UNDERSTANDING THE ROLES OF NUCLEAR RECEPTORS IN THE MAINTENANCE OF HIV PROVIRAL LATENCY USING NOVEL GENE EDITING TECHONOLOGY STEPHANIE C.

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1 UNDERSTANDING THE ROLES OF NUCLEAR RECEPTORS IN THE MAINTENANCE OF HIV PROVIRAL LATENCY USING NOVEL GENE EDITING TECHONOLOGY By STEPHANIE C. MILNE Submitted in partial fulfillment of the requirements for the degree of Master of Science Molecular Virology CASE WESTERN RESERVE UNIVERSITY August, 2015

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Stephanie C. Milne candidate for the degree of Master of Science*. Research Advisor Jonathan Karn Committee Chair John Tilton Committee Member Gary Landreth Committee Member Cornelia Bergmann Date of Defense March 12, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein.

3 TABLE OF CONTENTS List of Tables... iii List of Figures... iv List of Abbreviations... v Abstract... viii Specific Aims... 1 Research Strategy... 5 A. Background and Significance... 5 A.1. Proviral latency is a major hurdle for eradication and cure strategies... 5 A.1.1. Mechanisms of post-integration latency are complex among different cell types... 5 A.1.2. Proviral latency occurs in a number of cellular reservoirs... 7 A.1.3. Studying mechanism of latency using novel gene editing technology... 8 A.2. Significance B. Innovation C. Approach C.1. Specific Aim 1: Optimize the lentiviral CRISPR/Cas9 system for use in CD4 + T-cell and microglial proviral latency models C.1.1. Construction of lenticrispr vectors for enhanced selection and decreased off-target effects i

4 C.1.2. Producing efficient lenticrispr infection in primary cell latency models C.2. Specific Aim 2: Elucidate the roles of specific nuclear receptors in the maintenance of proviral latency C.2.1. Demonstrating the significance of nuclear receptors in proviral latency C.2.2. Generating CRISPR knockout cell lines of specific nuclear receptor in immortalized and primary T-cell and microglial cell lines C.2.3. In-depth characterization of the molecular mechanisms utilized by nuclear receptors to control proviral latency C.3. Specific Aim 3: Gain further understanding of the roles of epigenetic modifiers in proviral transcriptional silencing C.3.1. Utilizing the CRISPR/Cas9 gene editing system to produce complete gene knockouts of specific modifiers C.3.2. Elucidate the interactions between epigenetic modifiers and other binding partners used to control proviral latency Summary and Conclusions Bibliography ii

5 LIST OF TABLES Table 1. Nuclear receptors and their potential roles in regulating HIV latency Table 2. Epigenetic modifiers and their roles in proviral latency iii

6 LIST OF FIGURES Figure 1. Using the CRISPR/Cas9 system for protection and engineering Figure 2. Knocking out GFP using the lenticrispr construct Figure 3. Incorporation of the HcRed and Thy1.2 selection markers into the lenticrispr vector Figure 4. Unbiased shrna screen identified nuclear receptors as integral proteins in maintenance of proviral latency Figure 5. Nuclear receptor stimulation influence proviral reactivation in a cell type dependent manner Figure 6. ERα knockout sensitizes Jurkat/HIV (2d10) cells to proviral reactivation Figure 7. Comparing the roles of epigenetic modifiers in Jurkat/HIV (2d10) and CHME-5/HIV cells iv

7 LIST OF ABBREVIATIONS AIDS Acquired Immune Deficiency Syndrom Cas9 CRISPR associated protein 9 ChIP CHME CNS Co-IP Chromatin immunoprecipitation Rat microglial cells Central nervous system Coimmunoprecipitation Chicken ovalbumin upstream promoter COUP-TF transcription factor Clustered regularly interspaced short palindromic CRISPR crrna d2egfp Env ER FACS GR repeats CRISPR RNA destabilized enhanced green fluroescent protein Envelope Estrogen receptor Fluorescence-activated cell sorting Glucocorticoid receptor H3K4 Histone 3, lysine 4 H3K4me1 Histone 3, lysine 4 monomethylation H3K9 Histone 3, lysine 9 H3K9me3 HAART HAND Histone 3, lysine 9 trimethylation Highly active antiretroviral therapy HIV-associated neurocognitive disorders v

8 HAT HDAC HDACi HEK HIV HMT HRE Hμ Indel IRES Histone acteyltransferase Histone deacetylase Histone deacetylase inhibitor Human embryonic kidney Human Immunodeficiency Virus Histone methyltransferase Hormone respone elements Human microglia insertions/deletions Internal ribosomal entry site lenticrispr lentiviral CRISPR LTR MOI Nef NFAT NF-κB PAM PPAR ptefb RAR Rev RXR SAHA Long terminal repeat Multiplicity of infection Negative factor Nuclear factor of activated T cells Nuclear factor-κb Protospacer adjacent motif peroxisome proliferator-activated receptor Postive transcription elongation factor b Retinoic acid receptor Regulation of expression of viral proteins Retinoid X receptor Suberoylanilide hyroxamic acid vi

9 Seq sgrna shrna Tat Sequencing single guide RNA Short hairpin RNA trans-activator Thy1.2 Thymus cell antigen 1 TR tracrrna Vpu VSVG Thyroid hormone receptor Transactivating CRISPR RNA Viral protein U Vesicular stomatitis virus glycoprotein vii

10 Understanding the Roles of Nuclear Receptors in the Maintenance of HIV Proviral Latency Using Novel Gene Editing Technology Abstract by STEPHANIE C. MILNE The eradication of HIV-infected cells is complicated by the virus s ability to integrate within the host cell genome without subsequent expression. Although the administration of HAART can reduce viremia to levels below limits-of-detection, expression of latent provirus can be reactivated, leading to the production of new virus particles resulting in the infection of new cells. We have identified several nuclear receptors as potential candidates for maintaining proviral latency in either CD4 + T-cells or microglial cells. We will generate CRISPRs targeting diverse nuclear receptors to produce complete gene knockouts and perform subsequent experiments to understand their role in latency. Ultimately using this the CRISPR/Cas9 system, we will develop a more complete understanding of the molecular interactions leading to proviral latency. viii

