DUAL CONTROL OF HIV TRANSCRIPTION ELONGATION: VIRUS- SPECIFIC NEGATIVE CONTROL BY NELF-E IS COUNTERBALANCED BY POSITIVE TRANSCRIPTION FACTOR P-TEFb

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1 DUAL CONTROL OF HIV TRANSCRIPTION ELONGATION: VIRUS- SPECIFIC NEGATIVE CONTROL BY NELF-E IS COUNTERBALANCED BY POSITIVE TRANSCRIPTION FACTOR P-TEFb By JULIE K. JADLOWSKY Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisors: Jonathan Karn, PhD. Koh Fujinaga, PhD. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January 2009

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Julie K. Jadlowsky candidate for the Doctor of Philosophy degree *. (signed) Erik D. Andrulis (chair of the committee) Koh Fujinaga Zahra Toossi Jonathan Karn (date) November 13, 2008 *We also certify that written approval has been obtained for any proprietary material contained therein. 2

3 Table of Contents List of Tables 9 List of Figures 10 List of Abbreviations 13 Acknowledgements 16 Abstract 18 Chapter 1: Introduction 20 Introduction 21 HIV Transcription and Associated Cellular Factors 22 Negative Regulation of HIV Transcription 26 HIV Latency 33 Summary of Thesis Work 38 Chapter 2: The NELF-E Subunit of the Negative Elongation Factor Directly and Specifically Contributes to Post-integration Latency of HIV 46 Summary 47 Introduction 48 3

4 Results 49 A system for defining the role of NELF in latent HIV 49 Suppression of NELF-E partially activates latent proviral HIV 52 Silencing of NELF-E affects the rate and extent of reversion to latency 53 NELF-E silencing also has a more pronounced transcriptional effect on HIV than silencing of NELF-A 54 Silencing of NELF-E specifically enhances the recruitment of RNAPII to regions along the HIV proviral genome 55 Discussion 57 Materials and Methods 63 Luciferase Assays 63 Western Blots 63 ChIP Assays 63 FACS Analysis 64 Construction of shrna plasmids 64 Infection of H13L Jurkat cells 65 4

5 Activation and shutdown of H13L Jurkat cells 65 Chapter 3: Dominant Negative Mutant Cyclin T1 Proteins Inhibit HIV Transcription by Specifically Degrading Tat 94 Summary 95 Introduction 96 Results 99 Construction and screening of CycT1 mutants 99 An N-terminal CycT1 mutant exhibited the strongest dominant negative effect on Tat transactivation by promoting the degradation of Tat proteins 100 Expression of CycT1-U7 and Tat can be rescued by proteasome inhibitors 101 Discussion 103 Materials and Methods 107 Materials 107 Construction of CycT1 mutants 107 Generation of stable cell lines 108 Transfection and reporter assays 108 5

6 Ubiquitination assays 108 Proteasome inhibitor treatment 109 Immunofluorescence (IF) assay 109 Acknowledgements 110 Chapter 4: Dominant Negative Mutant Cyclin T1 Proteins that Inhibit HIV Transcription by Forming a Kinase Inactive Complex with Tat 124 Summary 125 Introduction 126 Results 127 A mutant CycT1 protein containing triple T to A mutations in the N-terminal region blocks HIV transactivation 127 CycT1-280 (T143, 149, 155A) proteins associate with Cdk9 and Tat as a kinase-negative complex 129 Discussion 131 Materials and Methods 134 Materials 134 Construction of CycT1 Mutants 134 6

7 Transfection and Reporter Assays 134 Tat-associated kinase (TAK) assay 135 Co-immunoprecipitations 135 Acknowledgements 136 Chapter 5: Discussion and Future Directions 144 Discussion 145 Future Directions 147 Hypothesis: NELF-E inhibits transcriptional elongation specifically by interaction with the TAR element of HIV 147 Hypothesis: The release of the NELF complex, upon phosphorylation of NELF-E, may enable NELF-E to become a positive elongation factor 148 Hypothesis: Knockdown of NELF-E in latently infected primary cells will result in partial activation of transcriptionally silent provirus 149 Hypothesis: Dominant-negative CycT1 molecules and related approaches can be applied to the development of novel anti-hiv therapeutics 150 7

8 Conclusion 151 Bibliography 152 8

9 List of Tables Table 2.1 Sequences of shrna targets 68 Table 3.1 Overview of CycT1 mutants used in this study 113 Table 3.2 Sequences of mutagenic oligonucleotides 123 9

10 List of Figures Figure 1.1 Transcription of the HIV provirus must occur for productive infection. 42 Figure 1.2. Establishment of latent HIV infection in CD4 + T cells. 44 Figure 2.1 Outlining the experimental approach to studying the role of NELF in latent HIV. 66 Figure 2.2 Construction of shrna lentiviral plasmids to target NELF 69 Figure 2.3 Activation and shutdown of H13L Jurkat clone 2D10 following TNF-α stimulation. 71 Figure 2.4 Initial infection of H13L Jurkat cells with mcherry shrna viruses. 72 Figure 2.5 Clones expressing mcherry-shrna suppress NELF expression. 73 Figure 2.6 NELF-E shrna clone is able to partially activate d2egfp expression in the absence of stimulation. 75 Figure 2.7 Multiple isolated NELF-E shrna clones show similar partial activation of d2egfp expression. 76 Figure 2.8 Spontaneous reactivation and shutdown of a latent H13L Jurkat clone

11 Figure 2.9 Suppression of NELF-E also exhibits spontaneous activation and shutdown. 78 Figure 2.10 Silencing NELF-E prevents cells from establishing a complete latent state following activation with anti-cd3. 80 Figure 2.11 Silencing of NELF-E prevents the establishment of a complete latent state following stimulation with TNF-α. 82 Figure 2.12 NELF-E silencing also has a more dramatic transcriptional effect than silencing of NELF-A. 83 Figure 2.13 Distribution of RNA polymerase II on the HIV and junb genomes, before and after treatment with TNF-α, when NELF subunits are suppressed. 85 Figure 2.14 Comparison of RNAPII at promoter and downstream regions of the HIV provirus and cellular genes in the absence of TNF-α stimulation reveals a specific elongation effect on HIV when NELF-E is suppressed. 87 Figure 2.15 Comparison of RNAPII at promoter and downstream regions of the HIV provirus and cellular genes in the presence of 60 minutes of TNF-α stimulation reveals a specific elongation effect on HIV when NELF-E is suppressed

12 Figure 2.16 Model for transcriptional regulation of cellular genes vs. HIV by NELF. 91 Figure 3.1. Construction of mutant CycT1 proteins. 111 Figure 3.2. N-terminal mutant CycT1 proteins (CycT1-U7) exhibit a strong dominant negative effect on HIV transactivation. 114 Figure 3.3 CycT1-U7 promotes the degradation of Tat. 116 Figure 3.4. Epoxomicin restores CycT1-U7 and Tat expression. 118 Figure 3.5. CycT1-U7 is ubiquitinated. 120 Figure 3.6. Proposed model for the mechanism of dominant negative effect elicited by CycT1-U Figure 4.1. Mutant CycT1 proteins containing triple T-to-A mutations in the N-terminal region also block HIV transactivation. 137 Figure 4.2. The dominant negative effect of CycT1-280 (T143,149,155A) is specific for Tat. 139 Figure 4.3. CycT1-280 (T143, 149, 155A) proteins associate with Cdk9 and Tat as a kinase- negative complex. 140 Figure 4.4. Tat can compete with HEXIM for binding to CycT Figure 4.5. Predicted conformational changes in CycT1 (1-280)

13 List of Abbreviations AIDS: Acquired Immune Deficiency Syndrome ARM: Arginine Rich Motif ATP: Adenosine Triphosphate CBF-1: C-Promoter Binding Factor CCR5: Chemokine (C-C motif) Receptor 5 CD4: Cluster of Differentiation 4 Cdk9: Cyclin Dependent Kinase 9 ChIP: Chromatin Immunoprecipitation CMV: Cytomegalovirus CTD: C-terminal Domain CycT1: Cyclin T1 CXCR4: Chemokine (C-X-C motif) Receptor 4 DNA: Deoxyribonucleic Acid DRB: 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole DSIF: DRB Sensitivity Inducing Factor d2egfp: Destabilized Enhanced Green Fluorescent Protein 13

14 EIAV: Equine Infectious Anemia Virus ELISA: Enzyme-Linked ImmunoSorbent Assay FACS: Fluorescent-Activated Cell Sorting GAPDH: Glyceraldehyde 3-phosphate dehydrogenase HAART: Highly Active Anti-Retroviral Therapy HDAC: Histone Deacetylase HEXIM1: Hexamethylene bisacetamide (HMBA)-induced protein 1 HIV: Human Immunodeficiency Virus IF: Immunofluorescence LTR: Long Terminal Repeat Luc: Luciferase NELF: Negative Elongation Factor NFAT: Nuclear Factor of Activated T cells NF-κB: Nuclear Factor-κB NTEF: Negative Transcription Elongation Factor PBMC: Peripheral Blood Mononuclear Cells PEST: Sequence rich in Proline (P), Glutamic Acid (E), Serine (S), and Threonine (T) 14

15 P-TEFb: Positive Transcription Elongation Factor b RNA: Ribonucleic Acid RNAPII: RNA Polymerase II RRM: RNA Recognition Motif shrna: Short Hairpin RNA TAK: Tat-Associated Kinase assay TAR: Trans-Activation Response element TFF1: Trefoil Factor 1 TNF- α: Tumor Necrosis Factor α TRM: Tat-TAR Recognition Motif WHSC2: Wolf-Hirschhorn Syndrome Candidate protein 2 15

16 Acknowledgements I would like to thank my advisors, Dr. Jonathan Karn and Dr. Koh Fujinaga for their help in guiding this project and aiding in seeing it to completion. Their expertise, advice, and direction have been invaluable. I would also like to thank the past members of the Fujinaga lab, especially Yehong Huang, Satsumi Roos and Renee Devor. Their assistance helped make my work easier, and my work environment much more pleasant. Additionally, I would like to thank all the members of the Karn lab, past and present, which welcomed me in and helped in any way they could, especially Richard Pearson, Kara Lassen, and Young-Kyeung Kim. I must also acknowledge Drs Michael Lederman and Scott Sieg for giving me opportunities in their laboratories to expand my research repertoire and be involved in other fantastic projects. It has been a pleasure to help them in any way I can. To the members of their laboratories, I extend huge thanks for all of their help, guidance, and friendship. They ve always included me as one of their own, continually being extremely supportive, understanding, and welcoming, and for that I will forever be grateful. I would like to thank Heather Pilch-Cooper who has become a great friend, confidant, and proofreader. I also need to thank Nick Funderburg, who has meant so much to me both personally and professionally; without his support and encouragement I would have been lost. I also really appreciate the hard work done on my behalf by the administrative staff of the Molecular Biology and Microbiology department, including Brinn Omabegho, Dorothy Canepari, Hollie Hurst, and Brad Fairfield. 16

