cells expressing
the C6.5 or B1D2 chimeric antigen receptors in the presence of select inhibitors
of T cell receptor signaling molecules indicate that TCR signaling through PI3
kinase and protein kinase C pathways may negatively and positively regulate HIV
transcription, respectively. Understanding the specific TCR signaling pathways
that lead to the initial establishment of latency within newly infected cells may
lead to the discovery of novel therapeutic targets aimed at eliminating the latent
reservoir.
v
TABLE OF CONTENTS
TITLE……………………………………………………………………………………...i
COPYRIGHT PAGE...…………………………………………………………………..ii
READER APPROVAL PAGE...………………………………………………………..iii
THE INFLUENCE OF T CELL RECEPTOR SIGNALING ON HUMAN
IMMUNODEFICIENCY VIRUS INFECTION ......................................................... iv
ABSTRACT ........................................................................................................... iv
TABLE OF CONTENTS ........................................................................................ vi
LIST OF TABLES ............................................................................................... viii
LIST OF FIGURES ............................................................................................... ix
LIST OF ABBREVIATIONS ................................................................................... x
INTRODUCTION ...................................................................................................1
HIV Structure and Life Cycle ...........................................................................2
Viral Latency ...................................................................................................9
Chromatin Remodeling .................................................................................11
Transcriptional Interference ..........................................................................12
Transcription Initiation ..................................................................................13
Transcription Elongation ...............................................................................15
T Cell Receptor Signaling and Its Influence on Latency ...............................17
vi
Specific Aims ................................................................................................22
METHODS ...........................................................................................................24
Establishment of Stable Cell Lines Expressing Chimeric Antigen Receptors
......................................................................................................................24
Cell Culture ...................................................................................................24
Cell Transfection ...........................................................................................25
Inhibitor Assays .............................................................................................25
RESULTS ............................................................................................................28
DISCUSSION ......................................................................................................34
REFERENCES ....................................................................................................40
CURRICULUM VITAE .........................................................................................44
vii
LIST OF TABLES
Table
1
Title
Plate Map for Inhibitor Assay
viii
Page
26
LIST OF FIGURES
Figure
Title
Page
1
Structure of Human Immunodeficiency Virus
5
2
Life Cycle of Human Immunodeficiency Virus
7
3
Plasma Viremia in HIV+ Individuals Treated with
Antiretroviral Therapy
10
4
Organization of the HIV 5’ LTR
14
5
Regulation of HIV Transcription Elongation
16
6
T Cell Receptor Signaling Cascade
20
7
Structure and Binding Affinities of Chimeric Antigen
Receptors
29
8
Effects of Chimeric Antigen Receptor Signaling on HIV
Transcription
31
9
Effects of Chimeric Antigen Receptor Signaling on HIV
Transcription in the Presence of Inhibitors
33
ix
LIST OF ABBREVIATIONS
APOBEC3G Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G
AIDS .......................................................... Acquired Immunodeficiency Syndrome
AP-1 ............................................................................................ Activator protein 1
ART ....................................................................................... Anti-retroviral therapy
CA .................................................................................................................Capsid
CBP ...................................................................................... CREB binding protein
CCR5 ............................................................... Chemokine (C-C Motif) Receptor 5
CDC ..................................................... Center for Disease Control and Prevention
CDK7 ............................................................................ Cyclin-dependent kinase 7
CDK9 ............................................................................ Cyclin-dependent kinase 9
cDMEM ............................... Complete Dulbecco’s Modification of Eagle’s Medium
CD3 ................................................................................ Cluster of Differentiation 3
CD4 ................................................................................ Cluster of Differentiation 4
CD28 ............................................................................ Cluster of Differentiation 28
CD4+................................................................. Cluster of Differentiation 4 positive
CTD .................................................................................Carboxy-terminal domain
CXCR4 .......................................................... Chemokine (C-X-C Motif) Receptor 4
DAG ...................................................................................................Diacylglycerol
DNA ...................................................................................... Deoxyribonucleic acid
DSIF ........................................................................ DRB sensitivity-inducing factor
Env ............................................................................................................ Envelope
x
ERK ............................................................... Extracellular signal-regulated kinase
FBS ........................................................................................... Fetal bovine serum
GDP ................................................................................... Guanosine diphosphate
gp41 ........................................................................................... 41 kD glycoprotein
gp120 ....................................................................................... 120 kD glycoprotein
gp160 ....................................................................................... 160 kD glycoprotein
GTP ...................................................................................Guanosine triphosphate
HAART ............................................................. Highly active anti-retroviral therapy
HAT ................................................................................. Histone acetyltransferase
HDAC ...................................................................................... Histone deacetylase
HEK293T ...................................................... Human embryonic kidney 293 T cells
HEXIM1 .................................... Hexamethylene bis acetamide inducible protein 1
HIV ........................................................................ Human Immunodeficiency Virus
HR1 ................................................................................................. Helical region 1
IKK .......................................................................................................... IκB kinase
IP3 .................................................................................. Inositol 1,4,5-triphosphate
ITAM ............................................ Immunoreceptor tyrosine-based activation motif
IκB ...................................................................................................... Inhibitor of κB
JNK ............................................................................. c-Jun amino-terminal kinase
LAT ........................................................................... Linker for activation of T cells
Lck ..................................................... Lymphocyte-specific protein tyrosine kinase
LTR ...................................................................................... Long Terminal Repeat
xi
MA ................................................................................................................. Matrix
MHC I ................................................................Major Histocompatibility Complex I
MHC II ..............................................................Major Histocompatibility Complex II
mRNA ........................................................................................... Messenger RNA
NC ...................................................................................................... Nucleocapsid
Nef ..................................................................................Negative regulatory factor
NELF .............................................................................. Negative elongation factor
NFAT ..................................................................Nuclear factor of activated T cells
NF-κB .......................................................................................... Nuclear factor- κB
NNRTIs ......................................... Non-nucleoside reverse transcriptase inhibitors
NRTIs ................................................... Nucleoside reverse transcriptase inhibitors
Nuc-0 ................................................................................................ Nucleosome 0
Nuc-1 ................................................................................................ Nucleosome 1
PKCθ ......................................................................................... Protein kinase Cθ
PLCγ ........................................................................................... Phospholipase Cγ
PI3 kinase ...................................................................... Phosphoinositide 3-kinase
PIP2 ............................................................. Phosphatidylinositol 4,5-bisphosphate
PR ............................................................................................................. Protease
P-TEFb ................................................. Positive transcriptional elongation factor b
RNA ...............................................................................................Ribonucleic acid
RNA Pol II ..................................................................................RNA polymerase II
RPMI ..................................................................... Roswell Park Memorial Institute
xii
RRE ..................................................................................... Rev response element
snRNP .................................................................. Small nuclear ribonucleoprotein
SPL-76 ..................................... SH2 domain-containing leukocyte protein of 76 kD
SP1 ...........................................................................................Specificity protein 1
SP1 ...............................................................................................Spacer peptide 1
SP2 ...............................................................................................Spacer peptide 2
TAR ................................................................................ Trans-activation response
Tat ........................................................................... Trans-activator of transcription
TCR .................................................................................................. T cell receptor
TFIIH ................................................................................... Transcription factor IIH
Vif ............................................................................................ Viral infectivity factor
Vpr .................................................................................................... Viral protein R
Vpu ................................................................................................... Viral protein U
VSV-G ............................................................. Vesicular Stomatitis Virus G protein
WHO ..............................................................................World Health Organization
ZAP-70 ................................................................. Zeta-associated protein of 70 kD
xiii
INTRODUCTION
The AIDS epidemic began in late 1981 with reports of several
previously healthy homosexual men in both California and New York suffering
from Kaposi’s sarcoma, Pneumocystis pneumonia, and other opportunistic
infections only seen in severely immunocompromised individuals (FriedmanKien, 1981; Gottlieb et al., 1981). Within months, this severe form of acquired
immunodeficiency became known as Acquired Immunodeficiency Syndrome
(AIDS). However, it was two years before the retrovirus responsible for the
disease, Human Immunodeficiency Virus (HIV), was discovered (BarréSinoussi et al., 1983) and another four years before the first antiretroviral
therapy (ART) became available (Ezzell, 1987). While it took six years to
develop a drug to treat the virus responsible for AIDS, it only took two years
for the virus to begin developing resistance to the drug (Bach, 1989). By the
time highly active anti-retroviral therapy (HAART) was introduced as the
standard for treating the virus and preventing drug resistance, more than 4.5
million people had been diagnosed with the disease worldwide (Centers for
Disease Control and Prevention (CDC), 1995).