11 SPECIFIC AIMS Human immunodeficiency virus type 1 (HIV-1) infection leads to the integration of proviral DNA into the host cell genome, allowing for continued virus production; however, transcriptional blocks can prevent proviral replication, leading to the production of latently infected reservoirs. Although the introduction of highly active antiretroviral therapy (HAART) has made HIV-1 infection a more manageable condition by blocking viral replication, HAART is unable to target latent proviruses, making complete eradication of the virus difficult. Although a patient may exhibit undetectable levels of viremia, release of transcriptional blocks, such as lack of transcription factors and chromatin modifications, incompliance with their HAART regimen, or general inflammatory responses can lead to transcriptional reactivation resulting in the production of infectious virus. The molecular mechanisms that are used to establish proviral latency are complex, involving a wide variety of factors to achieve transcriptional silencing. These mechanisms vary depending on the cellular reservoir, making complete eradication of the virus difficult. Both CD4 + T-cells and microglial cells serve as reservoirs for latent provirus, with CD4 + T-cell being the primary target for HIV infection, and microglia being implicated in the development of HIV-associated neurocognitive disorders (HAND). In order to understand complexities of the molecular mechanisms contributing to proviral latency in both CD4 + T-cells and microglial cells, we will examine a variety of proteins noted to contribute to transcriptional silencing in both cell types. In doing so, we will examine the similarities and differences in latency mechanisms among the two cell types, 1

12 ultimately using the mechanistic commonalities for potential cure strategies. We will examine the roles of nuclear receptors and epigenetic modifiers because the signaling pathways of these proteins are strongly interconnected. Additionally, many of these molecules were strong hits in genome wide shrna screens. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing system to produce complete gene knockouts of proteins of interest, we will gain a further understanding of the vast mechanisms of proviral latency by examining targets that are integral in the maintenance of proviral silencing and reactivation. Specific Aim 1: Optimize the lentiviral CRISPR/Cas9 system for use in CD4 + T-cells and microglial proviral latency models. The CRISPR/Cas9 system is a relatively new method of gene editing, therefore we must optimize the system for use in our latency cell models. We are currently using a lentiviral vector (lenticrispr) to introduce the CRISPR/Cas9 constructs into both immortalized and primary CD4 + T-cells and microglia. We will modify the current vector to improve selection of infected cells in our latency models. To prevent complications of permanent integration of the CRISPR/Cas9 construct and to limit off-target effects, we will also manipulate the lenticrispr vector to incorporate a lox/cre recombinase system to halt the constitutive expression of Cas9 after DNA cleavage has occurred. We will also optimize infection conditions for introduction into primary cell models. Specific Aim 2: Elucidate the roles of specific nuclear receptors in the maintenance of proviral latency. Nuclear receptors have been shown to 2

13 interact with the HIV 5 long terminal repeat (LTR) and act as transcription factors for proviral replication. However, little is understood about the contributions of nuclear receptors in regulation of proviral latency in different cellular reservoirs. Using the CRISPR/Cas9 system, we will generate complete gene knockouts of various nuclear receptors, and compare reactivation patterns in both immortalized and primary CD4 + T-cells and microglia, with the overall goal of understanding the complex mechanisms that these receptors use to reactivate or silence proviral transcription. Specific Aim 3: Gain further understanding of the roles of epigenetic modifiers in proviral transcriptional silencing. The roles of epigenetic modifiers in proviral transcription have been extensively studied. Targeting epigenetic modifiers may provide vast therapeutic prospects, however there is still more to be understood. In a similar method as described with the nuclear receptors, we will use the CRISPR/Cas9 gene editing system to generate complete gene knockouts of epigenetic modifiers, including various histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone methyltransferases (HMTs). This will provide information about the therapeutic prospects that could not be garnered using inhibitors or shrna. The proposed studies will not only provide vital information regarding the differences in mechanisms involved in proviral transcription in T-cells and microglia, but they will also help to determine the utility of the CRISPR/Cas9 system as a therapeutic strategy. There is great potential to generate a CRISPR/Cas9 construct to specifically target HIV-infected cells and knock out 3

14 specific transcription factors; however, more information is needed about the molecular mechanisms of proviral latency. By examining the roles of various nuclear receptors and epigenetic modifiers, we will gain a better understanding of both proviral transcriptional silencing and reactivation that could not be acquired previously, and better define potential therapeutic approaches to eradicate latent proviral reservoirs. 4

15 RESEARCH STRATEGY A. Background and Significance A.1. Proviral latency is a major hurdle for eradication and cure strategies When infection by HIV-1 was first discovered to be the cause of acquired immune deficiency syndrome (AIDS), the disease was considered a death sentence. The introduction of HAART made HIV infection more manageable, blocking viral replication at multiple stages of infection resulting in reduced levels of viremia to below limits of detection. Although viral levels in the peripheral blood are undetectable, integrated, transcriptionally inactive proviruses are harbored in both T-cells and myeloid cells. These latent proviruses pose issues for the treatment and complete eradication of HIV because they are not targeted by HAART. While the provirus may be transcriptionally inactive, incompliance with HAART treatment or general inflammatory responses can cause proviral reactivation, resulting in the production and propagation of infectious virus. A.1.1. Mechanisms of post-integration latency are complex among different cell types The molecular mechanisms of HIV latency are complex and not completely understood. Upon infection, two states of latency can occur. Pre-integration latency, which occurs due blocks in the viral life cycle prior to integration, such as poor reserve transcriptase activity, leads to inadequate production of proviral DNA. It contributes very little to viral persistence due to the limited half-life of the unintegrated virus (1). In contrast, post-integration latency occurs after viral 5