17 Additional thanks belong to the Cell and Molecular Biology Training Grant and the Case Center for AIDS Research for providing financial support. Finally, I would like to thank my family and friends for seeing me through all these years. Special and biggest thanks to Gam for always believing in me and my abilities and wanting and expecting nothing but the very best for me. Her steadfast support and encouragement in every aspect of my life have made me the person I am today. 17

18 Dual Control of HIV Transcription Elongation: Virus-Specific Negative Control by NELF-E is Counterbalanced by Positive Transcription Factor P-TEFb Abstract by JULIE K. JADLOWSKY Expression of the HIV provirus is regulated at the level of transcription by an intricate interplay between positive and negative factors. In the presence of positive transcription elongation factors such as the cellular cofactor Positive Transcription Elongation Factor b (PTEF-b), transcription proceeds efficiently, resulting in full-length HIV mrna. Alternatively, paused elongation produces an accumulation of short, abortive transcripts. Non-processive transcription occurs in the presence of negative factors, such as Negative Elongation Factor (NELF), which has been shown to suppress elongation through direct interaction between its NELF-E subunit and the HIV TAR. This work is intended to ascertain whether these factors can potentially be manipulated to benefit treatment of HIV, either through therapeutics to target active, ongoing infection, or by manipulating the latent viral reservoir which currently prevents eradication of virus from the body. This study used the development of dominant negative CycT1 mutants to attempt to specifically inhibit positive regulation of HIV transcription. Two separate CycT1 mutants were found to interfere with successful transcription of HIV mrna by distinct mechanisms. One mutant forms a kinase-inactive complex with Tat, while the other 18

19 causes specific degradation of Tat. On the opposing side of regulation, shrnaexpressing lentiviruses targeting NELF were applied to a re-activatable model of postintegration latency to better understand the associated transcriptional suppression. These results suggest the NELF-E subunit is essential for the suppression of transcription associated with latency, and that this NELF-E effect is specific to the Human Immunodeficiency Virus. These investigations demonstrate that the regulation of HIV transcription is a critical stage in the life cycle of the virus which can be exploited to not only gain a better understanding of the virus itself, but to possibly open new avenues for the future treatment of AIDS. 19

20 Chapter 1: Introduction 20

21 Introduction: Human Immunodeficiency Virus type 1 (HIV-1) was first isolated in 1983 [1], and was subsequently determined to be the causative agent of Acquired Immune Deficiency Syndrome (AIDS) [2-4]. HIV is a retrovirus composed of a 9kb RNA genome encoding 15 proteins from its 9 genes. This virus preferentially infects the CD4 + T lymphocytes of the immune system, using the CD4 molecule and either CXCR4 or CCR5 as a co-receptor to gain entry into the cell [5-7]. Once HIV has entered the cell, reverse transcription occurs, resulting in the production of double stranded DNA. The DNA irreversibly integrates into the host genome, thereby allowing cellular factors to direct the production of new viral particles and propagate infection (Fig. 1.1a). Infection with HIV is characterized by a rapid decrease in CD4 + T cell levels, chronic immune activation, and severe immune impairment, which leads to increased susceptibility to opportunistic infections. Current treatment for infection with HIV is most often a regimen of Highly Active Anti-Retroviral Therapy, or HAART. HAART uses a combination of drugs to target viral enzymes such as protease, reverse transcriptase, or integrase, or to interfere with the binding of the virus to the cell (fusion / entry inhibitors). Although when used in combination these drugs are able to suppress viral load, often to undetectable levels, HAART is only able to target actively replicating virus. Currently, no available therapy is effective in treating the reservoir of latent HIV proviruses which develop soon after initial infection. Therefore, gaining access to this reservoir is a key obstacle in the eradication of AIDS. Compounding the problem is the multi-factorial nature of 21

22 maintaining latency, which has made the determination of one uniquely defined and accepted molecular mechanism quite a challenge. Most evidence, though, indicates the phenomenon of HIV latency is regulated mainly at the level of transcription [8-12]. The work presented here will provide evidence for the contribution of a specific transcription factor, NELF, to the establishment and maintenance of latent HIV infection. HIV Transcription and Associated Cellular Factors Transcription of HIV is a precisely regulated process involving cellular factors and a small viral transactivator protein, Tat. Shortly after transcription is initiated from the HIV long terminal repeat (LTR), it enters the elongation stage. At this point, processivity of transcription depends on multiple regulatory factors provided by the host cell and the virus [13]. Depending on which factors are present, transcription can be paused, resulting in a predominance of short RNAs and thus low levels of gene expression, or transcription can proceed in a productive manner, resulting in long, fulllength transcripts with increased levels of gene expression. The manner in which transcription occurs is related to the processivity of RNA Polymerase II (RNAPII) and an intricate interplay between positive and negative transcription elongation factors [14, 15]. The mechanism behind this interplay appears to be the phosphorylation status of the RNAPII C-terminal domain (CTD). The transition to the elongation stage of transcription occurs following the phosphorylation of serine 5 in the heptad repeat sequence 1 YSPTSPS 7 of the RNAPII CTD by the general transcription factor TFIIH [16]. In addition to this phosphorylation of the CTD, HIV transcription also requires a viral protein to stimulate elongation. The transcriptional transactivator, Tat, is encoded by 22

23 HIV-1, and interacts with the transactivation response element (TAR) of HIV RNA and the cyclin T1 (CycT1) subunit of the cellular cofactor, positive transcription elongation factor b (P-TEFb). The TAR element is an RNA stem loop structure located at the 5 end of all nascent HIV transcripts. Once Tat binds to TAR, it then recruits the P-TEFb complex through interaction with CycT1. The kinase activity of the second subunit of P- TEFb, cyclin-dependent kinase 9 (Cdk9), is subsequently stimulated by Tat [17]. This leads to the hyperphosphorylation of the RNAPII CTD when Cdk9 phosphorylates the serine 2 residue of the RNAPII CTD. This hyperphosphorylation causes an increase in RNAPII processivity, and thus, productive transcriptional elongation. This processive elongation, in return, results in the generation of full length HIV transcripts [18, 19]. Additionally, is thought that the hyperphosphorylation which causes stimulation of transcription also disrupts the association of negative transcription elongation factors which can only associate with RNAPII in its hypophosphorylated form [14]. Tat, through its interaction with TAR, is necessary for RNA polymerase II to activate the inducible promoter within the viral LTR [20]. When Tat is present, processive RNAPII complexes are formed, which generate production of full length HIV transcripts [21-27] (Fig. 1.1b). In the absence of Tat, RNAPII prematurely terminates transcription, leading to an accumulation of short transcripts [24, 28, 29] (Fig. 1.1c). This low level of productive transcription in the absence of Tat may correspond to a possible mechanism of transcriptional latency for HIV. Because Tat is a viral protein, productive elongation must occur for appreciable levels of Tat to be made. However, if short transcripts are predominating due to a lack of Tat transactivation, the insufficient Tat levels produced by this non-processive transcription are thought to contribute to 23

24 transcriptional latency of HIV. In fact, these short, abortive transcripts are considered to be a marker of latent HIV infection [30]. In addition to the possibility that insufficient Tat may play a role in latency, another contributor may be the activity, or transactivation capabilities of the Tat. Similar to other proteins encoded by the HIV genome, Tat is susceptible to rapid mutation, as demonstrated by the isolation of several sequence variants from HIV positive individuals [31-33]. These attenuated Tat variants, with reduced transactivation abilities, could contribute to the latent state seen in quiescent cells, while later allowing for productive replication following cellular activation [34]. In support of this, it has been found that mainly short, promoter proximal transcripts are produced in cells expressing these variants [30]. As outlined above, in addition to Tat the transactivation of HIV transcription requires a host cell factor termed the positive transcription elongation factor b (P-TEFb). P-TEFb is a heterodimeric elongation factor comprised of a regulatory subunit, CycT1, and a catalytic subunit, Cdk9, and is involved in the production of most RNAPIIdependent transcripts [35]. Cdk9 is a cdc-2-like Ser/Thr kinase of 42 kda which is capable of autophosphorylation [36]. The CycT1 subunit, which is approximately 87 kda is a member of the cyclin family, although unlike most cyclins levels do not fluctuate along with stages of the cell cycle [37]. Of its 726 amino acids, the first 272 of CycT1 are sufficient for HIV transcription, being that they are responsible for interacting with Tat, TAR, and Cdk9 [38-41]. In actuality, three distinct P-TEFb complexes have been identified, although the CycT1/Cdk9 combination is the most common and the only 24

25 one able to support Tat transactivation [37, 38, 42, 43]. Cdk9 has also been found to complex with cyclin T2 and cyclin K [42, 44-46]. Because P-TEFb is involved in the regulation of a majority of cellular genes, it therefore must be tightly regulated to respond appropriately to the transcriptional needs of a cell during stages of growth or differentiation [46]. P-TEFb exists in cells in an active and inactive form. In the inactive form, P- TEFb is complexed with hexamethylene bisacetamide (HMBA)-induced protein 1 (HEXIM1) and 7SK small nuclear RNA (snrna). In this large complex, the kinase activity of Cdk9 is not functional. Both 7SK snrna and HEXIM1 associate with the C- terminal region of CycT1 and inhibit the kinase activity of Cdk9 [47-50]. Additionally, the 7SK-bound form cannot be recruited to the HIV LTR [51]. This inactive form of P- TEFb can be converted to an active form by multiple stimuli which dissociate HEXIM1 and 7SK snrna from P-TEFb. These stimuli include: ultraviolet light, stress, 5,6- dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), and actinomycin D [52]. The active form of P-TEFb has a positive regulatory component. This component, the bromodomain protein Brd4, binds P-TEFb which is not complexed with HEXIM1 and 7SK snrna, and acts to enhance the phosphorylation of serine 2 of the RNAPII CTD [53]. HIV-1 Tat has also shown to possess a similar recruitment function to that of Brd4, by directly recruiting the P-TEFb complex to enhance activated HIV-1 transcription [54]. Recently, it was demonstrated that Tat competes with HEXIM1 to increase the active pool of P-TEFb to facilitate HIV transcription [50]. 25