Although it has been thirty-five years since the beginning of the AIDS
epidemic, finding a cure for the disease remains elusive. While advancements
in antiretroviral therapy along with increased prevention efforts have led to a
steady decline in both the number of AIDS related deaths and the number of
1 individuals newly infected with HIV per year, HIV is still responsible for an
estimated 1.2 million deaths per year with 2 million individuals becoming
newly infected with the virus each year (“WHO | HIV/AIDS,” 2015). The region
most affected by the AIDS epidemic is sub-Saharan Africa, which accounts
for 70% of cases worldwide. However, even in developed countries where
HIV has become more of a chronic, treatable condition thanks to the success
of antiretrovirals, we are just beginning to see the adverse effects of long-term
antiretroviral treatment, such as the increased risk of cardiovascular disease,
liver disease, and cancer (Cahill & Valadéz, 2013). It is, therefore, just as
important as ever that a cure be found for this disease that affects an
estimated 36.9 million people worldwide.
HIV Structure and Life Cycle
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus composed of
two copies of positively stranded RNA encased in a cone-shaped core The
viral core consists of the capsid and its contents and is characterized by its
fullerene cone geometry of approximately 250 hexagomers and exactly
twelve pentamers. The virus is surrounded by a lipid envelope that is derived
from the phospholipid bilayer of the infected host cell and that has
approximately ten trimeric envelope proteins (Env) with subunits gp120 and
gp41 (Briggs & Kräusslich, 2011). Figure 1 shows the structure and
components of the virus.
2 The HIV-1 genome consists of two long terminal repeat (LTR)
regulatory regions located on either side of a coding region. Within the coding
region are the gag-pol and env genes along with the genes for several
accessory proteins. Gag-pol encodes both the Gag polyprotein precursor,
which is proteolytically processed into the structural proteins MA, CA, NC,
and p6 and the spacer peptides SP1 and SP2, and the Gag-pol polyprotein
precursor,
which
additionally
includes
the
protease
(PR),
reverse
transcriptase (RT), and integrase (IN) enzymes. Env encodes the gp160
precursor to the gp120 and gp41 envelope protein subunits (Sierra, Kupfer, &
Kaiser, 2005).
The tat, rev, nef, vpu, vif, and vpr genes each encode a corresponding
accessory protein. Tat is a transcriptional activator which binds to the transactivation response (TAR) element within the LTR and controls further HIV-1
gene transcription. Rev is an accessory protein which binds to the Rev
response element (RRE) found within singly spliced and unspliced viral
mRNA transcripts, facilitating their transport through the nuclear membrane
and allowing for their translation in the cytosol. Nef, as one of its many
putative functions, aids in both the endocytosis and degradation of CD4 coreceptors and MHCI and MHCII molecules found on the cell surface, a
function essential for viral assembly and budding. Vpu, along with inducing
the degradation of newly synthesized CD4 receptors within the endoplasmic
reticulum, counteracts the host restriction factor tetherin, which blocks viral
3 particle release by tethering them to the plasma membrane. Vif counteracts
the host restriction factor APOBEC3G, which blocks viral replication through
cytidine deamination which leads to guanine to adenosine hypermutations
during reverse transcription that render the viral genome highly unstable and
unable to integrate into the host genome. Vpr directs the preintegration
complex to the nuclear membrane, enabling it to enter the nucleus and
integrate into the host genome (Laguette & Benkirane, 2012; Sierra et al.,
2005).
4 Figure 1: Structure of Human Immunodeficiency Virus. The virus is
composed of two copies of RNA within a cone-shaped core and surrounded
by a lipid envelope derived from the host cell membrane. Figure taken from
the National Institute of Allergy and Infectious Disease.
5 The virus assembles and buds from an infected cell as an immature,
non-infectious particle. During assembly, the viral protease is activated,
leading to the proteolytic cleavage of the Gag polyprotein to release the
structural proteins MA, CA, NC, and p6 and the spacer peptides SP1 and
SP2. As the virus buds from the plasma membrane of the infected cell, it
acquires a lipid envelope derived from the host cell membrane. Upon
budding, the structural proteins then reassemble to form the matrix layer of
the inner viral membrane, the capsid layer encasing the nucleocapsid and the
enzymes reverse transcriptase and integrase, and the nucleocapsid layer
coating the viral RNA genome. This rearrangement results in the formation of
a mature, infectious virion capable of infecting a new cell (Ganser-Pornillos,
Yeager, & Sundquist, 2008). A new cell becomes infected when viral Env
binds to cell surface CD4 and either the CXCR4 or CCR5 chemokine
receptor, which function as co-receptors for HIV. Upon binding of CD4 and
CXCR4 or CCR5, the viral envelope fuses with the cell membrane. Following
fusion, the viral core is released into the cytosol and the capsid is uncoated,
enabling the viral genome to enter the cytosol. Within the cytosol, the viral
genome undergoes reverse transcription of its single-stranded RNA into
double-stranded DNA, forming a provirus that is incorporated into the host’s
genome by an integrase. Upon activation of the cell, the viral genome is
transcribed and translated, leading to protein synthesis and assembly of the
viral particles. Once the virus is enveloped by the host cell membrane, it is
6 released and capable of another round of infection (Ganser-Pornillos et al.,
2008; Gomez & Hope, 2005). The overall life cycle for HIV replication is
highlighted in Figure 2.