16 genome has been reverse transcribed and successfully integrated into the host genome. Despite successful integration, little to no proviral replication occurs (1). The lack of viral replication is the primary reason for establishment of latent reservoirs and can result from many mechanisms that vary among different cell types. These include transcriptional interference, which results from viral integration within a highly expressed host gene leading to transcription at one promoter interfering with transcription at another, and limited recruitment of transcription elongation factors, namely ptefb (2). Mechanisms of proviral latency that are of particular interest in the proposed studies include sequestration of transcription factors and epigenetic modifications. Proviral transcription is restricted by the availability of transcriptional factors including NF-κB and NFAT, which are important for transcriptional initiation (2). Other proteins, such as various nuclear receptors, may also play a significant role in the maintenance of HIV latency. Nuclear receptors can influence expression of genes involved in metabolic and immune responses through ligand-dependent signaling (3). Several hormone receptors, including estrogen, thyroid hormone, glucocorticoid receptors, and non-steroid receptors, including retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs), have been implicated as possible factors for the maintenance of proviral latency due to their strong interactions with the HIV 5 LTR (4). Differences in nuclear receptor expression among cell types may influence reactivation and silencing of proviral transcription. Through the proposed studies, we will elucidate the mechanisms used by these receptors in the maintenance of proviral latency. 6

17 The chromatin environment within the HIV 5 LTR plays an integral role in the maintenance of HIV latency. Specific epigenetic modifications can influence chromatin structure in ways that either promote or prohibit proviral transcription. Modifications such as histone acetylation result in chromatin relaxation leading to the recruitment of transcriptional machinery to the promoter and transcription initiation (5). In contrast, histone deacetylation leads to chromatin constriction, thus halting transcription (5). Histone deacetylases (HDACs) have become a popular target for potential eradication strategies. Treatment with HDAC inhibitor vorinostat resulted in proviral reactivation and increased histone acetylation in vivo, allowing latently infected cells to be targeted for killing (6, 7). Additionally, histone methylation may have a role in maintaining HIV latency; however, it is less well defined. By studying the interplay between nuclear receptors and epigenetic modifiers, we will gain a greater understanding of maintenance mechanisms in both T-cells and microglial cells. A.1.2. Proviral latency occurs in a number of cellular reservoirs The primary reservoir for latent provirus is resting CD4 + T cells, including both naïve and memory populations (8-10). Early infection of activated CD4 + T- cells with CCR5-tropic viruses most often results in rapid cell death; however, in rare instances activated CD4 + T-cells can be infected while reverting back to a resting state (9). Because of the differences in the transcriptional environment in resting versus activated CD4 + T-cells, HIV-1 gene expression is repressed resulting in proviral latency (9). Naïve CD4 + T-cells have also been reported as 7

18 reservoirs for latent provirus. These cells are still readily infectable despite very low expression of the CCR5 co-receptor (11). In addition to T-cells, marcophages and other myeloid cells have been demonstrated to harbor latent provirus. Microglial cells, which serve as the primary immune cells of the central nervous system (CNS), serve as another primary proviral reservoir. HIV-1 infection of microglia and perivascular macrophages has been attributed to the development of HAND. Activation of latently infected microglia results in the secretion of viral proteins as well as inflammatory cytokines leading to neurodegeneration (12). Both resting memory CD4 + T-cells and microglia cells serve as ideal proviral reservoirs due to low cell turn-over, allowing stable integration of the provirus without active transcription. Due to the lack viral production, HIV-1 latently infected cells cannot be targeted for killing by HIV-1 activated lymphocytes, allowing them to remain viable within the host. A.1.3. Studying mechanisms of latency using novel gene editing technology To study proviral latency, our lab has developed latent cell models in both CD4 + T-cells and microglia. Immortalized Jurkat T-cells and human microglial cells (Hμ), cells were infected with a single-round, VSVG-pseudotyped HIV bearing a fragment of HIV-1pNL4-3, containing Tat, Rev, Env, and Vpu, cloned into the phr backbone, plus a d2egfp reporter gene inserted next to Env (13). Upon infection, the virus is allowed to enter latency. Latent cells are then sorted by FACS based on reactivation profiles, and clonal populations are produced. Similar methods are 8

19 also used to produce latently infected primary CD4 + T-cell subsets and microglial cells While our lab has studied the relevance of specific proteins involved in proviral latency using shrna, issues associated with RNAi prevent us from completely understanding the roles of particular proteins. Although shrna is capable of producing gene knockdowns, these knockdowns are not permeant with uncertain efficiencies (15). shrna is also known to produce off target effects with limited target confirmation. In order to combat these issues, we will produce complete gene knockouts in our latently infected cell systems by employing the CRISPR/Cas9 gene editing system. The CRISPR/Cas9 system was originally identified as an adaptive immune mechanism used by bacteria to protect against foreign nucleic acids (Figure 1). Bacteria utilizing type II CRISPR/Cas9 systems, which are widely utilized for gene editing, integrate sequences from foreign DNA (protospacers), such as a plasmid or virus, between CRISPR repeat sequences. Transcription of the CRISPR repeat sequence and its protospacer produces CRISPR RNA (crrna), which when hybridized with a transactivating CRISPR RNA (tracrrna), can complex with the Cas9 nuclease (14). The crrna directs Cas9 to complementary sequence of protospacers within the foreign DNA. If the foreign DNA target sequence is directly 5 of necessary protospacer adjacent motif (PAM) sequence, Cas9 can produce a double-stranded break within the target sequence, which is then repaired by non-homologous end-joining or homologous recombination (14, 16). These repair mechanisms can be taken advantage of for gene editing purposes; non-homologous end-joining will produce 9