26 Negative Regulation of HIV Transcription Just as HIV transcription can be regulated by positive transcription elongation factors to result in the production of full length transcripts, conversely, regulation can occur in a negative fashion to impede efficient transcriptional elongation. Following initiation, RNAPII encounters negative transcription elongation factors (NTEFs) which impose a restriction to processive transcription, and thus cause abortive elongation. DRB-sensitivity-inducing factor (DSIF) and Negative Elongation Factor (NELF) are multi-subunit cellular factors which act together to lower the elongation rate by increasing the time RNAPII spends at pause sites, thereby causing production of short transcripts [55-57]. The results of in vitro transcription analyses suggest DSIF and NELF associate with RNAPII shortly after the initiation of transcription, but prior to the polymerase reaching the pause sites [58, 59]. The repression achieved by the cooperative action of these factors is alleviated by P-TEFb [19, 56, 60]. The complex interplay between positive and negative transcription elongation factors and their role in the transition from abortive to productive elongation began to unfold following experiments with the protein kinase inhibitor DRB. DRB is an ATP analog which inhibits transcription elongation by RNAPII through inhibition of CTD kinases such as Cdk7 or Cdk9 [61]. The identification and purification of negative-acting DSIF and NELF, as well as positive-acting P-TEFb was a result of experiments intended to identify factors required to reconstitute DRB sensitivity in in vitro transcription assays [57, 60, 62-64]. DSIF is a transcription elongation factor which has a dual role as both and activator and repressor of transcription. In cooperation with NELF, DSIF exerts a 26

27 negative effect on elongation. In the absence of NELF, however, evidence supports a role for DSIF as an activator of elongation following phosphorylation of Spt5, when nucleotide concentrations are low [59, 62, 65]. DSIF has 2 subunits in humans: p160 and p14. These subunits are homologs of the Spt4 and Spt5 proteins which are important for transcription elongation in Saccharomyces cerevisiae [62, 66]. DSIF is required for sensitivity to DRB, and its purification was based on its capacity to return DRB sensitivity to a partially purified transcription system [62]. Following purification of DSIF in the laboratory of Hiroshi Handa, in an effort to further characterize the mechanism of DRB-sensitive elongation using a more defined transcription system, the requirement for an additional factor became apparent [57]. This factor was termed Negative Elongation Factor (NELF). Negative Elongation Factor (NELF) is a multi-subunit complex of approximately 300 kda. This four subunit complex is composed of NELF-A, NELF-B, either NELF-C or D, and NELF-E, also known as RD because of its 24 tandem repeats of arginineaspartic acid (or glutamic acid) dipeptides. In addition to the central RD repeated sequence, NELF-E also contains a carboxy-terminal leucine zipper through which it associates with NELF-B, as well as a C-terminal RNA recognition motif. NELF-B is a cofactor of Breast Cancer 1 Gene, BRCA1. NELF-C and NELF-D are TH1-like products of alternatively spliced forms of a common mrna species [67]. NELF-A, also known as the Wolf-Hirschhorn syndrome candidate protein 2 (WHSC2), has weak sequence similarity to hepatitis delta antigen, and binds RNAPII [67, 68]. Although both NELF-B and NELF-C/D are essential for the formation of a functional NELF complex, their biochemical functions in the complex are not yet clearly defined [69]. Initially, studies 27

28 had reported an estrogen-dependent role for NELF-B in breast cancer [69]. Recent studies have additionally indicated an estrogen-independent role for NELF-B in primary Upper Gastrointestinal Adenocarcinomas [70]. These, and future studies, will hopefully soon serve to better clarify a role for NELF-B. Two of the four subunits, NELF-A and NELF-E, contain RNA recognition motifs (RRM). Through the RRM of the NELF-E subunit, the NELF complex has been shown to bind the RNA of various sequences, in a manner sensitive to mutation of the RRM [71]. Previously, it has been shown that NELF interacts with the Transactivation Response Element (TAR) of HIV RNA [71, 72]. More specifically, it is the NELF-E subunit which binds to the lower stem region of TAR. NELF-E does not, however, bind the TAR element of equine infectious anemia virus (EIAV) which possesses a shorter stem sequence than that of HIV [72]. The majority of the initial understanding of the NELF complex, and its role in transcription, can be attributed to the work of Dr. Hiroshi Handa and collaborators. In the original identification and purification of NELF from HeLa nuclear extract, it was found that in the absence of P-TEFb, NELF was able to strongly repress transcription regardless of the presence of DRB. Addition of P-TEFb, though, was able to partially reverse this repression [57]. This report also found that DSIF and NELF work cooperatively to repress transcription by acting on hypophosphorylated, but not hyperphosphorylated, RNAPII [57]. This suggested a role for phosphorylation in alleviating the repression of NTEFs. Subsequently, in conjunction with the laboratory of Dr. David Price, kinetic studies were performed which indicated that the cooperative action of DSIF and NELF increased the time RNAPII spent at pause sites [56]. The first indication of DSIF having a stimulatory effect on transcription elongation was also reported. This activity was 28

29 independent of NELF, and despite increasing concentrations of NELF, no effect on the processivity of elongating RNAPII was seen in the absence of DSIF [56]. To date, there has been no published evidence of NELF promoting elongation. It was also determined by the Handa group, that NELF does not appear to substantially bind to RNAPII or DSIF alone, but does, however, bind to a preformed DSIF/RNAPII complex [71]. In regards to the RNA-binding ability of NELF, mutations in the RRM of NELF-E still permitted complex formation and association with DSIF and RNAPII, however transcriptional repression was impaired [71]. It was also concluded that each subunit of NELF is important, and together these subunits are sufficient for functioning in vitro [71]. Therefore, the proposal was made that within cells, the four subunits exist primarily in the context of the NELF complex. This proposal was based upon data of Flag-tagged NELF-E co-immunoprecipitating with near stoichiometric amounts of the other subunits, arguing against the existence of NELF-E in the context of other protein complexes. Additionally, immunodepletion of HeLa nuclear extract with antibodies directed against NELF-E depleted not only that subunit, but NELF-A as well. This implied that the majority of endogenous NELF-A was stably associated with NELF- E [71]. Interestingly, NELF is not highly conserved among species. It appears to be expressed in vertebrates and insects, however, no homologs in yeast, nematodes, or plants have been identified [67]. NELF studies by Dr. David Gilmour s group have been conducted in Drosophila. In characterizing Drosophila NELF (dnelf), some differences from the human form were found. Notably, dnelf-a is twice the size of 29

30 human NELF-A. Surprisingly, the region corresponding to the alternating asparagines and aspartic acid residues, as well as serines corresponding to those at positions 181, 185, 187, and 191 in hnelf-e are absent in dnelf-e. These serines have been identified as phosphorylation targets for the dissociation of mammalian NELF [72]. In spite of these differences, results of their studies are generally quite consistent with previous NELF findings. In particular, it was determined that depletion of NELF-E from salivary glands resulted in a reduction in paused polymerases found on hsp70 heat shock gene in live cells [73]. They also implicate DSIF and NELF in this promoter proximal pausing [73]. Chromatin Immunoprecipitation (ChIP) analysis was subsequently used to detect the NELF complex at the promoters of both the hsp70 and the β1-tubulin gene, where pausing had previously been detected [74]. Based on the available data, it was proposed that promoter proximal pausing occurs with the emergence of the nascent transcripts from the RNA exit channel of RNAPII allowing for interaction with the NELF-E subunit [73]. This proposal corresponds with previous results of in vitro transcription analysis indicating elongation cannot be inhibited by NELF and DSIF until the nascent transcript reaches a length of approximately 30 nucleotides [57]. More recent studies using measurement of pause strength suggest that inhibition begins when the RNA chain is approximately 18 nucleotides long. This nucleotide length corresponds roughly to the position where the nascent RNA strand begins to emerge from RNAPII [75]. This same study also suggested a 2 nd cooperative activity of DSIF and NELF. The transcription cleavage factor TFIIS stimulates cleavage of backtracked transcripts that are a result of RNAPII pausing and arrest. This cleavage provides for productive elongation. This study 30

31 proposes that DSIF and NELF inhibit the cleavage mediated by TFIIS. Inhibition of this cleavage may be another way to negatively regulate transcription elongation [75]. Recently an additional, novel function has been proposed for NELF. Narita et al have provided evidence to suggest that NELF is able to coordinate several mrna processing steps, in particular, the 3 end processing of replication-dependent histone mrnas [76]. In regards to the more traditional view of DSIF and NELF working as inhibitors to regulate elongation by RNAPII, co-immunoprecipitation experiments indicate a strong association of NELF and DSIF with hypophosphorylated RNAPII, but not with hyperphosphorylated RNAPII [57, 77]. This suggests a role for phosphorylation in alleviating the repression of these negative factors. DSIF and NELF do not interact with the CTD of RNAPII directly, but most evidence supports the idea that phosphorylation of the RNAPII CTD by the Cdk9 subunit of P-TEFb functions in the release of these factors, thus reversing the inhibition they impose [57-59]. Currently, P-TEFb is known to phosphorylate both Spt5 of DSIF and the E subunit of NELF [58, 72]. These phosphorylation events are also believed to contribute to the relief of inhibition. Prior to the determination of the subunits composing NELF, the function of the NELF-E protein was unknown. In an attempt to elucidate the function of the NELF complex, it was found that depleting NELF-E increased transcription levels in the presence of DRB, resulting in a clear reduction of DRB sensitivity. With the addition of crude or purified NELF back to the NELF-E-depleted extract, transcription was once again repressed in the presence of DRB, and thus DRB sensitivity was restored [57]. This was an indication that the NELF-E subunit was important for the activity of the 31