Figure 2: Life Cycle of Human Immunodeficiency Virus The HIV life cycle
is comprised of the following steps: fusion with the host membrane, entry into
the host cell, reverse transcription, integration, transcription and translation,
formation of immature virion, and budding of mature virion from the host cell
surface membrane. Figure taken from the National Institute of Allergy and
Infectious Diseases.
7 There are currently six major classes of antiretroviral drugs, each
targeting a specific stage of the viral life cycle. Both the CCR5 antagonists
and the fusion inhibitors target viral entry; the CCR5 antagonists by
interacting with the host CCR5 co-receptor, preventing recognition and
binding of the viral gp120, and the fusion inhibitors by binding to the HR1
sequence of the viral gp41, inhibiting fusion of the viral and host cell
membranes. The two classes of reverse transcriptase inhibitors, the
nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleoside
reverse transcriptase inhibitors (NNRTIs) work by blocking the reverse
transcription of viral RNA into DNA. As nucleoside analogues that lack a 3’hydroxyl group, NRTIs work by competitively inhibiting reverse transcriptase
and, once incorporated into the growing DNA strand, blocking the addition of
subsequent nucleotides. NNRTIs, on the other hand, work by binding to a
hydrophobic pocket near the active site of reverse transcriptase and
noncompetitively inhibiting the enzyme’s activity. The integrase inhibitors or
“strand transfer inhibitors” competitively inhibit integrase activity and prevent
the integration of the proviral DNA into the host genome. The protease
inhibitors are substrate analogues that bind to the active site of the enzyme,
preventing the proteolytic processing of the Gag proteins and, therefore, the
assembly of the mature virion (Sierra-Aragón & Walter, 2012). Standard
antiretroviral treatment regimes, which incorporate multiple drugs with
8 different targets, have proven effective in both suppressing viremia and
decreasing the risk of developing drug resistance.
Viral Latency
Although HAART and its predecessor ART have done wonders in
suppressing patient viremia and in delaying the progression of HIV infection
to AIDS, both have been unsuccessful at completely eliminating the virus
from infected individuals. While antiretrovirals are capable of preventing
ongoing viral replication, they are unable to prevent replication within already
infected cells. Therefore, even when patient viremia is suppressed to
undetectable levels (<50 copies/mL), residual viremia (~1 copy/mL) remains
present in the blood, as is evident by the rapid rebound of patient viral loads
seen upon cessation of antiretroviral therapy (Siliciano, 2010; Van Lint,
Bouchat, & Marcello, 2013). Figure 3 details the changes in patient viremia
over the course of antiretroviral therapy and the rapid rebound in viremia
upon its cessation.
9 Figure 3: Plasma Viremia in HIV+ Individuals Treated with Antiretroviral
Therapy. Upon initiation of antiretroviral therapy, patients show an initial rapid
decline in plasma viremia followed by a second phase, in which the rate of
decline slows, and a third phase, in which viremia is suppressed to clinically
undetectable levels. Upon cessation of antiretroviral therapy, patient viremia
rebounds rapidly. Figure amended from (Van Lint et al., 2013).
This viral rebound is due in part to the establishment of viral reservoirs
within latently infected cells. The most significant contributors to the latent
reservoir are the resting CD4+ T cells. Although resting CD4+ T cells are
resistant to infection by HIV-1, activated CD4+ T cells are highly susceptible
to infection. Although most die quickly from either cytotoxic effects of the virus
or from the immune response, a small population returns to a resting state
10 and survives as memory T cells, resulting in virus that is integrated into the
host genome but is transcriptionally silent. This transcriptionally silent virus
can persist within the host for years or even decades, hidden from both the
immune system and from antiretroviral drugs. However, once a memory cell
becomes reactivated through antigen exposure, the cell is capable of
producing virus. If the patient is on antiretroviral therapy, the virus will be
unable to infect new cells, but, if treatment has been suspended, the virus
released from this latent reservoir will be capable of infecting new cells,
leading to a rebound in plasma viremia. (Siliciano & Greene, 2011) Although
the exact mechanism for the establishment and maintenance of latency in
CD4+ T cells is unclear, it is thought to involve a combination of chromatin
remodeling, transcriptional interference, and the regulation of transcription
initiation and elongation.
Chromatin Remodeling
Although it was initially suggested that HIV-1 integrates into condensed
heterochromatic regions of the host genome, it has since been shown that the
provirus tends to integrate into euchromatic regions of actively transcribed
genes (Han et al., 2004). Nevertheless, even when integrated into regions of
euchromatin,
the
integrated
provirus
is
associated
with
positioned
nucleosomes that form within the 5’ LTR (Verdin, Paras, & Van Lint, 1993).
These nucleosomes, nuc-0 and nuc-1, are regulated by SWI/SNF remodeling
11 complexes and posttranslational modifications of the histone tails, particularly
histone tail acetylation (Siliciano & Greene, 2011). Histone tail acetylation by
histone acetyltransferases (HATs), along with SWI/SNF remodeling complex
recruitment, leads to the displacement of nuc-1 and to the activation of HIV-1
transcription. Repression of transcription is associated with the recruitment of
SWI/SNF remodeling complexes and histone tail deacetylation by histone
deacetylases (HDACs), resulting in a more condensed chromatin structure
that interferes with efficient gene transcription. Thus, posttranslational
modifications of nucleosomes are hypothesized to contribute to the
maintenance of HIV latency (Schiralli Lester & Henderson, 2012).
Transcriptional Interference
The findings that the HIV-1 provirus preferentially integrates into
actively transcribed host genes suggest that transcriptional interference may
play a role in the maintenance of viral latency. Transcriptional interference
occurs when ongoing host genome transcription prevents formation of the
pre-initiation complex at the HIV 5’ LTR, thereby interfering with viral
transcription. Two different forms of transcriptional interference can occur,
depending upon the direction of proviral integration with regard to the host
transcriptional regulatory elements. If the viral genome integrates in the same
orientation as the host gene, this can result in transcription of upstream host
genes by RNA polymerase II through the HIV 5’ LTR, leading to the
12 displacement of key transcription factors on the HIV LTR, the prevention of
pre-initiation complex assembly on the viral promoter, and consequently, the
inhibition of effective proviral transcription (Lenasi, Contreras, & Peterlin,
2008; Siliciano & Greene, 2011). On the other hand, if proviral integration is in
the opposing direction to that of host gene transcription, collisions between
the two RNA Pol II complexes can occur, resulting in premature transcription
termination (Han et al., 2008; Siliciano & Greene, 2011; Schiralli Lester &
Henderson, 2012). Thus, regardless of the orientation of the integrated
provirus within the host gene, transcriptional interference seems to be a
significant mechanism of viral persistence.
Transcription Initiation
Another possible mechanism for establishing and maintaining latency
is through the regulation of transcription initiation. This regulation is
accomplished through the HIV 5’ LTR, which is divided into the Tat activating
region (TAR), the promoter, the enhancer, and the modulatory element.