20 insertions/deletions (indel) mutations within the target sequence, while homologous recombination can be used to introduce specific sequences into the gene of interest. We will use the CRISPR/Cas9 gene editing system in order to study proviral latency by examining the phenotypes resulting from various nuclear receptor and epigenetic modifier knockouts as well as understanding their interactions with other proteins. Figure 1. Using the CRISPR/Cas9 system for protection and engineering. CRISPR/Cas9 systems utilized by (a) bacteria for defense against foreign nucleic acids and (b) those used for genetic engineering (14). 10

21 A.2. Significance Identifying and comparing important targets for maintenance of proviral latency in CD4 + T-cells and microglia cells will provide valuable insight into the complex molecular mechanisms of latency. In the following studies, we will to identify nuclear receptors and epigenetic modifiers that when specifically knockedout will lead to either permanent proviral silencing or constitutive reactivation in both cell types. Achieving permanent proviral silencing could provide a potential functional cure by permanently blocking proviral transcription, preventing the production of new virus. In contrast, causing constitutive proviral reactivation would open doors for the popular shock and kill strategy, allowing latently infected cells to be targeted by HIV-1activated lymphoid cells without having to use other drug treatments. B. Innovation The interruption of HAART and general inflammatory stimuli are enough to reactivate transcription of latent proviruses, leading to the production of newly infectious virus. Current strategies used to the purge latent reservoirs such as shock and kill, have been a topic of much discussion. This strategy works by reactivating latent provirus using drugs such as histone deacetylase inhibitors (shock) and the immune system destroying HIV infected cells (kill) (7). Although there has been success in showing the potential of HDACi vorinostat in shock and kill strategies, there are still many questions and potential issues that need be 11

22 addressed, including the inability of the drug to reactivate all latently infected cells and the negative side effects associated with the drug. Rather than using generalized inhibitors that may have adverse effect on uninfected cells, we hope that the proposed studies will ultimately lead to the use of CRISPRs or similar gene editing technologies in eradication strategies. CRISPRs have previously been utilized to disrupt the HIV 5 LTR in Jurkat T-cells and CHME-5 rat microglial cells (17, 18), however this strategy have yet to be used to study specific proteins that are involved in maintaining HIV latency. Our lab has identified proteins of interest using shrna library screens. While these studies provide information about potential targets, using shrna to study the mechanism of latency is limited in that the production of complete knockdowns is not possible. We propose to use CRISPRs to completely disrupt target gene, allowing us to gain an understanding that would be possible otherwise. After identifying potential therapeutic targets in primary cells, we hope to generate a CRISPR/Cas9 that can specifically target latently infected cells, which is currently not possible with generalized inhibitors of HAART. The inability to target latent HIV reservoirs poses issues for complete eradication of the virus. Further complicating complete eradication is the diversity of the molecular mechanisms used to maintain latency within the different reservoirs. While understanding the mechanisms of latency in the resting CD4 + T- cell primary reservoirs is important, it is also essential to make comparisons between CD4 + T-cells and other reservoirs, such as microglial cells. It is important to compare the mechanisms of the different reservoirs in order to develop the most 12

23 comprehensive cure strategy. We have previously shown that mechanisms utilized by T-cells and microglial cells do differ. The unbiased shrna screens performed in our lab have demonstrated these differences, including the utilization of different estrogen receptor subsets in microglia versus T-cells. By performing pseudoscreens using CRISPRs, we will better understand the differences between both reservoirs and identify potential therapeutic targets common among microglia and T-cells. Much of the focus in HIV cure strategies has been targeting latent reservoirs by reactivating proviral transcription. While we plan to investigate nuclear receptors and epigenetic modifiers that are involved in transcriptional repression, we also want to examine proteins that activate proviral transcription. For example, histone acetyltranferases (HATs), such as p300, promotes proviral transcription through chromatin relaxation. By knocking out specific HATs, we would potentially eliminate important chromatin acetylation within the proviral LTR, thereby preventing transcription from occurring. Identifying similar targets would open doors to finding a functional cure HIV by permanently repressing latent reservoirs. C. Approach The main goal of the proposed studies is to further the understanding of the mechanisms of proviral latency by examining connections between nuclear receptor signaling and epigenetic modification in the context of transcriptional repression. Multiple nuclear receptors have been shown to interact with proviral 5 LTR to yield transcriptional activation or silencing; however, 13

24 these events have also been shown to involve epigenetic modification, including DNA histone methylation and acetylation. Additionally, epigenetic modifying proteins have been shown to interact with various nuclear receptor proteins. By examining the dynamics between nuclear receptors and epigenetic modifiers, we will gain a greater understanding of the complex mechanism of proviral latency. C.1 Specific Aim 1: Optimize the lentiviral CRISPR/Cas9 system for use in CD4 + T-cell and microglial proviral latency models. Rationale: Many of the proteins that we wish to investigate in the proposed studies were originally discovered as important regulators of proviral latency through genome-wide, unbiased shrna screens. While these screens provide pertinent information about specific proteins and their potential role in proviral latency, their limitations, including incomplete loss of gene activity and short-lived nature, pose issues in evaluating the roles of particular proteins of interest (15). In order to combat these limitations, we propose to perform pseudoscreens of various nuclear receptors and epigenetic modifiers by creating total gene knockouts using the CRISPR/Cas9 gene editing system. Guided by target sgrna sequences, Cas9 can specifically produce doublestranded breaks in the DNA sequence of the protein of interest, ultimately producing a permanent gene knockout. Using CRISPR/Cas9 will not only confirm the results of the shrna screens, but also provide novel information regarding specific cell signaling pathways in the context of proviral transcription. Because of the novelty of this gene editing technology, it is imperative that the system be optimized for use in our particular immortalized 14