32 NELF complex. Therefore, with the knowledge that phosphorylation plays a key role in the regulation of elongation in transcription, and that NELF is an important negative regulator of transcription elongation which also binds to the TAR element of HIV through the NELF-E subunit, it was logical to then look for a relationship between NELF, phosphorylation, and the role they play in the elongation of HIV transcripts. It was shown by Fujinaga et al (2004), that NELF-E is phosphorylated by P-TEFb, and once phosphorylated, is incapable of binding to TAR [72]. The NELF-E subunit has shown to be necessary for the inhibitory activity of the NELF complex, and previous findings indicate that interaction of NELF with nascent RNA through its RRM is important for the repression of transcription elongation [71]. Knowing that NELF-E is phosphorylated by P-TEFb and suppression is alleviated by P- TEFb, this may indicate that NELF performs an important role in the transition from nonproductive transcription, in which only short transcripts are produced, to productive transcription elongation [57, 72] This transition from non-productive to productive transcription is a key aspect to the re-activation of latent provirus in HIV infection. In latently infected cells, nonproductive transcription predominates. However, upon activation of these cells, the transition is made to allow for the production of full-length transcripts. Given the evidence of a dynamic relationship between the TAR element of the HIV transcript and the E subunit of NELF, logic suggests that NELF can suppress transcriptional elongation through direct interaction with nascent HIV RNA. This suppression may lead to the establishment or maintenance of the post-integration form of latent HIV infection. 32

33 Supporting this idea, recent work by Zhang et al has provided direct evidence that NELF does inhibit transcription from an integrated HIV provirus [78]. Because of its importance to the ability of NELF to negatively regulate transcription elongation and its known association with the TAR structure of nascent HIV transcripts, NELF-E therefore became the primary focus of this investigation into deciphering a specific role for the NELF complex in post-integration latency of HIV infection. HIV Latency HIV is a retrovirus able to persist in the body in a latent form. This phenomenon was first identified in the patient population in 1995, and described again in patients with HAART-suppressed virus in 1997 [79, 80]. Latency is a non-productive reversible, infectious state which allows the virus to persist long-term within the host. Viral replication is essential for virion production, but latency allows HIV-1 to remain quiescent, evading the immune response, as latently infected cells lack any distinguishing cell markers. Latency in HIV is a natural result of the pathology of infection by the virus. HIV-1 infects mainly CD4 + T lymphocytes, and replication of the virus occurs preferentially in activated CD4 + T cells. Activated T cells are those which have encountered and responded to antigen. Subsequent to antigenic challenge, a small subset of the responder cell population reverts back to a resting state and continues on as memory cells. These memory cells have a long life span, and are primed to quickly respond to antigen in the future. HIV replication cannot occur in these quiescent CD4 + T cells. 33

34 Although resting CD4 + T cells cannot actively replicate HIV, it is possible for them to harbor latent provirus, even in infected individuals with clinically undetectable viremia receiving Highly Active Anti-Retroviral Therapy (HAART) [13, 80-82]. As stated, in activated CD4 + T cells, HIV infection leads to viral replication and soon after results in the death of the CD4 + T cell. If, however, an activated CD4 + T cell is infected during the transition to a memory state, the infection can become latent. In this case, two forms of latency are possible. In one form, the HIV DNA, which is a product of the reverse transcription step of the viral life cycle, is unable to integrate into the DNA of the host s resting CD4 + T cell. This pre-integration latency is predominantly found in individuals with untreated HIV infection [83, 84]. The alternative form of latency occurs after HIV has integrated into the host DNA, and is referred to as post-integration latency (Fig. 1.2). Pre-integration latency is characterized by the presence of linear, full-length, unintegrated HIV DNA in a resting CD4 + T cell. This is thought to occur when the virus infects a quiescent T cell, but given the low metabolic activity in such a cell, required processes like reverse transcription, nuclear import, and integration are inhibited. This inhibition results in the viral DNA remaining in the cytoplasm for days [13, 84, 85]. This phenomenon is found to occur primarily in recently infected CD4 + T cells, and if such cells become activated before the unintegrated material has decayed, integration and replication can be triggered, and productive infection can ensue. Although this form of latency is dominant in untreated individuals, these cells have been found to decay within three months of starting therapy [86]. Because therapy is able to eliminate this form of latently infected cells, they are not deemed to be clinically important. 34

35 Latency in the context of integrated provirus, or post-integration latency, is characterized by defective transcription of HIV, and the production of short viral transcripts [30]. This latency reflects a situation in which the virus is stably integrated into the host, but transcription is silenced. In 1997, a reservoir of latently infected cells was identified in patients on HAART [80]. It has been estimated that the size of this reservoir is approximately 10 5 to 10 6 infected memory CD4 + T cells, and that given current therapy, it would take more than 60 years for clearance of this reservoir [87]. Because of this unrealistic timeframe for clearance, and the inaccessibility of the reservoir to current drugs, post-integration latency is the major obstacle to eradicating AIDS, and is therefore of tremendous clinical significance. Based on existing therapies, eliminating HIV from the body would require a purging, or clearance of the latent reservoir. Most recent approaches propose using various stimuli to activate the T cells [13, 88-92]. However, global activation of T cells can cause an additional set of problems, resulting from cytokine inductions in individuals with faulty immune systems. Therefore, in order to develop a more safe and targeted approach, it is important to understand the molecular mechanism(s) responsible for this type of latent infection. The molecular mechanism behind this silencing has been a topic of great debate during recent years. Several plausible methods have been proposed, and ultimately it appears that the phenomenon of post-integration latency is caused by multiple factors, suggesting that a combination of mechanisms may be responsible. Most hypotheses center on a cause related to the regulation of transcription. Mechanisms thought to contribute to proviral latency are: insufficient Tat or host transcription factors, 35

36 accessibility of transcriptional machinery due to the site of integration, and the presence of transcriptional repressors [13, 93]. A deficiency in the amount of Tat or the availability of cellular transcription factors may be responsible for the lack of gene expression seen in post-integration latency. The lack of productive transcription may be a result of unfavorable conditions inside the memory cell for the expression of HIV genes. For instance, in resting memory CD4 + T cells, two host transcription factors which are important for HIV gene expression, nuclear factor-κb (NF-κB) and nuclear factor of activated T cells (NFAT), are excluded from the nucleus. Both factors continue to be restricted from the nucleus until cellular activation occurs [13]. Under these restricted conditions, the production of functional transcripts is extremely low. This compounds the problem, because with such a low level of functional transcripts, it is difficult to attain the necessary levels of Tat. As noted earlier, Tat is required, not only for the transactivation of transcription, but also the recruitment of P-TEFb, whose levels are also low in resting cells [94]. In essence, exclusion of NF-κB and NFAT from the nucleus in a resting CD4 + T cell inhibits production of sufficient Tat. Insufficient Tat, along with low levels of P-TEFb, prevents processive transcription from occurring, and thus results in an accumulation of the short transcripts characteristic of latent HIV infection. This supports a role for low levels of Tat and host transcription factors in the mechanism driving HIV latency. Another potential contributing factor to proviral latency may be the site within the chromosome where integration occurs. Quiescent T cells have been found to exhibit large-scale chromatin condensation, transition of genes to these heterochromatic regions, 36

37 and a tightly compacted nucleus [95, 96]. These restrictive characteristics lead to the idea that the state of the chromatin near the site of integration may regulate HIV gene expression. It was thought that a heterochromatic state was excluding necessary transcription factors, thus interfering with processive transcription [8, 10]. However, contradicting evidence has emerged concerning the importance of chromatin at the integration site. Some groups have concluded that proviral integration occurs frequently in heterochromatin, a tightly packed form of DNA which is restrictive to transcription [97-99]. Others, however, have found preferential integration in highly expressed or actively expressed regions referred to as euchromatin [ ]. Integration into active regions may affect the availability of, or cause interference among transcription factors. For example, if the provirus integrated into the chromosome with the same polarity as a surrounding host gene, which was being actively transcribed, read-through transcription from the upstream promoter of the host gene could displace important transcription factors which are required by the HIV promoter. This could also lead to suppression if termination from the upstream promoter did not occur. In this case, termed Transcriptional Interference, when termination of the upstream transcription fails, and reads through into the downstream gene, initiation from the downstream promoter is prevented [87, 103]. Alternatively, if the provirus integrates in the opposite polarity, competition between host and viral promoters, or a collision of polymerases may cause silencing of both host and viral genes [104]. Based on the conflicting reports concerning the accessibility of the integration site, it seems the state of the chromatin may be important, but most likely is not the primary factor behind latent infection. 37

38 Transcriptional repressors, which have the ability to negatively regulate transcription, may also function in the molecular mechanism responsible for postintegration latency of HIV. The transcription factors YY1 and LSF have been shown to cooperatively work to recruit histone deacetylase 1 (HDAC-1) to the HIV LTR to repress transcription [11, 12]. HDACs work to decrease acetylation, and this hypoacetylation is known to correlate with repression of transcription. Another transcription factor, the C- promoter binding factor (CBF-1) has also been shown to inhibit the HIV LTR to induce transcriptional silencing [105]. As previously mentioned above, NELF is also a transcription factor which functions to negatively regulate transcription. The work presented here is intended to demonstrate a role for NELF in the establishment and maintenance of post-integration HIV latency. Summary of Thesis Work The NELF complex is an important transcriptional regulator which functions in blocking elongation in the absence of Tat. This negative regulation contributes to the production of short, abortive transcripts. In instances of latent HIV infection, there is a block to productive transcription of integrated provirus, and short transcripts predominate [30]. Given that short transcripts like those produced as a result of NELF s effects on RNAPII transcription are also produced in cells latently infected with HIV, this investigation aimed to discern whether NELF plays a contributing role in the establishment and maintenance of post-integrational latency. 38

39 Supporting a potential role for NELF in latent HIV is the fact that NELF has a direct interaction with the TAR element in nascent HIV transcripts. This interaction, which appears to be structure specific, occurs between the NELF-E subunit and the stem of TAR s stem-loop structure [71, 72]. When unphosphorylated, NELF-E maintains its interaction with TAR, allowing the complex to continue to negatively regulate by inducing a pause in transcription and preventing the production of full length transcripts [55]. The short transcripts seen in this negative regulation suggest a possible role for NELF in production of the short transcripts observed in peripheral blood mononuclear cells (PBMCs) of HIV-infected persons [106]. If the short transcripts observed in PBMCs from HIV-1 infected individuals are due, in part, to the negative regulation of transcriptional elongation imposed by NELF, then this would implicate NELF as a critical factor in controlling the conversion from non-productive to productive transcription of HIV-1, and confirm a role in the mechanism behind latent infection. In an effort to confirm NELF is contributing to HIV proviral latency, an shrnabased approach has been applied to an inducible Jurkat T cell model of latency. This model allows for the detection of a state of activation using d2egfp as an indicator. Following the identification of clones stably producing mcherry-expressing shrna designed to silence either NELF-E or NELF-A, analysis of uninduced activation revealed that silencing of the NELF-E subunit resulted in partial activation of the latent provirus cells. This was in contrast to silencing of NELF-A, which had no effect on the activation of transcription. This was the first evidence to suggest that the individual subunits of NELF were behaving distinctly, and that transcriptional repression was not simply due to the complex functioning as a whole. Time course experiments designed to assess the 39