Inducible host transcription factors bind to sites within these regions of 5’ LTR
to regulate the initiation of HIV-1 transcription (Figure 4). The transcription
factor SP1, for example, binds to three sites within the core promoter to
activate transcription, whereas the factors AP-1, NFAT, and NF-κB bind to
sites within the enhancer to regulate transcription initiation (Siliciano &
Greene, 2011; Schiralli Lester & Henderson, 2012).
13 Figure 4: Organization of the HIV 5’ LTR. The 5’ LTR is divided into the
TAR, promoter, enhancer, and modulatory element. Transcription factors bind
to sites within the LTR to regulate initiation of HIV transcription. Figure
amended from (Schiralli Lester & Henderson, 2012)
The NF-κB/Rel family of transcription factors is a perfect example of a
transcription factor that has both activating and inhibitory effects on the HIV 5’
LTR. NF-κB is composed of two subunits, RelA (p65) and p50. In resting
cells, the RelA subunit is sequestered in the cytosol by the inhibitory complex,
IκB. Not only does this limit the amount of active NF-κB available in the
nucleus to bind to the 5’ LTR and activate transcription initiation, thereby
repressing transcription, but it also enables homodimers of the p50 subunit to
bind to the NF-κB sites within the LTR. These p50/p50 homodimers recruit
HDACs,
which
promote
histone
tail
deacetylation
and
chromatin
condensation, leading to further transcriptional repression. However, once the
cell is activated, RelA is released from IκB, enabling it to from heterodimers
with p50, which translocate to the nucleus and displace the p50/p50
homodimers(R. F. Siliciano & Greene, 2011; Schiralli Lester & Henderson,
2012). Bound p50/RelA heterodimers recruit p300/CBP, leading to histone tail
acetylation and increased binding of RNA Pol II and TFIIH/CDK7. Binding of
14 TFIIH/CDK7 leads to increased promoter clearance and phosphorylation of
serine 5 residues on the carboxy-terminal domain (CTD) of RNA Pol II,
resulting in transcriptional activation (Kim et al., 2006; R. F. Siliciano &
Greene, 2011).
Transcription Elongation
Transcription elongation presents another possible regulatory step in
the establishment and maintenance of latency (Figure 5). Once transcription
has been initiated, the negative elongation factors NELF and DSIF bind to the
HIV 5’ LTR and cause RNA Pol II pausing, resulting in limited proviral
transcription but nevertheless allowing for the production of Tat. Once
sufficient amounts of Tat have been produced, it binds to the TAR element to
form the Tat-TAR complex, which in turn recruits P-TEFb, a kinase
sequestered in an inactive ribonucleoprotein complex composed of 7SK RNA
and HEXIM1 (7SK snRNP complex). Tat interacts with both the cyclin T1 and
CDK9 subunits of P-TEFb, causing its release from the 7SK snRNP complex.
P-TEFb subsequently phosphorylates both DSIF and NELF, converting DSIF
to a positive elongation factor that prevents premature release of RNA Pol II
at termination sequences and causing dissociation of NELF, and coupled with
the phosphorylation of serine 2 residues on the CTD of RNA Pol II allows for
efficient transcription elongation (Siliciano & Greene, 2011; Schiralli Lester &
Henderson, 2012). The high levels of both Tat and P-TEFb activity seen in
15 activated cells compared to the low levels seen in resting cells likely indicate
that Tat and P-TEFb may be key regulators of viral latency.
Figure 5: Regulation of HIV Transcription Elongation. Top: Following
transcription initiation, NELF and DSIF bind to the 5’ LTR, resulting in RNA
Pol II pausing just downstream of the transcriptional start site. Bottom: Tat
binds to the TAR element and recruits P-TEFb, which phosphorylates serine
2 residues on the CTD of RNA Pol II, NELF, and DSIF, enabling transcription
elongation to proceed. Figure amended from (Schiralli Lester & Henderson,
2012).
An increased understanding of the mechanisms involved in viral
latency has led to the search for compounds capable of reactivating latently
infected cells in the hope that this would lead to their destruction by the
immune system or by the cytotoxic effects of the virus. Early trials using
16 HDAC inhibitors to reactivate latent cells showed promising results (Lehrman
et al., 2005) but subsequent studies failed to show significant reductions in
the size of the latent reservoir (Siliciano et al., 2007; Sagot-Lerolle et al.,
2008; Archin et al., 2010).
The failure of HDAC inhibitors to significantly reduce the latent
reservoir suggests that the role of transcriptional regulation in latency is more
complex than previously thought and highlights the need for additional
research. As most latency research has focused on the transcriptional
regulatory events that act to maintain latency, there is a particular need for
research into the initial establishment of latency early in infection. Recent
evidence indicates that the activation state of the cell at the time of infection
may influence whether or not latency is established within the cell.
T Cell Receptor Signaling and Its Influence on Latency
Activation of CD4+ T cells by engagement of the T cell receptor (TCR)
and CD4 co-receptor and the CD28 co-stimulatory molecule results in a
signaling cascade of events, beginning with the activation of Lck, a Src kinase
attached to the cytosolic tails of the CD4 co-receptor. Lck phosphorylates
both the tyrosine kinase ZAP-70 and the tyrosine residues of the
immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD4associated CD3ζ chains. Activated ZAP-70 binds to the phosphorylated
ITAMs and phosphorylates a host of adaptor proteins, most important of
17 which are LAT and SLP-76 (Huang & Wange, 2004; Brownlie & Zamoyska,
2013). These adaptor proteins activate PLCγ1, an enzyme that catalyzes the
hydrolysis of PIP2 into the second messangers IP3 and DAG. The hydrolysis
of PIP2 presents a branch point in the signaling cascade, as IP3 and DAG
each activate one or more separate pathways.
The formation of IP3 results in the release of calcium from the
endoplasmic reticulum into the cytosol. The rise in cytosolic calcium leads it to
bind to calmodulin, and the calcium-calmodulin complex activates the
phosphatase
calcineurin.
Activated
calcineurin
dephosphorylates
the
transcription factor NFAT, enabling its translocation to the nucleus. DAG
formation leads to the activation of both protein kinase Cθ (PKCθ) and Ras.
Activated PKCθ activates IκB kinase (IKK), which phosphorylates the NF-κB
inhibitory protein IκB, targeting it for degradation. The degradation of IκB
leads to NF-κB release and translocation to the nucleus. Another kinase
involved in NF- κB activation is PI3 kinase (PI3K). PI3K phosphorylates PIP2
to form PIP3. The serine-threonine kinase Akt binds to PIP3 and becomes
activated. Akt interacts with PKCθ to enhance activation of NF- κB. DAG
activation of Ras involves the exchange of bound GDP for GTP and leads to
activation of the MAP kinase pathway, at the end of which are the kinases
ERK and JNK. Activation of ERK leads to the upregulation of c-Fos
expression while activation of JNK leads to the phosphorylation of c-Jun.