25 latency cell models. Using similar strategies, we plan to produce complete gene knockouts in primary CD4 + Tcells and microglial cells. C.1.1. Construction of lenticrispr vectors for enhanced selection and decreased off-target effects The CRISPR/Cas9 system has previously been utilized to modify the HIV LTR in both Jurkat T-cell and CHME-5 latently infected cell lines (17, 18). In both cases, cells were transiently transfected with CRISPR/Cas9 constructs containing sgrnas targeting various sequences within the LTR and proviral reactivation was measured. For the proposed studies, rather than using transient transfection, we have chosen to infect cells with a lenticrispr virus, a lentiviral vector encoding Cas9, the sgrna target sequence of interest, as well as a puromycin reporter (Addgene #49535). By using a lentiviral vector, we can more effectively introduce the CRISPR/Cas9 construct into cells that are difficult to transfect, such as Jurkat T-cells and primary cells. To ensure the efficacy of infection with lenticrispr viruses, we first performed infections of latently infected Jurkat/HIV (2d10) cells and the immortalized human microglial cell clone, Hμ/HIV (C13) using a lenticrispr targeting GFP (Figure 2). As expected, infections in both cell lines resulted in significant loss of GFP expression. Following infection, when Jurkat/HIV (2d10) cells were reactivated with TNF-α and HDACi SAHA, minimal GFP expression resulted (Figure 2A). Conversely, following the infection of the constitutively activated Hμ/HIV (C13) cells, GFP expression was dramatically reduced within three days post infection (Figure 2B). 15

26 Figure 2. Knocking out GFP using the lenticrispr construct. (A) Jurkat/HIV (2d10) were infected with a VSVG pseudotyped lenticrispr sgrna GFP virus. Infected cells were selected using puromycin 1 day post infection. Following selection, cells were treated with suboptimal levels of TNF-α and HDACi SAHA for 16 hours and GFP expression was measured by flow cytometry. (B) Hμ/HIV (C13) were infected with increasing volumes of lenticrispr sgrna GFP virus. GFP expression of the mixed population was measured 3 days post infection. The original lenticrispr vector used to create the GFP knockouts possesses a puromycin resistance gene for selection of infected cells. Although puromycin can be an effective method of selection, we fear its toxicity may affect successfully infected cells should the knockout produce cell fragility. In order to improve the selection of infected cells and prevent unwanted toxicity, we will enhance the current lenticrispr vector by substituting the puromycin resistance 16

27 gene for additional selection markers. We will incorporate both a fluorescent HcRed reporter as well as the glycophosphatidylinositol (GPI)-linked surface protein, thymus cell antigen 1 (Thy1.2), into the lenticrispr vector (Figure 3). In doing so, we will have two separate markers that we can use to detect infected cells by flow cytometry, microscopy, and magnetic isolation. Figure 3. Incorporation of the HcRed and Thy1.2 selection markers into the lenticrispr vector. Using a multi-stage cloning strategy, HcRed and Thy1.2 sequences will be incorporated in the lenticrispr backbone. The 2kb filler is utilized for the insertion of sgrna sequences. Adapted from Addgene. To incorporate both HcRed and Thy1.2 into the lenticrispr vector, we will use a multi-stage cloning strategy. We will first isolate the HcRed DNA sequence and its SV40 promoter from the pbabehcred plasmid (Addgene #10677) and insert it upstream of internal ribosomal entry site (IRES) sequence within the pmithy1.2 plasmid (Bioss #232). Doing so will allow simultaneous translation of both proteins in the final construct (19). We will replace the puromycin resistance gene within the lenticrispr vector with the Hc-Red-Thy1.2 fragment resulting in the construct shown in Figure 3. We will be able to use HcRed as a fluorescent reporter to monitor lenticrispr infection. Additionally, we will be able use both the HcRed fluorescent reporter and the Thy1.2 surface antigen for sorting by FACs, ultimately yielding cleaner populations and less harmful selection. 17

28 One of the main concerns when using the CRISPR/Cas9 system is the frequency of off-effects. Previous data show that even single and double nucleotide mismatches between the guide RNA and target DNA sequences can result in Cas9 cleavage (20). While we can control what guide RNAs are chosen to minimize the risk of off-target effects, infection with the lenticrispr virus results in integration of the construct and constitutive expression of the Cas9 nuclease, which may result in off-target gene cleavage. In order to reduce these risks, we will produce lenticrispr vector containing loxp sites to remove Cas9 following gene cleavage. To achieve this, we will clone a loxp site into the SIN U3 region of the 3 LTR, allowing for production of the same loxp sequence in the 5 LTR following reverse transcription. This will produce loxp sites that flank the lenticrispr proviral DNA within the cellular genome. Following Cas9 cleavage, we will introduce Cre recombinase through a second infection using a self-deleting Cre lentivirus carrying a fluorescent reporter to prevent the cytotoxic effects associated with prolonged Cre expression (21). Cre expression will lead to the removal of the lenticrispr DNA, halting the constitutive expression of the Cas9 and its guide RNA. Data from our lab has shown that in Jurkat/HIV (2d10) cells, GFP expression decreases within approximately 48 hours post infection with lenticrispr GFP virus. Knowing this, when infecting with lenticrispr-loxp viruses, we will infect with a second virus carrying Cre-recombinase and a fluorescent reporter at 36 hours post infection to shut down Cas9 expression. We can then assess the efficacy of the superinfection by microscopy and flow cytometry. By eliminating Cas9 expression following cleavage of the target, we should minimize cleavage of 18

29 off-target genes, ensuring that any resulting phenotype is due to cleavage of the gene of interest. C.1.2. Producing efficient lenticrispr infection in primary cell latency models While producing complete gene knockouts in the immortalized cell models will provide information about molecular mechanisms of latency, the ultimate goal is to be able to utilize the lenticrispr system in primary CD4 + T-cells and microglia because it the most similar environment to in vivo systems. Because primary cells are generally more difficult to infect than immortalized cell lines, we will utilize our lenticrispr sgrna GFP virus to optimize infection in both primary CD4 + T-cells and primary microglia. To generate the latent primary T-cell model, naïve CD4 + T-cells isolated from patient peripheral blood mononuclear cells (PBMCs) obtained from leukopacks are polarized using specific cytokines to generate four effector T-cell subsets: TH1, TH2, TH17, and Treg cells. Once polarized, cells are infected with phr -Nef(+)CD8a/GFP virus. The CD8a/GFP fusion protein allows for monitoring of infection by GFP and selection of positively infected cells by magnetic isolation for CD8a. Once isolated, CD8a + cells are quiesced by gradually lowering the concentration of IL-2 in the media, as well as IL-23 for the TH17 cells. Quiescence markers, including ps175 and CycD1, as well GFP and Nef are monitored by flow cytometry while cells enter latency. Primary microglia are isolated from epileptic patient brain tissue samples procured by neurosurgeon, Dr. Jonathan Miller. From both the epileptic and 19

