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Boston University OpenBU http://open.bu.edu 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. 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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