Together c-Fos and phosphorylated c-Jun form the transcription factor AP-1
18 (Huang & Wange, 2004). Through these separate pathways, TCR signaling
ultimately leads to the upregulation of the transcription factors NFAT, NFkB,
and AP-1, resulting in the regulation of gene expression (Figure 6). These
pathways also converge to regulate HIV transcription by binding to elements
within the HIV 5’ LTR.
19 Minireview: T Cell Receptor Signaling
constitutively targeted to the lipid rafts by virtue of
Cytoskeletal Reorganization and Formation of
Immunological Synapse
of two juxtamembrane cysteine residues (8, 13, 14).
ein interaction domains Figure
other than
the
multiple
A
new
layer
of
complexity
in the Engagement
process of TCR signaling
has
6: T Cell Receptor Signaling Cascade.
of the TCR
ues that are phosphorylated by ZAP-70 following come to light in recent years in the form of the immunological
results in a signaling cascade of events that ultimately leads to the
. When phosphorylated, these tyrosines serve as synapse (IS), which has also been referred to as the supramolecular
upregulation
of the
transcription
factors(SMAC).
NFAT, This
NF-κB,
and AP-1.
Figureordered
taken
activation complex
is a dynamic
yet highly
or specific SH2 domain-containing
proteins.
There
(Huang
& Wange, structure
2004). that forms at the site of T cell contact with an antigen8 tyrosine residues in from
LAT that
are conserved
ns, mice, rats, and bovines (14). The C-terminal 5 presenting cell (19). The mature IS is characterized by a central
ues play critical roles in the ability of LAT to bind region (c-SMAC) that is enriched in clustered TCR and PKC#,
surrounded by a peripheral ring (pSMAC) of adhesion factors such
the linker proteins Gads and Grb2. SLP-76 assoAT via Gads. SLP-76 can also directly bind to as LFA-1 and a distal ring (dSMAC) containing proteins such as
ting the existence of higher order interactions be- the tyrosine phosphatases CD148 and CD45. The IS, although not
nd possibly other signaling proteins in the signa- required for initiating TCR signaling, is required for sustained
and proliferation (19). It also has the
ition to the proteins already mentioned, phospho- signaling, IL-2 production,
20 ability
to
act
as
a
positive
or
negative servo, either amplifying weak
lso binds to PI3K, Grap, 3BP2, Shb, SOS, c-Cbl,
TCR
signals
or
attenuating
strong signals (20).
ocalizing these molecules in close proximity and a
Clustering of lipid rafts at the contact site and formation of the
ation to one another within the lipid raft domain of
IS is an active process that requires several upstream signaling
embrane. Jurkat T cells deficient for LAT expresevents to occur. Most important of these are the signals that lead to
vere signaling defects to Ca2! mobilization, mito-
Downloaded from http://www.jbc.org/ by guest on March 30, 2016
ell receptor signaling
g to activation of tranors. This figure presents
some of the key signaling
the binding of peptideell antigen receptor (TCR/
CD4 costimulatory recepce of key phosphorylation
ted on some of the signalsmall gray circles labeled
all proteins undergoing
phosphorylation are lanates ubiquitination. The
fferent interactions is deaccompanying text. For
al clarity some important
s are not depicted; most
all molecular weight linkrb2 are excluded from the
gnalosome complex. Also
the signals contributed
costimulatory receptor toion of NF"B and the Vavfeedback loop.
HIV is able to co-opt the components of the TCR signaling pathway in
order to promote effective viral transmission and replication. It has long been
established that signals emanating from the TCR can upregulate HIV
transcription (Gruters et al., 1991), however, recent reports implicate TCR
signaling molecules in the regulation of HIV entry, viral assembly, and
release. The tyrosine kinase ZAP-70, for instance, has been demonstrated to
regulate cell-to-cell transmission of HIV through formation of the virological
synapse, a structure similar to the immunological synapse that forms between
the infected cell and an uninfected target cell (Sol-Foulon et al., 2007). PIP2
formation by the lipid kinase PI4P5 kinase Iα has been found to be a
requirement for HIV entry and infection (Barrero-Villar et al., 2008). The Src
kinase Lck has been demonstrated to aid in both viral assembly and release
at the host cell plasma membrane (Strasner et al., 2008). In addition,
activation of the MAP kinase ERK has been found to be necessary for HIV
replication and crucial for the prevention of HIV-mediated apoptosis (Furler &
Uittenbogaart, 2010). Furthermore, it has been demonstrated that signaling
from the CD28 co-stimulatory receptor can positively and negatively regulate
HIV transcription (Cook, August, & Henderson, 2002; Cook, Albacker, August,
& Henderson, 2003; Natarajan, August, & Henderson, 2010). However,
whether specific stimulatory and co-stimulatory signals at the time of infection
bias towards productive or latent infection has yet to be investigated.
21 There is, therefore, a need for further elaboration of the signaling
pathways that lead to HIV transcriptional regulation and the establishment of
a productive or latent infection within CD4+ T cells. The current study aims to
identify the role of TCR activation in determining latency as well as the
specific pathways through which TCR signaling is regulating transcription. Specific Aims
One of the most significant obstacles to finding a cure for AIDS is the
presence of latent reservoirs of HIV infection. Antiretroviral drugs are able to
prevent ongoing viral replication but are unable to eliminate the virus from
already infected cells. Therefore, patients’ viral loads are suppressed to
undetectable levels while on antiretroviral therapy, but, upon cessation of
therapy, viral loads rebound as the virus is released from latent reservoirs of
infection. In order to completely eradicate HIV, it is essential to understand
the mechanisms that contribute to the establishment and maintenance of viral
latency in the hope of discovering a mechanism for reactivating and
eliminating the latent reservoir. The purpose of the present study is to
investigate the mechanisms early in HIV infection that direct the cell towards
productive or latent infection.
Specifically, this study aims to:
22 1. Determine whether the strength of signaling at the time of HIV infection
is correlated with the level of HIV transcription.
2. Characterize the T cell receptor signaling pathways that regulate HIV
transcription.
Understanding the signaling events that lead to the initial establishment of
latency within newly infected cells is an important step in eliminating the latent
reservoir and curing AIDS.
23 METHODS
Establishment of Stable Cell Lines Expressing Chimeric Antigen
Receptors
The laboratory of Dr. Wilson Wong had previously provided chimeric
antigen receptors (CARs) expressing one of five single-chain variable
fragments (scFvs) with differing affinities for a shared ligand packaged in phR
lentiviral vectors driven by a spleen focus-forming virus (SFFV) promoter. The
CAR-containing lentiviral vectors had then been transduced into a Jurkat E6.1
T cell line by spinoculation. Three days post-transduction, the cells had then
been sorted based upon positive expression of the mCherry reporter within
the CAR using a Legacy MoFlo Cell Sorter by Beckman Coulter. Once sorted,
cell stocks had been immediately frozen down in order to minimize
downregulation of the receptors.