30 healthy tissue samples we receive, separate single-cell suspensions are produced using the Neural Tissue Dissociation Kit (Miltenyi). From the neuronal cell suspension, microglia cells are selected through magnetic isolation using CD11b (Microglia) MicroBeads (Miltenyi). Once the microglia are isolated, they can either be used in their primary state, or they can be immortalized by infecting the SV40 virus for other experiments. We have also shown that these cells are readily infectable with replication competent HIV virus. To optimize lenticrispr infection in both primary CD4 + T-cell subsets and primary microglia, we will utilized the lenticrispr targeting GFP. Because superinfection in primary cells can be difficult, we plan to perform co-infections by spinoculation with the lenticrispr sgrna GFP virus with the phr -Nef(+)- CD8a/GFP. Both viruses will be titered in Jurkat/HIV (2d10) and uninfected Jurkat (E6) cells respectively prior to infection in the primary T-cells, and in the constitutively active Hμ/HIV (C13) prior to infection in the primary microglia. Successful infection will eliminate GFP expression. Because the CD8a sequence of the CD8a/GFP fusion protein is upstream of GFP, it should be unaffected by indel mutations within GFP. We will monitor HIV infection by flow cytometry using a CD8a antibody. While we expect GFP levels to remain very low throughout infection, CD8a should be detectable and vary based on the success of the infection. Genomic DNA will isolated and sequence using the Ion Torrent to detect indel mutations in GFP. We will also confirm the knockouts by western blot and qrna PCR. 20

31 C.2. Specific Aim 2: Elucidate the roles of specific nuclear receptors in the maintenance of proviral latency Rationale: One of the main classes of proteins that we are interested in studying in the context of HIV latency is nuclear receptors. Nuclear receptors are ligand-regulated transcription factors composed of three major domains that help to control transcriptions of their target genes (22). The amino-terminal transactivation domain recognizes other co-activators and transcription factors and vary among the different receptors in the family. The carboxy-terminal ligandbinding domain allows for specific ligand binding to occur. The central DNAbinding domain is the most conserved on the three domains, recognizing specific target sequences and activating genes (3). Binding of nuclear receptor monomers, homodimers, or heterodimers to hormone response elements (HREs) within the DNA in response of ligand-binding regulates transcription of specific target genes. In our case, we are interested in nuclear receptors that interact with the HIV 5 LTR, including those listed in Table 1. Our goal is to understand how different nuclear receptors control HIV latency in both microglia and CD4 + T-cells and to compare and contrast their roles in the different reservoirs. Based on shrna screens performed in both CHME-5/HIV and Jurkat/HIV (2d10) cells, we demonstrated that different proteins may play a more pertinent role in regulating one cell type than the other (Figure 4). Generating knockout cell lines of these receptors will not only help us to comprehend the complexities of latency pathways, it will also provide potential therapeutic targets for future eradication strategies. 21

32 22

33 o Figure 4. Unbiased shrna screen identified nuclear receptors as integral proteins in maintenance of proviral latency. CHME-5/HIV and Jurkat/HIV (2d10) cells were transduced with the Human Genome-Wide shrna Library (Cellecta) and GFP positive cells were sorted by FACs. The identifying barcode region of integrated shrna provius was sequenced. The ranks and corresponding percentiles were determined based on number of sequencing reads (the lower the rank, the higher the number of reads). C.2.1. Demonstrating the significance of nuclear receptors in proviral latency Prior to generating complete gene knockouts, we want to gain an idea of whether specific nuclear receptors are essential for proviral transcriptional reactivation or silencing in our latent cell models. While research has indicated interaction of various nuclear receptors with HIV 5 LTR, characterization in both CD4 + T-cells and microglial cell has yet to be explored. By performing preliminary studies to gain a greater general understanding of these receptors, we will have better idea of what kinds of phenotypic changes to expect in the knockouts. One important step will be to quantify both mrna and protein levels of the nuclear receptors of interest in both the Jurkat/HIV (2d10) cells and Hμ/HIV. Nuclear receptor mrna levels in both untreated and activated cell will be quantified by RTqPCR, while protein expression will be measured by western blot. These data will allow for comparison of expression in microglia versus T-cells. 23

34 In order to gain a more general understanding of the roles of specific nuclear receptors, we will perform reactivation studies using different receptor agonists and antagonists (Table 1). Jurkat/HIV (2d10) cells and Hμ/HIV cells will be pretreated with various concentrations of the receptor agonists and antagonists for 2 to 4 hours. The same type of experiment will also be performed in the primary T- cell subsets and microglia. This will provide an interesting comparison of the effects of the nuclear receptor agonist and antagonists on the different cell types. Following pre-treatment, cells are stimulated with sub-optimal concentrations TNFα and SAHA for 16 hours. Changes in GFP expression will observed by flow cytometry. We have studied various nuclear receptors using specific agonists and antagonists in Jurkat/HIV (2d10) and CHME-5/HIV cells (Figure 5). We have demonstrated that ERα plays a prominent role in maintaining proviral latency in the Jurkat/HIV (2d10) cells, while ERβ signaling is more significant in CHME- 5/HIV cells. Pre-treating Jurkat/HIV (2d10) cells with the ERα specific antagonist, fulvestrant, resulted in sensitization of proviral reactivation in response to TNF-α stimulation, which does not occur in CHME-5/HIV cells (Figure 5A). Additionally, pre-treatment of CHME-5/HIV cells with ERβ specific agonists inhibits proviral reactivation (Figure 5B). PPARs and RXRs have also shown to be significant in both the Jurkat/HIV (2d10) and CHME-5/HIV cells (Figure 5C). When treated with PPARγ specific agonists (troglitazone) and antagonist (bisphenol A), Jurkat/HIV (2d10) cells were sensitized to TNF-α stimulation. A similar effect is also seen RXR antagonist HX531 followed by TNFα stimulation. In contrast, reactivation in 24