40 rate at which NELF-silenced cells would return to latency after activation with external stimuli, revealed that shutdown in the context of NELF-A silencing mimicked that of wild type cells. However, NELF-E silenced cells were found to be unable to completely reach a latent state. They instead shut down only to the degree of the basal level at which they were activated without additional stimulation. Given these distinctions between the E and A subunits of NELF and the involvement in latency, it was then important to look closely at differences at the level of chromatin. Using Chromatin Immunoprecipitation (ChIP) assays, it was found that each subunit had a distinct impact on transcription, with the NELF-E subunit exclusively exerting an effect on transcription elongation. In comparison with cellular genes, both those known to be regulated by NELF and those which are NELF-independent, it was determined that the exclusivity of the NELF-E silencing effect was specific to HIV transcription. Although not surprising, given the direct association of NELF-E with HIV RNA transcripts, this was a novel finding, which may potentially have implications for understanding the mechanisms underlying post-integration latency of HIV. In addition to the work on NELF s contribution to latency, a separate project was undertaken to attempt to inhibit HIV transcription elongation by employing dominant negative CycT1 mutants. This work resulted in the identification of two dominant negative mutants capable of inhibiting HIV transcription through distinct mechanisms. One mutant acts by specifically degrading Tat, while the other forms a kinase-inactive complex which inhibits HIV transcription. Results of this work may potentially facilitate 40

41 the development of a therapeutic strategy designed to target host transcription factors in an HIV-specific manner. 41

42 A B Ab Absence of Tat N-TEFs (negative elongation factors) TAR NFkB SP1 TATA RNAPII Abortive Transcripts C Presence of Tat Presence of Tat P P P P-TEFb Cdk9 CycT1 Tat Full length transcripts 42

43 Figure 1.1 Transcription of the HIV provirus must occur for productive infection. (A) In the life cycle of HIV, the virus attaches to a T-cell by binding to the cluster of differentiation 4 (CD4) receptor on the surface of the cell and subsequently binding to either the CXCR4 or CCR5 co-receptor. Following attachment, the virus fuses with the cell membrane, and viral uncoating occurs. With the release of viral RNA, reverse transcription produces DNA, which is imported into the cell nucleus and integrated into the host genome. In activated cells, this DNA is transcribed, generating HIV mrna. This mrna is translated into viral proteins which are then assembled at the cell membrane into new virions. These new viral particles bud from the membrane, and are released to propagate infection in other cells. This figure was adapted from Reeves, JD [107]. (B) Transcription elongation of HIV can occur in two manners. As highlighted in (A), transcription of HIV is required to produce viral mrnas for packaging into new virions. In the absence of the viral protein Tat, negative transcription elongation factors act to impede the processivity of RNAPII. This results in the production of short, abortive transcripts which are insufficient for the continuation of the viral life cycle. (C) Conversely, in the presence of Tat, P-TEFb is recruited to TAR, which then elicits hyperphosphorylation the CTD of RNAPII. This promotes the dissociation of negative elongation factors and allows for productive transcription of full length HIV mrnas. 43

44 Ag HIV HIV X HIV Pre-Integration Latency X Post-Integration Latency Ag HIV Ag Ag Ag CD4 + T cells HIV Naive Resting Memory Activated Virion RNA DNA Host Chromosome Figure 1.2. Establishment of latent HIV infection in CD4 + T cells. Naïve CD4 + T cells are not susceptible to infection by HIV. When cells become activated by interaction with antigen they become effector cells. Small populations of these cells survive and transition back to a resting state as memory T cells. Activated CD4 + T cells are the primary target for HIV infection. When these effector cells become infected the virus readily progresses through its life cycle and eventually releases progeny virus, killing the cell. The potential for the establishment of latency exists prior to the integration of viral DNA into the host chromosome and after integration. Pre-integration latency arises when a resting memory cell becomes infected. Because reverse transcription is slow and 44

45 nuclear export is blocked, the HIV DNA quickly becomes non-functional. If, however, activation is induced by antigen before the decay, the virus is capable of reproducing. Post-integration latency can occur when activated CD4+ T cells infected with HIV transition back to a resting memory state carrying provirus in their chromosomal DNA. Upon exposure to antigen, these cells become re-activated, and then can begin to express the viral genes necessary to complete the viral life cycle. This figure was adapted from Lassen, K [13]. 45

46 Chapter 2: The NELF-E Subunit of the Negative Elongation Factor Directly and Specifically Contributes to Post-integration Latency of HIV 46

47 Summary The ability of HIV to enter a latent state represents a major obstacle to the eradication of AIDS. Latency in HIV is characterized by a defect at the stage of viral transcription, although the molecular mechanism(s) involved have yet to be definitively identified. The purpose of this investigation is to determine what role the NELF transcription elongation factor plays in the establishment and maintenance of latent HIV infection, and how this contribution arises. Using an shrna-knockdown approach against 2 of the 4 subunits of NELF (NELF-A and NELF-E) applied to a re-activatable Jurkat T cell model of latency has demonstrated that NELF is an important factor used to maintain proviral latency, and has also led to the identification of a novel role for the NELF-E subunit in HIV transcription regulation. FACS analysis in NELF-E suppressed cells led to the finding that silencing of NELF-E causes partial activation of latent provirus. Furthermore, this absence of NELF-E slows the rate and extent of reversion back to a latent state following cellular stimulation. ChIP analysis also revealed knockdown of NELF-E specifically enhanced the recruitment and distribution of RNAPII to regions all along the HIV proviral genome. These results are the first to suggest the negative regulation imposed by NELF in proviral latency is specific to the NELF-E subunit, whereas the NELF-A subunit appears to be dispensable in the case of HIV transcription elongation. 47

48 Introduction Latency associated with infection by the Human Immunodeficiency Virus (HIV) is a major obstacle to the eradication of AIDS. Currently, the most widely used form of treatment, Highly Active Anti-Retroviral Therapy (HAART), is only effective against actively transcribed virus. However, in the case of latent infection, the virus has integrated into the host chromosome but remains transcriptionally silent. In order to combat this dilemma imposed by latency, it is important to fully understand the complexities involved in post-integration latency. Currently, though, the mechanisms responsible for development and maintenance of latency are not well defined or understood. Many potential mechanisms have been proposed to explain this phenomenon. Reasons attributed to proviral latency include: insufficient Tat or host transcription factors, inaccessibility of transcriptional machinery due to the site of integration, and the presence of transcriptional repressors [13, 93]. In any case, it is generally agreed upon that latency is regulated and maintained at the level of transcription. However, transcription of HIV itself is a very complex process. Transcription of HIV requires a complex interplay between positive and negative cellular factors. In the absence of the viral Tat protein, the presence of negative transcription factors DSIF and NELF at the LTR act cooperatively to repress transcription and contribute to the production of short, abortive transcripts [57, 62, 72]. When Tat is 48

49 present, however, it acts to recruit the positive transcription elongation factor, P-TEFb. P-TEFb, which is composed of Cdk9 and Cyclin T1, induces phosphorylation of both DSIF and NELF, as well as RNA polymerase II [19, 58, 72]. This allows for release of the inhibition, enhances the processivity of polymerase, and thus results in production of full length transcripts. In cells latently infected with HIV, it has been documented that there is an accumulation of abortive transcripts in the cytoplasm [30]. These transcripts are similar to those produced when negative factors such as NELF are present. Given the apparent connection, and the great need for a better understanding of the mechanisms underlying latent HIV infection, this study aims to elucidate a role for NELF in contributing to latency. Additionally, this work should provide insight into the magnitude of NELF s contribution, enabling safe manipulation of the latent reservoir to gain access for therapeutics. Results A system for defining the role of NELF in latent HIV. NELF is an important cellular factor whose regulation of HIV transcription prevents efficient elongation in the absence of Tat. This negative regulation is believed to result from the association of the NELF-E subunit with the TAR element of nascent HIV transcripts, an association which is relieved upon phosphorylation [71, 72]. Although NELF, in conjunction with DSIF, has been found to associate with RNAPII 49

50 shortly after the initiation of transcription, it is unclear as to exactly how it is recruited. The presence of NELF in the transcription complex prevents processive transcription and results in the accumulation of short, abortive transcripts. Latent HIV infection is a phenomenon in which the integrated provirus remains transcriptionally silent, and production of short, incomplete transcripts is predominant [30]. Given the characteristic short transcripts of latent infection, and the production of such transcripts in the presence of the NELF complex, it has become important to determine what, if any, contribution NELF provides to latency. While this work was in progress, Henderson and colleagues also obtained evidence confirming NELF can inhibit transcription from an integrated provirus [78]. To determine the exact contribution of the NELF complex to establishing or maintaining latent infections with HIV, an shrna-based approach has been employed (Fig. 2.1). Target sequences intended to suppress the NELF-A and NELF-E subunits, as well as corresponding scrambled controls (Table 2.1) were constructed and cloned into the psuper vector, which provides the RNAP III- transcribed H1-RNA promoter for expression of small hairpin RNA [108]. The target sequences and H1 promoter were then subcloned into the plvthm lentiviral vector [109], which has been modified to express the mcherry fluorescent protein, in place of green fluorescent protein (GFP), to allow for monitoring infection (Fig. 2.2). A Jurkat T cell line, developed in the Karn laboratory, expressing an attenuated Tat along with a fluorescent reporter gene (Fig. 2.3) serves as an inducible model of latent HIV infection [110]. This model uses an integrated provirus containing the H13L 50