Cell Culture
Jurkat E6.1 cells, a CD4+ T cell line, were cultured in RPMI medium
(Life Technologies) supplemented with 5% Fetal Bovine Serum (FBS), 100
units/mL of penicillin, 100 µg/mL of streptomycin, and 2mM of L-glutamine in
250 mL flasks and incubated at 37°C with 5% CO2. Human Embryonic Kidney
293T (HEK293T) cells were cultured in complete Dulbecco’s Modification of
Eagle’s Medium (cDMEM, Cellgro) supplemented with 10% FBS, 100
24 units/mL of penicillin, 100 µg/mL of streptomycin, and 2mM of L-glutamine in
100 mm cell culture dishes and incubated at 37°C with 5% CO2.
Cell Transfection
HEK293T cells were plated in 6-well plates at a density of 3 x 105 cells
per well in cDMEM and incubated overnight at 37°C with 5% CO2. Cells were
subsequently transfected with calcium phosphate, 10 µg/mL of NL4-3.Luc
DNA plasmid, 6 µg/mL of VSV-G DNA plasmid, 2 µg/mL of Rev DNA plasmid,
and 2 µg/mL of Tat DNA plasmid to produce Vesicular Stomatitis Virus G
protein (VSV-G) pseudotyped NL4-3.Luc, an Env negative and Nef negative,
replication incompetent strain of HIV-1. Cells were transfected for five hours,
the media was replaced with fresh cDMEM, and the cells were incubated
overnight at 37°C with 5% CO2. Approximately twenty-four hours posttransfection supernatant was collected and frozen down.
Inhibitor Assays
A 96-well plate was coated with 1 µg/mL of Her2 in a solution of
phosphate buffered saline (PBS) and incubated overnight at 37°C with 5%
CO2. On day two, the wells were washed three times with PBS. The plate was
treated with a 5% FBS-PBS solution and incubated at 37°C with 5% CO2 for
one hour. 2 x 105 Jurkat cells expressing either the C6.5 or B1D2 chimeric
antigen receptor were plated in triplicate in VSV-G pseudotyped NL4-3.Luc
25 and one of the following conditions: alone, with 1 µM efavirenz, with 10 µM
PP2, with 10 µM LY294002, or 10 µM staurosporine (Table 1).
Table 1: Plate Map for Inhibitor Assay. Jurkat E6.1 cells expressing either
the C6.5 or B1D2 CAR were plated in triplicate in the presence of VSV-G
pseudotyped NL4-3.Luc and an inhibitor. Efz: efavirenz, LY: LY294002, SS:
staurosporine
1
2
3
4
5
A
C6.5 C6.5 C6.5 C6.5 C6.5
B
+
HIV
C
D
E
F
+
HIV
+
HIV
+
HIV
+
HIV
+
Efz
+
PP2
+
LY
+
SS
6
B1D2 B1D2 B1D2 B1D2 B1D2
+
HIV
+
HIV
+
HIV
+
HIV
+
HIV
+
Efz
+
PP2
+
LY
+
SS
G
H
26 7
8
9
10
11
12
The plate was incubated overnight at 37°C with 5% CO2. On day three,
the wells were washed with PBS and treated with a 20% Cell Lysis BufferPBS solution. The plate was spinoculated for one minute, and the contents of
each well were transferred to a black Costar plate. The plate was treated with
luciferase and luciferase activity was measured with a Luminometer.
27 RESULTS
The purpose of our experiment was to determine whether the strength
of signaling at the time of HIV infection is directly correlated with level of HIV
transcription and to characterize the T cell receptor signaling pathways that
regulate HIV transcription. We hypothesized that the strength of signal at the
time of infection determines the magnitude of early HIV transcription.
To test our hypothesis, we used a panel of engineered chimeric
antigen receptors composed of an extracellular Her2 binding domain,
intracellular CD28 and CD3ζ signaling domains, and an mCherry reporter,
packaged in a lentiviral vector and expressing differing affinities for the Her2
ligand (Figure 7). The use of chimeric antigen receptors provided us with a
model of T cell receptor engagement at the time of HIV infection. The CD28
and CD3ζ signaling domains enabled us to mimic the activation and costimulation of the T cell receptor while the range of Her2 binding domains
enabled us to modulate the strength of the signal given to the TCR at the time
of infection.
28 Figure 7: Structure and Binding Affinities of Chimeric Antigen
Receptors. A) Representation of Her2 chimeric antigen receptors showing
extracellular Her2 receptor and intracellular CD28 and CD3ζ with key binding
domains and tyrosine kinase residues. B) Dissociation constants of Her2 for
CARs used in experiment.
Jurkat E6.1 T cells that had been previously transduced with either the
C6.5 or B1D2 chimeric antigen receptor were simultaneously activated with
Her2 ligand and infected with VSV-G pseudotyped NL4-3.Luc single round
virus either alone or in the presence of 1 µM efavirenz, 10 µM PP2, 10 µM
LY294002, or 10 µM staurosporine. Cells infected with virus but not
stimulated with Her2 served as controls in the first set of experiments, while
29 cells infected with virus and stimulated with Her2 in the absence of an
inhibitor or in the presence of efavirenz served as controls in the second set
of experiments. Twenty-four hours post-infection, cells were lysed and
measured for luciferase activity as an indicator of HIV transcription.
To test whether the strength of signal at the time of infection directly
correlates with HIV expression, Jurkat T cells expressing the C6.5 and B1D2
CARs were infected with NL4-3.Luc in the presence and absence of Her2
stimulation and HIV expression was measured (Figure 8). In the Her2- control
condition, there was no difference in the HIV expression of cells expressing
the low affinity receptor compared with cells expressing the high affinity
receptor. However, in the presence of Her2 stimulation, there was a 1.5-fold
increase in the HIV expression of cells expressing the low affinity receptor
compared to the unstimulated control and a nearly 3-fold increase in the HIV
expression of cells expressing the high affinity receptor compared to the
unstimulated control.
30 Figure 8: Effects of Chimeric Antigen Receptor Signaling on HIV
Transcription. Jurkats expressing C6.5 or B1D2 CARs were infected with
NL4-3.Luc in the presence and absence of Her2 ligand. 24 hours postinfection, cells were measured for luciferase activity. Data is representative of
three separate experiments.
To begin to characterize the signaling pathways that regulate HIV
transcription, Jurkat T cells expressing the C6.5 and B1D2 CARs were
simultaneously infected with NL4-3.Luc and stimulated with Her2 in the
absence and presence of select inhibitors, and HIV-LUC transcription was
measured with a luciferase assay. When infected in the presence of the nonnucleoside reverse transcriptase inhibitor efavirenz, HIV transcription was
31 reduced by 91% in Jurkats expressing the C6.5 CAR and by 96% in Jurkats
expressing the B1D2 CAR. When infected in the presence of the Src kinase
inhibitor PP2, HIV transcription was reduced by 61% in Jurkats expressing
the C6.5 CAR and by 71% in Jurkats expressing the B1D2 CAR. When
infected in the presence of the PI3K inhibitor LY294002, HIV transcription
showed no reduction and, in fact, showed a 30% increase in Jurkats
expressing the C6.5 CAR, while showing a 15% reduction in Jurkats
expressing the B1D2 CAR. When infected in the presence of the PKC
inhibitor staurosporine, HIV transcription was reduced by 26% Jurkats
expressing the C6.5 CAR and by 30% in Jurkats expressing the B1D2 CAR.