35 the CHME-5/HIV cells was strongly inhibited by triglitazone. Similar effects were also seen when CHME-5/HIV cells were treated with PPARα and PPARδ specific antagonists. When CHME-5/HIV cells were treated RXR agonist, bexarotene, proviral reactivation was inhibited. We will test all of the nuclear receptor agonists and antagonist in the newly developed Hμ/HIV cell line to confirm the findings we have seen in the rat microglia. While many of the nuclear receptors that we plan to screen known have identified ligands, ligands for COUP-TFI and II have yet to be identified. Therefore, to studies these receptors we will over-express the receptors and examined the reactivation profiles. Previous studies have shown that signaling through COUP-TF activates HIV expression in both microglia and T-cells (33). Therefore, we hypothesize that COUP-TF overexpression will increase basal reactivation as well TNF-α induced reactivation. 25

36 Figure 5. Nuclear receptor stimulation influence proviral reactivation in a cell type dependent manner. (A) Pre-treatment of Jurkat/HIV (2d10) cells, but not CHME-5/HIV cells, with ERα specific antagonist fulvestrant results in increased proviral reactivation in response to TNF-α stimulation. (B) Pretreatment with ERβ specific agonists, ERB 041 and FERb 033, inhibits proviral reactivation in response to TNF-α stimulation in CHME-5/HIV cells. (C) Pretreatment with PPAR, RAR, RXR specific agonists (dark blue bars) and antagonists (light blue bars) effect proviral reactivation in a cell type dependent manner. C.2.2. Generating CRISPR knockout cell lines of specific nuclear receptor in immortalized and primary T-cell and microglial cell lines While performing studies using nuclear receptor agonists and antagonists will provide some information of whether these receptors are involved in controlling latency, our goal is to understand the complete mechanism of their involvement. 26

37 To begin to understand these mechanisms, we will generate CRISPR knockout cell lines targeting specific nuclear receptors. Using CRISPRs rather than inhibitors or shrna will provide beneficial answers to questions regarding the molecular mechanisms of latency because the resulting phenotype will be permanent. Not only will we be able to understand what happens during the absence of the protein of interest, but we will be able to use these knockout cell lines to understand the recruitment of other host factors involved in proviral latency. To generate the knockout cell lines, we will use CRISPR Design software made available by the Zheng Lab at MIT to determine potential RNA guide sequences within the gene of interest. Inputting the nucleotide sequence of the first exon of the target protein yields a list of potential guide RNA sequences within that region, each with a corresponding score between 0 and 100 related to the number of off-target sequences. The greater the score of the guide sequence, the lower the likelihood of off-target binding. Off-target sequences are also scored based on the number of mismatches between the guide and off-target sequence; the greater the number of mismatches, the lower the score. The program also provides information about the off-target sequence, including the gene locus, allowing the investigator to better assess the severity of off-target binding. For our purposes, we will only use guide sequences with scores greater than 80 to minimize off-target binding. We will then clone the guide sequence oligonucleotides into the filler sequence of the lenticrispr-hcred-thy1.2 plasmid and generate VSVG-pseudotyped virus through transfection in HEK 293T cells with the pspax2 packaging plasmid. 27

38 The specific viral stocks for each nuclear receptor lenticrispr virus will be titered based on HcRed fluorescence in HEK-293T cells prior to infection in the latent cell models in order to ensure that similar MOIs are used in each infection. In order to obtain efficient infection in both CD4 + T-cell and microglial latency models, spinoculations will be performed at very high cell concentrations ( cells/ml for primary and immortalized CD4 + T-cells, cells/well in a 24-well plate for primary and immortalized microglial) using an MOI of 1 in order to reduce the risk of multiple virus particles infecting one cell. The MOI will be increased should infection efficiency be low, and off-target effects will be monitored closely. In the immortalized Jurkat/HIV (2d10) and Hμ/HIV cell lines, we will perform superinfections using the titered lenticrispr viruses. Following infection, infection efficiency will be measured based on HcRed fluorescence for 3 days post infection. The positively infected cells will then be sorted by magnetic isolation using CD90.2 MicroBeads (Miltenyi Biotec). Using magnetic isolation rather than selecting based on antibiotic resistance will prevent unnecessary stress to the knockout cells. Single-cell clones expressing both GFP from the HIV infection and Hc-Red from the lenticrispr infection will be obtained through serial dilution and will be sequenced along with the mixed population using the next-generation sequencing to detect indel mutation resulting from Cas9 cleavage. We will also confirm the knockouts by western blot and qrna PCR. Preliminary studies using the original lenticrispr vector targeting the promoter of ERα showed increased reactivation in Jurkat/HIV (2d10) in response to TNFα and SAHA stimulation, recapitulating what was seen using the ERα antagonists (Figure 6). 28

39 Figure 6. ERα knockout sensitizes Jurkat/HIV (2d10) cells to proviral reactivation. Jurkat/HIV (2d10) cells were infected with a lenticrispr virus targeting the promoter of ERα. Puromycin selected cells were treated with suboptimal levels of TNF-α and HDACi SAHA for 16 hr. ERα knockout results in increased reactivation when compared to control cells. Once we have confirmed the presence of indel mutations in our single-cell clones, we will characterize each nuclear receptor knockout cell line in terms of proviral reactivation. These studies will provide preliminary data regarding the significance of each receptor in CD4 + T-cell and microglial cells, allowing us to further understand the latency mechanisms in the different reservoirs. We will examine the proviral reactivation by examining both basal GFP expression and stimulation by sub-optimal concentrations of TNF-α and HDACi SAHA and measure proviral reactivation by flow cytometry. Stimulation with TNF-α will allow us to examine nuclear receptor interactions with the NF-κB signaling pathway. GRs, ESRs, and PPARα have been shown to interact and interfere with NF-κB signaling, therefore, we predict that knocking out these receptors reduce this interference, resulting in increased proviral reactivation when compared to 29