51 mutation in Tat, a variant first identified in the U1 latently infected cell line, which has been documented to have reduced transactivation capabilities and lead to a progressive reduction of HIV expression [110, 111]. These cells also express the d2egfp fluorescent marker, in place of Nef, and its short half-life allows for more accurate assessment of transcription rates. Specifically, these cells contain an NF-κB-responsive integrated LTR that can be stimulated with TNF-α to activate d2egfp expression. By monitoring d2egfp expression using FACS analysis, over time, it has been documented that after stimulation with TNF-α, these cells return to a latent state when NF-κB is removed (Fig. 2.3). For all results described herein, a single clone of these H13L Jurkat cells, termed clone 2D10, has been used. As published by Pearson et al. 2008, this clone has a high level of transcriptional restriction prior to NF-κB induction, as the unstimulated cells are more than 95% d2egfp-negative. Following TNF-α treatment, appreciable levels of d2egfp can be detected in over 98% of the population [110]. This H13L Jurkat clone was infected with VSV-G pseudotyped lentiviruses made from the plvthm-mcherry shrna constructs intended to stably suppress the NELF-E or NELF-A subunits. Additionally, this clone was also used for infection with viruses expressing scrambled control shrnas. Expression of the mcherry reporter was measured by flow cytometry 48 hours post infection and was used to assess and identify successful integration. Based on FACS analysis, it was determined that a sizable portion of cells were infected (Fig. 2.4). Cells in the mcherry-positive population were further sorted to obtain clonal populations. Following single cell sorting, cells were expanded for approximately 3-5 weeks before levels of mcherry expression were once again monitored. These shrna clones were also used for Western blot analysis to confirm 51

52 suppression of NELF-E or NELF-A protein levels (Fig. 2.5). Although several clones were found to both express mcherry and suppress NELF expression, it was decided to focus on one clone for each of the target and scrambled sequences for the remainder of this study. NELF-A shrna clone 1F6, and its corresponding control clone 3F11, as well as NELF-E shrna clone 2G10 and NELF-E scrambled clone 2F2 have been chosen based upon robust expression of mcherry and strong suppression of NELF, or lack thereof in the case of the scrambled controls. Suppression of NELF-E partially activates latent proviral HIV. Following the determination of ideal clones, it was then necessary to identify any changes in proviral expression due to suppression of NELF subunits. By looking at d2egfp expression in unstimulated shrna clones, it was found that suppression of NELF-A caused no change in d2egfp expression, as compared to both uninfected and scrambled control cells. However, knockdown of NELF-E induced d2egfp in 23.1% of cells, again compared to a lack of expression in controls (Fig. 2.6). This increase in d2egfp levels corresponds to activation of the latent provirus. In order to confirm that the result seen when NELF-E is suppressed is not an isolated phenomenon, several additional clones were analyzed for d2egfp expression. As represented in Figure 2.7, multiple clones showed similar activation profiles, with d2egfp expression ranging from 18.8% to 37.2% of the total population. Although only a partial activation occurs with suppression of NELF-E, these levels are comparable to activation seen with several other drugs or treatments. Activation with prostratin, a phorbol ester, was able to induce viral expression in 8.3% of the population in a model of latency, whereas costimulation with 52

53 antibodies to CD3 and CD28 activated 9% of those cells [112]. Both IL-7 and IL-2 have also been reported to induce activation of latent provirus. In experiments using murine CD24 as a virus-encoded reporter of proviral activation in a SCID-hu mouse model, IL-7 treatment resulted in proviral activation of 5.22% of cells, while IL-2 activated provirus in 2.72% of cells, compared to 4.94% in cells costimulated with anti-cd3 and anti-cd28 [113]. To confirm specificity of the effect seen with NELF-E suppression, additional clones suppressing NELF-A were also evaluated; none were shown to activate expression of d2egfp (data not shown). The existence of a sub-population of cells within a clonal population has been previously documented in this model system [110]. As shown in Figure 2.8, Pearson et al documented an H13L Jurkat clone (Clone 6) in which individual cells of the population were d2egfp + without stimulation. To determine if these cells were constitutively active, they were sorted into d2egfp positive and negative populations and monitored by FACS analysis. Over time, these cells failed to maintain distinct populations, but instead experienced a spontaneous reactivation and shutdown to once again reach a state of equilibrium [110]. To determine if a similar phenomenon was occurring in the NELF-E clones, 2 clones were chosen and sorted into d2egfp positive and negative populations, and monitored by FACS. These cells were also found to spontaneously activate and shutdown in a reversion back to a dual state of activation (Fig. 2.9). This dynamic reversion between activation states may indicate an inherent requirement for the cell to balance positive and negative factors. Silencing of NELF-E affects the rate and extent of reversion to latency. 53

54 Given that suppression of NELF-E resulted in a basal level of activation, it was important to explore what the effect would be on the reversion to latency following cellular stimulation. To do this, uninfected H13L Jurkats, NELF-A and E shrna clones, and their respective scrambled control cells were activated overnight with 5µg/ml anti- CD3 and monitored for d2egfp expression via FACS analysis. Results indicate that although each cell line became activated to a similar level, the subsequent shutdown rate of the NELF-E silenced clone was much slower than all others (Fig. 2.10b). Both scrambled controls and the NELF-A knockout exhibited shutdown profiles which closely mimicked the uninfected H13L Jurkats and each other (Fig a,b). Additionally, the NELF-E silenced cells were found to never reach a degree of shutdown beyond their initial activation level, even when the experiment was carried out 14 days post- activation with anti-cd3 (Fig. 2.10b, and data not shown). Identical experiments performed using an mcherry-positive mixed population stimulated with 10ng/ml of TNF-α as a stimulus showed similar results (Fig. 2.11). Collectively, these results suggest the repression of NELF-E is necessary for latency. NELF-E silencing also has a more pronounced transcriptional effect on HIV than silencing of NELF-A. The same shrna viruses initially developed for infection of the H13L Jurkat cells, were then applied to another cell model commonly used to measure transcriptional effects. Although not a model of latency, HeLa phr luc cells can be used to measure transcriptional activity in HIV. These cells are an actively transcribing, constitutively active model of HIV transcription which harbors a luciferase gene under the control of 54

55 the HIV LTR (Fig. 2.12a). These cells also do not express any form of Tat. Assay of cell lysates results in a readout of luciferase activity, which correlates with transcriptional activity. These HeLa phr luc cells were infected with the NELF shrna viruses and scrambled control viruses, and assayed after 48 hours. Results indicate that silencing of NELF-E again shows a greater increase in transcription levels than silencing of NELF-A, from an increase of fold over uninfected when NELF-A is silenced, to an increase of 1.7- fold over uninfected when NELF-E is suppressed (Fig. 2.12b). This suggests further a specific role for the NELF-E subunit in negatively regulating HIV. Silencing of NELF-E specifically enhances the recruitment of RNAPII to regions along the HIV proviral genome. Given the apparent role NELF-E plays, specifically, in contributing to the suppression of transcription which is characteristic of latency, it became necessary to obtain a more detailed analysis of transcription along the genome when NELF-E is absent. Chromatin Immunoprecipitation (ChIP) assays using an RNAPII antibody were performed in the same NELF shrna and scrambled clones, as well as the uninfected H13L Jurkat clone. Distribution of RNAPII was then determined using various primer sets along the proviral genome. Additionally, as a control, it was important to evaluate another gene which has been documented to be NELF-regulated. For this purpose, recruitment of RNAPII along junb, an immediate-early gene which functions as a component of the AP-1 transcription factor complex, was also measured. Work by Aida et al has shown that NELF is involved in regulating both basal and activated levels of transcription of junb [114]. ChIP assays were performed in the absence of TNF-α, and 55

56 after 60 minutes of stimulation with 2ng/ml of TNF-α. Prior to this work, enhanced expression of junb has been shown in the presence of TNF-α [115], indicating that similar to HIV, it is also NF-κB responsive. Additional control genes also evaluated included the cellular GAPDH and IκBα genes. These genes serve as internal controls which differ in NF-κB-responsiveness. GAPDH is NF-κB-independent, and is typically stable and well-expressed in most cells. Transcription of IκBα is responsive to NF-κB, and therefore also acts as an important control gene. In the absence of TNF-α stimulation, H13L Jurkat-derived cells essentially represent a Tat-negative condition, due to the lack of sufficient NF-κB levels required to stimulate the transcription required for production of appreciable levels of Tat. ChIP assay results reveal that in the absence of stimulation with TNF-α, suppression of both NELF-E and NELF-A can enhance recruitment of polymerase in promoter-proximal regions (Fig. 2.13a, right panel). However, the suppression of NELF-E shows a more dramatic effect, which is sustained, to some degree, along the entire length of the genome (Fig 2.13a, right panel, Fig top row, right). In contrast, RNAPII distribution along the junb genome in the absence of stimulation shows a similar profile among knockout, scrambled, and uninfected clones (Fig 2.13b, right). Silencing of neither NELF-E nor A alters the trend in polymerase recruitment to regions of junb. In comparison to the other control genes, the downstream effect on transcription seen with silencing of NELF-E appears to be specific to HIV (Fig. 2.14, right column). This specific impact on transcription seen with NELF-E suppression becomes even more apparent following stimulation with TNF-α. Although after one hour of TNF- 56

57 α treatment Tat and its effects are still somewhat restricted, there was a significant and dramatic increase in the amount of polymerase present at all points along the HIV genome (Fig. 2.13a, left). Especially noteworthy is the sustained elongation effect, which becomes more pronounced with stimulation (Fig, 2.13a, left and Fig top row, right). Once again the RNAPII profile along the junb gene appears to be similar among all NELF conditions, with only a slight increase seen in the middle of the gene when NELF- E is absent (Fig. 2.13b, left). This effect diminishes at more distal regions of the genome, in contrast to findings along HIV (Fig. 2.13b, left, and Fig. 2.15, right column). As compared to the outlined controls, the ability of NELF-E suppression to stimulate polymerase recruitment, and thus transcription, is specific for HIV. Although junb has been documented to be regulated by the NELF complex, it appears that both the NELF-E and NELF-A subunits contribute to the regulation at some point along the genome (Figs. 2.13, 2.14, 2.15). Interestingly, Aida et al have indicated that the GAPDH gene is not affected by NELF-E silencing [114]. However, in the absence of TNF-α, at the downstream region, it appears that the NELF elongation factor may be regulating basal transcription (Fig. 2.14). Once again, though, this regulation is not NELF-E specific, as increased polymerase levels are found in cases of both NELF-E and NELF-A suppression, adding further support to a specific role in HIV. Discussion Latent infection of HIV is a major obstacle to the eradication of the virus. Postintegration latency, and its preservation, is a complex and multifactorial problem most 57