32 Figure 9: Effects of Chimeric Antigen Receptor Signaling on HIV
Transcription in the Presence of Inhibitors. Jurkats expressing C6.5 or
B1D2 CARs were infected with NL4-3.Luc and stimulated with Her2 in the
absence or presence of an inhibitor. 24 hours post-infection, cells were
measured for luciferase activity. Data is representative of three separate
experiments, expect for LY294002 data, which is representative of two
separate experiments, and staurosporine data, which is preliminary. Efz:
efavirenz, LY: LY294002, SS: staurosporine
33 !
DISCUSSION
The AIDS epidemic has claimed the lives of more than 34 million
individuals worldwide. Although advancements in prevention strategies and
treatment have led to a steady decline in the rate of AIDS-related deaths in
recent years, nevertheless, more than a million deaths are attributed to AIDSrelated causes each year. Furthermore, while antiretroviral treatment can
significantly prolong the lives of individuals with HIV, it is not a cure. Even with
complete viral suppression to clinically undetectable levels, residual viremia
remains present in the blood. Upon treatment failure or cessation, viral loads
rebound to pre-treatment levels within weeks, resulting in a drop in CD4
counts and the reappearance of symptoms. Therefore, individuals with HIV
must remain on antiretroviral therapy for the rest of their lives. Yet, the
problems posed by lifelong treatment, including non-adherence due to the
high pill burden and tremendous side effect of antiretroviral drugs and,
conversely, severe adverse effects associated with long-term antiretroviral
use, make the discovery of a cure essential.
A significant barrier to curing AIDS is the presence of a latent reservoir
of cells infected with integrated but transcriptionally silent provirus. Although
attempts have been made to reactivate the latent reservoir using compounds
that inhibit key molecules indicated in transcriptional repression leading to the
34 maintenance of latency, none have shown a significant reduction in the latent
reservoir. The unsuccessfulness of efforts to reactivate the latent reservoir
highlights the need for a better understanding of transcriptional repression
and its role in latency. In particular, insight into the molecular events that lead
to the initial establishment of latency at the time of infection could lead to the
discovery of novel therapeutic targets aimed at elimination of the latent
reservoir and eradication of the virus. Preliminary research from our lab has
indicated CD4+ T cell activation at the time of infection as a possible
determinant to the establishment of productive or latent infection, which led to
the current study investigating the influence of T cell receptor signaling on
HIV transcription.
Initial infection of Jurkat E6.1 T cells expressing the C6.5 or B1D2
chimeric antigen receptor revealed that stimulation of the C6.5 low affinity
receptor at the time of infection led to a 1.5-fold increase in HIV transcription
compared to the unstimulated control, whereas stimulation of the high affinity
receptor at the time of infection led to a nearly 3-fold increase in HIV
transcription compared to the unstimulated control. These findings support
our hypothesis that the strength of signaling at the time of infection
determines the magnitude of HIV transcription as increased signaling leads to
the activation of pathways that positively regulate HIV transcription shortly
after infection. Future experiments to determine whether the differences in
HIV transcription correspond with latency will be conducted by equally
35 reactivating Jurkats expressing C6.5 and B1D2 CARs with CD3/28 antibody
beads. We hypothesize that signaling through the low affinity receptor biases
cells toward latent infection while signaling through the high affinity receptor
biases cells toward productive infection. Therefore, we expect reactivated
Jurkats expressing the C6.5 CAR to show a greater percentage increase in
HIV transcription than reactivated Jurkats expressing the B1D2 CAR.
Furthermore, additional experiments will be conducted with Jurkats
expressing mid-range affinity CARs in order to determine whether the
strength of signaling directly correlates with HIV expression. However,
preliminary data from Jurkats expressing the ML3-9 and H3-B1 mid-range
affinity receptors revealed HIV transcription levels comparable to those from
Jurkats expressing the C6.5 low affinity receptor. These preliminary results
suggest the possibility of a signaling threshold, whereby sub-optimal
stimulation below the threshold biases the cell towards transcriptional
repression while stimulation above the threshold biases the cell towards
transcriptional activation. Further experiments need to be conducted to test
the hypothesis of a signaling threshold.
Simultaneous infection and stimulation of Jurkat E6.1 T cells
expressing the C6.5 or B1D2 chimeric antigen receptor in the presence of the
Src kinase inhibitor PP2 revealed a 61% reduction in HIV transcription in
Jurkats expressing the C6.5 receptor compared with the uninhibited control
and a 71% reduction in HIV transcription in Jurkats expressing the B1D2
36 receptor compared with the uninhibited control. These findings indicate that
the Src kinase inhibitor PP2 diminishes the transcriptional effects of the CARs
and support the hypothesis that HIV transcription is regulated through TCR
signaling, as the Src kinase Lck is an early activator in the TCR signaling
pathways. Therefore, if TCR signaling is indeed mediating the regulation of
HIV transcription, Lck inhibition would lead to the inhibition of Lck-activated
downstream pathways within the TCR signaling cascade. The reduction in
HIV transcription upon addition of the Src kinase inhibitor PP2 seen in our
experiments is consistent with this hypothesis.
Simultaneous infection and stimulation of Jurkat E6.1 T cells
expressing the C6.5 or B1D2 chimeric antigen receptor in the presence of the
PI3 kinase inhibitor LY294002 revealed a 30% increase in HIV transcription in
Jurkats expressing the C6.5 receptor compared with the uninhibited control
and a 15% reduction in HIV transcription in Jurkats expressing the B1D2
receptor compared with the uninhibited control. These findings indicate that
PI3 kinase inhibition enhances HIV transcription in the Jurkats expressing the
low affinity receptors while slightly repressing HIV transcription in the Jurkats
expressing the high affinity receptors. The upregulatory effects of PI3 kinase
inhibition are consistent with previous findings from our lab that PI3 kinase
recruitment to CD28 represses HIV transcription while inhibition of PI3 kinase
leads to enhanced HIV transcription (Cook et al., 2002). This would suggest
that the TCR and CD28 co-stimulatory receptor are signaling through the PI3
37 kinase pathway to negatively regulate HIV transcription. The mechanism for
the repressive effects on HIV transcription seen in the Jurkats expressing the
high affinity receptors is less obvious. It is possible that signaling through the
high affinity receptor is producing a saturating effect on the upregulation of
HIV transcription, and, therefore, inhibition of a transcriptional repressor is
unable to further increase HIV transcription. The mechanisms by which PI3
kinase lead to a decrease in HIV transcription in this situation is unclear and
needs to be investigated further.