40 uninfected cells treated with TNF-α, but not necessarily when treated with SAHA (34). Conversely, unliganded RARs and TRs have been shown to repress transcription of target genes through interactions with the NcoR/SMRT complex and HDACs, therefore we predict that knocking out these receptor may relieve this repression, synergistically increasing GFP expression when combined with SAHA (35). These preliminary studies will help us to determine which nuclear receptors are most important to focus on for deducing the complete mechanism of latency. When the effects of the nuclear receptor knockouts have been characterized in the immortalized cell lines, we will perform infections in the primary CD4 + T-cell subsets and primary microglial cells to confirm our findings. Unlike the infections in the immortalized cells lines, co-infections with phr -Nef(+)- CD8a/GFP HIV and specific lenticrispr viruses will be performed to maximize infection efficiency of both viruses. We will sort cells that are positive for both HcRed and GFP by FACS to isolate cells that have been successfully infected with both viruses. The cells will then be allowed to enter latency through the process of quiescence. Because of the short-lived nature of primary cells, we will not obtain single-cell clones from these infections; rather, we will confirm the presence of indel mutations by next generation sequencing and perform characterization studies on the mixed population of double-positive cells. C.2.3. In-depth characterization of the molecular mechanisms utilized by nuclear receptors to control proviral latency. While studies involving reactivation with TNF-α and HDACi SAHA in the nuclear receptor knockouts will provide a general indication as to which nuclear 30

41 receptors play a significant in proviral latency, we ultimately want to under the molecular mechanism through which these receptors activate or inhibit proviral latency. By understanding these mechanisms, we can develop new potential therapeutic targets to eradicate latent reservoirs. Knocking out specific nuclear receptors may result in a number of effects on cellular biochemistry by interrupting the formation of protein complexes or reducing the catalytic activity of the complex. Therefore it is important to not only understand that nuclear receptor knockout effect proviral reactivation, but also how exactly these effects are achieved. In order to gain a more complete understanding of these mechanisms, we will perform a variety of studies to determine the different proteins that interact with the specific nuclear receptor. In order to study specific protein-protein interactions of the nuclear receptors, we will perform co-immunoprecipitation (Co-IP) assays using antibodies targeting specific nuclear receptors of interest using the Co- Immunoprecipitation Kit (Pierce). These initial studies will be performed in both the Jurkat/HIV (2d10) and Hμ/HIV (C01) cell lines which still express the nuclear receptor of interest. To ensure the specificity of the nuclear receptor antibodies, we will perform western blot analysis on untreated lysates from both cell lines. Once specificity is confirmed, we will perform Co-IP experiments on cell lysates from untreated cells, as well as cells treated for 16 hours with TNF-α (10 ng/ml) and HDACi SAHA (1 μm). Protein-protein interactions will be analyzed by mass spectrometry. If antibodies to a particular nuclear receptor are unavailable or bind non-specifically, we will perform pull-down assays by over-expressing the tagged nuclear receptor protein under the same conditions as the Co-IP assays. Once we 31

42 have determine what proteins are interacting the nuclear receptor, we will perform similar Co-IP studies in the nuclear receptor knockout cell lines, examining the ability of the protein complexes to form in the absence of the nuclear receptor. These data will provide information about how the absence of the specific nuclear receptor affects protein complex formation. Additionally, we will perform chromatin immunoprecipitation (ChIP) assays to understand how specific nuclear receptors and their binding partners interact with the proviral 5 LTR. ChIP-Seq experiments will be performed using Jurkat/HIV (2d10) and Hμ/HIV (C01) cell nuclear extracts obtained at different time points post TNF-α stimulation (0, 0.5, 4, 8, and 16 hours) as well as from unstimulated nuclear extracts. Antibodies targeting the nuclear receptor and its binding partners, as well as RNAPII as a control, will be used for immunoprecipitation. DNA products will then be analyzed though next-generation sequencing to determine the interactions of the nuclear receptor protein complexes with proviral 5 LTR. In order to study DNA binding in the nuclear receptor knockout cells, we will perform DNA pull-down assays. Using the tagged DNA binding sequence within the 5 LTR for the specific nuclear receptor, we will determine if the protein complexes in the absence of their specific nuclear receptors can still bind to DNA. These assays will provide valuable information regarding the interactions of nuclear receptors and their binding partner and their roles in proviral reactivation. 32

43 C.3. Specific Aim 3: Gain further understanding of the roles of epigenetic modifiers in proviral transcriptional silencing. Rationale: The roles of epigenetic modifiers in the context of proviral latency have been extensively studied due to their potential as therapeutic targets for eradication strategies. These proteins, which include histone acetyltransferase, histone deacetylases, and histone methyltransferases, are important for modifying the chromatin environment of the HIV-1 promoter, influencing transcriptional activation or silencing. While inhibitors, such as vorinostat (HDAC class I and II inhibitor), chaetocin (H3K9 methyltransferase inhibitor), and curcumin (HAT inhibitor), and protein knockdowns can provide valuable insight, they are often nonspecific and not permeant, making it difficult to completely understand how these modifiers influence latency. By producing complete gene knockouts of specific epigenetic modifiers identified in the literature and through unbiased shrna screens performed in our lab (Table 2), we will not only gain an understanding of what happens chemical when these proteins are absent, but also begin to determine the potential for using CRISPRs and genetic therapy for the eradication of HIV. 33

44 34