58 often characterized by defects or restrictions in transcription. Negative Elongation Factor, NELF, is a cellular factor known to function in the negative regulation of HIV transcription. Recent work has shown that NELF is able to inhibit transcription from an integrated HIV provirus [78]. In order to gain a better understanding of the intricacies involved in latency, it has therefore become necessary to investigate the role NELF plays in latency, and the specifics of its involvement. Prior to this work, it has been generally assumed that all subunits of NELF are necessary and work interdependently to repress transcription. It was first reported by the Handa group that each of the 4 subunits is important, and that they co-exist primarily as a complex, citing that immunodepletion of 1 subunit causes a reduction in others [67, 71]. Additionally, depletion of either NELF-B or NELF-E resulted in similar transcriptional effects, suggesting a tight functional interdependence among subunits [69]. NELF-E and NELF-B have been shown to directly interact with one another [67]. In one of the most compelling studies, albeit in Drosophila, a genome-wide location analysis showed NELF- B and E distribution overlapping each other by a minimum of 1kb at least 80% of the time [116]. This inability to distinguish unique behavior and the closeness of proximity between subunits is consistent with NELF functioning primarily as a complex. To discern NELF s contribution to latency, an shrna-based approach was applied to a re-activatable model of latency based in Jurkat T cells. H13L Jurkat cells were infected with lentiviruses expressing both mcherry and shrna to stably suppress expression of the NELF-E and NELF-A subunits. Subunit-silenced clones, as well as scrambled control clones were subsequently evaluated for transcriptional effects on latent 58

59 proviruses, as compared to wild type, uninfected H13L Jurkats. Results indicated that knockdown of NELF-E, but not NELF-A can partially activate latent provirus, without any extracellular stimulation (Fig. 2.6). Analysis of several clones confirmed that this is not an isolated phenomenon, and that it appears specific to the NELF-E subunit (Fig. 2.7). This is an indicator that, at least in the case of HIV, each subunit of NELF is not contributing equally to repression of proviral transcription. It may be that the NELF-A subunit is dispensable functionally, yet necessary for the integrity, or proper interaction among subunits to form the complex. Regardless, it appears that the NELF-E subunit contributes specifically to HIV latency. It can also be inferred that the transcriptional block imposed by NELF-E is necessary for post-integration latency. By evaluating the shutdown rate in these cells following activation, it was discovered that, although all clones were capable of activating to the same extent, NELF-E silenced cells displayed a much slower return to latency (Fig. 2.10). Additionally, the NELF-E suppressed cells were unable to completely shut down, and interestingly never reverted beyond their initial level of basal activation. The fact that these cells never completely shut down suggests that NELF-E is a necessary contributing factor to establishing a latent infection. Confirming that this NELF-E effect is unique to HIV required comparison to other genes using ChIP analysis to track RNA polymerase II distribution along genomes. Both with and without TNF-α treatment, suppression of NELF-E was distinctively able to enhance processivity of RNAPII, with the most dramatic accumulation of polymerase seen as the transcription complex was able to overcome the pause site invoked by the 59

60 TAR stem-loop structure (Fig. 2.13a). Maintenance of the elongation effect was most notable at the distal regions of the HIV genome in the presence of TNF-α stimulation (Fig. 2.13a, left, and Fig right column), although a similar, more subtle effect could be detected without stimulation (Fig. 2.13a, right, and Fig right column). Comparison to the junb gene, which is also negatively regulated by NELF, confirmed further that this NELF-E effect is exclusive to HIV. Both in the presence and absence of TNF-α, the polymerase profiles along the junb genome are very uniform (Fig. 2.13b). There is no distinct contribution of one subunit over the other affecting junb transcription. Analysis of the cellular genes GAPDH and IκBα, and their lack of a notable change in polymerase recruitment due explicitly to suppression of NELF-E adds further strength to the suggestion that NELF-E functions expressly and specifically in contributing to HIV latency. This is consistent with the model presented in Fig In Fig. 2.16a, cellular genes such as IκBα and GAPDH are regulated by the general association of NELF and DSIF with the polymerase to increase the time the transcription complex stalls at pause sites. Following the recruitment of P-TEFb, Cdk9 phosphorylates the RNAPII CTD and the Spt5 subunit of DSIF, allowing for enhancement of elongation, whereas the phosphorylation of NELF-E dissociates the complex from the transcriptional machinery to remove the inhibition. The NELF-free transcription complex is then able to fully transcribe the gene. In the case of HIV, though, it appears that the role of NELF is more specific, and this specificity is imparted by the NELF-E:TAR interaction. In the absence of Tat (Fig. 2.16b), the NELF-E:TAR interaction is maintained, which efficiently prevents RNAPII from elongating the transcript, resulting in production of premature 60

61 mrnas. In Figure 2.16c, when Tat is present in the HIV system, its recruitment to TAR, along with P-TEFb, stimulates elongation. The Cdk9 subunit again phosphorylates Spt5 and the RNAPII CTD, allowing for enhanced elongation activity. It also phosphorylates NELF-E, enabling its dissociation from TAR. Combined, these events allow the transcription complex to proceed along the genome. Although unknown, it is possible, that the NELF-E subunit may become unphosphorylated at a later stage and again become associated with TAR to allow for regulation at additional downstream pause sites. If this were the case, whether the NELF complex maintains its integrity, or whether NELF-E can dissociate from the complex and regulate as a monomeric factor is yet to be determined. When NELF is absent from the system, as outlined in Fig. 2.16d, the ability to only partially activate transcription suggests that both short and full-length transcripts are made. Short transcripts could be due to the negative regulation imparted by DSIF. Full-length transcripts most likely represent DSIF s inability to maintain complete repression of transcription without NELF s contribution, thereby allowing some successful elongation. Throughout this study, results seen with suppression of NELF-A mimic those of uninfected cells or scrambled controls. This is suggests the NELF subunits do not exhibit close functional interdependence in the transcription of HIV. Although challenging to the widely accepted idea that all subunits work cooperatively to repress transcription, this is not the first report of one subunit having a greater, more dominant impact. In work published by McChesney et al, they found that NELF-B regulation of the TFF1 gene is independent of NELF-E in gastric cancer cells [70]. This does not exactly agree with the work in Drosophila showing NELF-B and E migrate together, although 20% of the time, 61

62 they did not [116]. This provides support for the hypothesis that different subunits can regulate to varying degrees depending on the particular gene being transcribed. Although there are no indications as to specific factors or motifs which would impose a requirement for one subunit over another, the RNA Recognition Motif of NELF-E could be one explanation for its apparently distinct role in HIV and latency. All nascent HIV transcripts contain the TAR stem loop structure. NELF-E has the ability to bind to double-stranded RNA through its RRM, and thus binds the stem portion of TAR [72]. This viral element may be responsible for inferring specificity of NELF-E. Although the work first implicating NELF as an inhibitor of proviral transcription was performed using suppression of NELF-B, suggesting the B subunit was imposing the negative regulation [78], this is not necessarily the case. It has already been suggested that the NELF-B and NELF-E subunits are intimately linked and directly interacting [67, 71]. It has also been shown that suppression of one subunit oftentimes results in suppression of another [117]. It is therefore possible that the effect seen with suppression of NELF-B could be resultant of indirect suppression of NELF-E. Further studies will be necessary to clarify this issue, as well as to gain a better understanding of the impact NELF imparts on the establishment and maintenance of post-integration latency in HIV infection. 62

63 Materials and Methods Luciferase Assays. HeLa phr P-Luc cells were maintained in Dulbecco s Modified Eagle s Medium (DMEM) supplemented with 10% fetal bovine serum at 37 C with 5% CO 2. Cells were transfected using Lipofectamine 2000 (Invitrogen) and harvested after 48 hours. Cells were washed with phosphate-buffered saline, and lysed using 1x Passive Lysis Buffer (Promega) containing Protease Inhibitor Cocktail (Sigma) for 1 hour at 4 C, with rocking. 15μl of lysate was assayed using 50μl of substrate (Promega) added by the injection unit of a microplate luminometer (Bio-Rad). Luciferase Activity was normalized to total protein concentration using the Protein Assay kit (Bio-Rad). Western Blots. Western Blot analysis was performed in accordance with standard protocols. Anti- NELF- E, anti-nelf- A (Santa Cruz Biotechnology), and anti-β actin (Cell Signaling Technology) primary antibodies were used. Anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad) was used along with Immobilon Western Chemiluminescent HRP Substrate (Millipore) to detect proteins by chemiluminescence on X-ray film (Denville Scientific). ChIP Assays. Jurkat cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 100 μg/ml Normocin (Invivogen) at 37 C with 5% CO 2. Cells were stimulated with 2ng/ml TNF-α as indicated. Cells were crosslinked for 10 minutes using 0.5% formaldehyde, followed by the addition of glycine at a final concentration of 125mM for 5 minutes to quench unreacted 63

64 formaldehyde. Nuclei were sonicated for 10 pulses of 20 seconds each (Misonix Sonicator 3000) to generate DNA fragments averaging between 200 and 1000 base pairs each. Immunoprecipitations were performed using anti-rnapii (Santa Cruz Biotechnology) at a dilution of 1:500, incubating overnight at 4 C, with rotation. Washed Protein A Sepharose beads (50 μl), which had been pre-incubated with BSA and Calf thymus DNA, were added for 2 hours, with rotation at 4 C. The beads were pelleted and washed with Low Salt Immune Complex Buffer, High Salt Immune Complex Buffer, and RIPA Buffer, each kept on ice, one time for 5 min each with rotation at 4 C, followed by 2 washes with TE buffer at room temperature. The immunocomplexes were then eluted from the beads, 20 μl of 5M NaCl was added, and samples were reverse crosslinked at 65 C overnight. The following day, 10 μl of 0.5M EDTA and 10 μl 2M Tris Cl, ph 6.5, and 2 μl of 10mg/ml of proteinase K were added to the eluates, with incubation for 2 hours at 50 C. DNA was recovered by phenol/chloroform extraction and ethanol precipitation and used for real-time PCR analysis. FACS Analysis. Cells were analyzed by flow cytometry using the LSRII flow cytometer (Becton Dickinson) and data were analyzed using the FacsDIVA program. Construction of shrna plasmids. Using the Oligoengine RNAi Design Software tool, complementary oligonucelotides containing the BglII and HindIII restriction enzyme sites were designed to target the NELF-A and NELF-E genes, as well as corresponding scrambled controls containing randomized identical nucleotides (Table 2.1). Following annealing, the oligonucleotides were cloned into the corresponding sites in the psuper 64