Simultaneous infection and stimulation of Jurkat E6.1 T cells
expressing the C6.5 or B1D2 chimeric antigen receptor in the presence of the
protein kinase C inhibitor staurosporine revealed a 26% reduction in HIV
transcription in Jurkats expressing the C6.5 receptor compared with the
uninhibited control and a 30% reduction in HIV transcription in Jurkats
expressing the B1D2 receptor compared with the uninhibited control. These
findings indicate that PKC inhibition modestly represses HIV transcription and
suggest that the TCR is signaling, at least in part, through the PKC pathway
of the TCR signaling cascade. The lesser effect of PKC inhibition on HIV
transcription as compared to the effect of Src kinase inhibition could be due to
the fact that, while the Src kinase Lck is upstream within the signaling
cascade and common to multiple signaling pathways, PKC is downstream
within one of several signaling pathways. Therefore, inhibiting Lck would
inhibit multiple Lck-activated pathways within the signaling cascade while
38 inhibiting PKC would only inhibit the PKC activated pathways. This suggests
the possibility that signaling has a combinatorial effect on HIV transcription
regulation, involving multiple pathways within the TCR signaling cascade.
Further experiments to determine the roles of different TCR signaling
pathways on HIV transcription will be conducted using inhibitors to
downstream targets within the PKC pathway, MAP kinase inhibitors, and
inhibitors to targets within the IP3/calcium mobilization pathway. In addition,
experiments using combinations of inhibitors from multiple pathways should
be conducted in order to test the combinatorial effects of signaling on HIV
transcription.
Overall, the current study provides evidence for the regulation of HIV
transcription through T cell receptor signaling pathways and suggests that
TCR signaling at the time of HIV infection may be a determinant of latency.
Further characterization of the specific TCR signaling pathways that lead to
activation or repression of HIV transcription could lead to novel strategies
aimed at eliminating the latent reservoir.
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43 CURRICULUM VITAE
MELISSA B. HERRING
43 Saint Mary’s Street #6 • Brookline, MA 02446
(318) 773-1118 • [email protected] • Birth year: 1986
Education:
Boston University School of Medicine, Boston, MA
• Master of Science: Medical Sciences, May 2016
Sept. 2014—present
Boston University School of Education, Boston, MA May 2011—Sept. 2014
• Master of Education: Curriculum & Teaching, September 2014
Louisiana State University, Baton Rouge, LA
Aug. 2004—May 2009
• Bachelor of Science: Biological Sciences, May 2009
• Minor: French
Work Experience:
Boston University School of Medicine, Boston, MA
Sept. 2015—present
Graduate Research Assistant, Principle Investigator: Andrew Henderson,
PhD
• Participating in a project to determine how cell signaling pathways affect
HIV transcription in infected cells
• Perform the lab techniques of cell culture, cell transfection, and virus
collection
Boston University School of Medicine, Boston, MA
Sept. 2015—Dec. 2015
Biochemistry Tutor
• Served as a tutor for a graduate course in Biochemistry and Cell Biology
• Worked with students to provide them with a deeper understanding of the
material and to sharpen their problem solving and critical thinking
strategies
East Baton Rouge Parish School System, Baton Rouge, LA Aug. 2010—May
2011
Substitute Teacher
• Had full responsibility of kindergarten through fifth grade classrooms in
diverse urban elementary schools when teacher was absent
• Responsible for completing all learning objectives and lesson plans left by
teacher
44 Our Lady of the Lake RMC, Baton Rouge, LA
Oct. 2007—April 2010
Medical Scribe, Emergency Department
• Conducted patient interviews to obtain medical histories from patients
admitted into the emergency room
• Communicated with specialists on behalf of emergency physician
• Verified patient contact information for billing purposes
• Compiled patient discharge paperwork
Volunteer Experience:
Medical Mission Trip, Santiago, Dominican Republic
Aug. 2014
Medical Team Volunteer
• Volunteered with an organization that sets up medical clinics to provide
treatment to impoverished villages in and around Santiago
• Interviewed patients in triage to determine “chief complaint,” sorted
medications, filled prescriptions, and provided patients with discharge
instructions
• Served as translator for triage nurses, nurse practitioners, and in the
pharmacy.
Bolivia Mission Trip, Santa Cruz, Bolivia
Spring Break 2009, Summer
2009—2010
Campus Crusade for Christ Volunteer
• Organized fundraising events to raise money for the trip
• Built churches in impoverished villages outside the cities of Santa Cruz
and La Paz
• Taught English classes at one of the local universities
• Assisted with college and children’s ministries
Medical Centers of West Africa, Meskine, Cameroon Sept. 2009—Nov. 2009
Hospital Volunteer
• Shadowed physicians and physician’s assistant during rounds in the
hospital wards, outpatient clinic, and operating room
• Assisted nurses in the pediatric ward with writing orders and administering
medications
• Assisted the community health physician in running a vaccination clinic
once every two weeks
Medical Mission Trip, Nueva Rosita, Mexico
Spring Break 2005
Medical Team Volunteer
• Accompanied a team of physicians, nurses practitioners, and pharmacists
to provide medical treatment to rural villages of Mexico
• Took vital signs, filled prescriptions, and entertained children in the
“waiting area”
45 Extracurricular Experience:
Kids Hope USA, Wildwood Elementary, Baton Rouge, LA Sept. 2008—May
2011
Student Mentor
• Met with an at-risk student in an urban elementary school once a week to
provide him with individual attention and instruction in reading and math
as well as spending time reading and playing games with him
Common Grounds, Baton Rouge, LA
January 2008—May 2011
Volunteer Barista
• Served as a volunteer at a non-profit coffee shop as part of the church’s
college ministry to provide students with free coffee and desserts and a
place to study
• Served as a barista for two hours a week and as a dessert baker once a
month
Up ‘til Dawn, Louisiana State University, Baton Rouge, LA Aug. 2005—May
2008
Volunteers’ Coordinator, August 2006—May 2007
• Part of a team of ten executive board members that planned and
organized an annual Letter Writing Party and Finale Event to raise money
for St. Jude Children’s Research Hospital
• Responsible for recruiting and coordinating volunteers to help during the
organization’s events
Delta Gamma, Louisiana State University, Baton Rouge, LA
Aug. 2004—
May 2008
Vice Party Chair, Philanthropy Round of Recruitment, Summer 2007
• Assisted the Vice President of Philanthropy in planning and organizing a
philanthropy event attended by more than 600 girls that benefited and
raised awareness for Service for Sight for the Blind and the Baton Rouge
Eye Bank
• Volunteered at the Louisiana School for the Blind and at events held for
the children of Our Lady of the Lake hospital, such as Arts and Crafts Day
and the annual Tri Delta and Delta Gamma Easter Egg Hunt, throughout
my four years as a member and was the first recipient of the Serving
Sister Award for exceptional service
46