65 vector. Target sequences and the H1 promoter were subsequently cut out of the psuper vector using ClaI and EcoRI, and inserted into the plvthm plasmid, which has been modified by excising the GFP sequence and replacing it with mcherry. Infection of H13L Jurkat cells. VSV-G pseudotyped shrna viruses were produced by transient co-transfection of plvthm shrna plasmids with the packaging plasmids pmd.g and pcmvδr8.9i into 293T cells using Lipofectamine 2000 (Invitrogen) [118]. mcherry expression was assessed 48 hours later by flow cytometry to confirm expression, and Western blots were performed to evaluate NELF expression. The mixed populations were then sorted into 100% mcherry positive populations, and subsequently sorted and grown into clonal populations. Clones were again assessed for NELF expression levels by Western Blot analysis. Activation and shutdown of H13L Jurkat cells. Cells were activated with either 10ng/ml TNF-α or 5µg/ml plate-bound anti-cd3 (BD Biosciences) for 18 hours and analyzed for d2egfp expression by FACS analysis. To initiate shutdown, cells were washed with PBS and resuspended and maintained in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 100 μg/ml Normocin (Invivogen) at 37 C with 5% CO 2. In subsequent days, cells were analyzed by FACS to determine the percentage of d2egfp positive cells. 65

66 Figure 2.1 Outlining the experimental approach to studying the role of NELF in latent HIV. An shrna-based approach originating in the psuper RNAi expression vector has been applied in elucidating the role of NELF in HIV latency. Target sequences were designed using the Oligoengine RNAi Design Software and cloned into psuper, providing the H1 promoter. The target sequences were subsequently cut from psuper and cloned into the plvthm-mcherry lentiviral vector. The constructs were used to infect the H13L Jurkat T cell clone serving as a model of latent infection. Infections was confirmed and assessed 48 hours later by FACS analysis for mcherry 66

67 expression. Cells were then sorted for, and later cloned by mcherry expression. Suppression of NELF subunit expression was confirmed by Western Blot analysis. 67

68 Target Nucleotide Sequence NELF-A shrna NELF-A scramble GCAGCCTAAGAAGAACCTG AGAACAAACGCACGTGCAC NELF-E shrna NELF-E scramble CAGCCAAGGTGGTGTCAAA GAGCTTAGACTCGAACGGA Table 2.1 Sequences of shrna targets. Target sequences for shrna were identified using the RNAi Design Software tool on the Oligoengine website ( Scrambled sequences are composed of the same nucleotide composition as their target gene counterparts; however the order of nucleotides has been randomized. 68

69 Figure 2.2 Construction of shrna lentiviral plasmids to target NELF. The H1 promoter and sequences targeting NELF-E and NELF-A subunits, as well as corresponding scrambled controls, were excised from previously confirmed psuper constructs using the ClaI and EcoRI restriction sites and inserted into the plvthm 69

70 lentiviral vector. The plvthm vector had been modified to express the mcherry fluorescent protein in place of GFP. Vector map is taken from 70

71 d2egfp Figure 2.3 Activation and shutdown of H13L Jurkat clone 2D10 following TNF-α stimulation. A Jurkat T cell clone expressing the H13L variant of the Tat protein and d2egfp as a marker of activation serves as a model system for HIV latency. When stimulated with TNF- α, these cells become activated, as reflected in d2egfp expression. Over time, this expression decreases, and cells return to a latent, or shutdown state. 71

72 Figure 2.4 Initial infection of H13L Jurkat cells with mcherry shrna viruses. H13L Jurkat cells were infected with lentiviruses expressing both the mcherry fluorescent protein and shrna against NELF target sequences or controls. FACS analysis 48 hours later revealed a sizable percentage of cells were infected, allowing for stable expression of the shrna. 72

73 mcherry 73

74 Figure 2.5 Clones expressing mcherry-shrna suppress NELF expression. Clones were isolated by single-cell FACS sorting for mcherry expression, followed by Western Blot analysis to assess knockdown of the target NELF subunit. Expression of β-actin serves as the comparative loading control. 74

75 d2egfp Figure 2.6 NELF-E shrna clone is able to partially activate d2egfp expression in the absence of stimulation. Silencing NELF-E in H13L Jurkat cells causes partial (23.1%) activation of the latent provirus in the absence of cellular stimulation, as observed by expression of d2egfp, as determined by FACS analysis. In contrast, cells harboring shrna against scrambled control sequences or NELF-A do not result in proviral activation. 75

76 Figure 2.7 Multiple isolated NELF-E shrna clones show similar partial activation of d2egfp expression. Suppression of the NELF-E subunit in H13L Jurkat cells is consistently able to partially activate expression of latent provirus, to varying degrees, as demonstrated in several clones isolated based upon mcherry expression. 76

77 Figure 2.8 Spontaneous reactivation and shutdown of a latent H13L Jurkat clone. Clone 6 (upper panel), an H13L Jurkat clone, displayed phenotype representing a dual activation state prior to sorting. These cells were sorted into d2egfp - (lower left) and d2egfp + (lower right) populations. FACS analyses immediately following sorting are represented by the dark blue lines. Grey lines represent the same populations after 8 days, and indicate a trend reverting back to the unsorted profile. (As published by Pearson, et al [110]) 77

78 B Figure 2.9 Suppression of NELF-E also exhibits spontaneous activation and shutdown. Two NELF-E suppressed clones, 2G10 (A) and 1G9 (B), which showed appreciable populations of spontaneously activated cells (upper boxes) were sorted into 78

79 d2egfp negative (dark) and positive (light) populations, and subsequently monitored by FACS analysis. Assessment at days 3 and 7 post-sort generally indicate a spontaneous reversion toward the original state of dual activation. 79

80 A B Figure 2.10 Silencing NELF-E prevents cells from establishing a complete latent state following activation with anti-cd3. (A) Clones stably expressing either shrna targeting NELF-A or its corresponding scrambled sequence, in the H13L Jurkat 80

81 background, were activated overnight with 5µg/ml anti-cd3. Anti-CD3 was then removed to allow for examination of the shutdown rate. Activation (represented by d2gfp expression) was monitored by flow cytometry before anti-cd3 stimulation (Day - 1), upon activation (Day 0), and in days subsequent. As illustrated above, NELF-A knockdown cells shutdown at a similar rate to both uninfected H13L Jurkats and the scrambled control. (B) An identical shutdown timecourse was performed in the NELF-E shrna clones as done in (A). The NELF-E suppressed clone does not shutdown beyond the initial basal activation level. 81

82 Figure 2.11 Silencing of NELF-E prevents the establishment of a complete latent state following stimulation with TNF-α. H13L Jurkat cells (clone 2D10) were infected with an mcherry-expressing shrna lentivirus targeting NELF-E or a scrambled control and sorted for mcherry-positive populations. These sorted populations, as well as the original H13L clone were stimulated overnight with 10ng/ml TNF-α overnight. TNF-α was then removed to allow for assessment of the shutdown rate. Activation (measured by d2egfp expression) was monitored by flow cytometry before TNF-α treatment (Day -1), upon activation (Day 0), and in subsequent days. NELF-E knockdown cells fail to shutdown beyond initial basal activation levels, unlike the scrambled or uninfected controls. 82

83 A B Figure 2.12 NELF-E silencing also has a more dramatic transcriptional effect than silencing of NELF-A. HeLa cells stably expressing a luciferase reporter gene under the control of the HIV LTR (A) were infected with the mcherry shrna viruses. (B) In assays of the cell lysates, greater luciferase activity was observed in the NELF-E knockdown cells, which indicates an increase in transcription levels. The above results 83

84 are representative of assays done in quadruplicate, with error bars representative of standard deviations. 84

85 A HIV B LTR TAR junb 85

86 Figure 2.13 Distribution of RNA polymerase II on the HIV and junb genomes, before and after treatment with TNF-α, when NELF subunits are suppressed. ChIP assays were performed on the HIV proviral genome (A) and the junb genome (B) using RNAPII (N-20) antibody, in the presence or absence of 60 minutes of TNF-α stimulation in clones expressing NELF-shRNA or controls. Suppression of NELF-E specifically results in increased polymerase levels along the HIV genome. 86

87 Promoter Downstream HIV (-4 to +116) HIV ( ) junb (-3 to +133) junb ( ) GAPDH (-125 to -10) GAPDH ( ) IκBα ( ) IκBα ( ) 87

88 Figure 2.14 Comparison of RNAPII at promoter and downstream regions of the HIV provirus and cellular genes in the absence of TNF-α stimulation reveals a specific elongation effect on HIV when NELF-E is suppressed. ChIP analyses were performed in clones expressing NELF shrna or scrambled controls using antibodies to RNA polymerase II at the promoter and downstream regions of the HIV proviral genome and the cellular genes junb, GAPDH, and IkBα in the absence of stimulation. Suppression of NELF-E specifically causes an increase of RNAPII at the downstream region of HIV. Results are representative of assays done in triplicate, with error bars representing standard deviations. 88

89 Promoter Downstream HIV (-116 to +4) HIV ( ) junb (-3 to 133) junb ( ) GAPDH (-125 to -10) GAPDH ( ) IκBα ( ) IκBα ( ) 89

90 Figure 2.14 Comparison of RNAPII at promoter and downstream regions of the HIV provirus and cellular genes in the presence of 60 minutes of TNF-α stimulation reveals a specific elongation effect on HIV when NELF-E is suppressed. ChIP analyses were performed in clones expressing NELF shrna or scrambled controls using antibodies to RNA polymerase II at the promoter and downstream regions of the HIV proviral genome and the cellular genes junb, GAPDH, and IkBα following 60 minutes of TNF-α stimulation. Suppression of NELF-E specifically causes a significant increase of RNAPII at the downstream region of HIV. Results are representative of assays done in triplicate, with error bars representing standard deviations. 90

91 A B C 91

92 D Figure 2.16 Model for transcriptional regulation of cellular genes vs. HIV by NELF. In the general transcription of cellular genes (A) the NELF complex works cooperatively with DSIF to increase the time RNAPII spends at pause sites. P-TEFb relieves this repression when Cdk9 phosphorylates NELF-E, the Spt5 subunit of DSIF, and the CTD of RNAPII. These phosphorylation events enhance processivity of transcription and allow for successful elongation and production of full length mrna. (B) In the case of HIV, in the absence of Tat, NELF-E binds to the TAR RNA structure via its RRM, and along with DSIF, represses transcription by pausing the polymerase complex in the promoter proximal region. This results in an accumulation of short, abortive transcripts. (C) When Tat is present, along with P-TEFb, it is recruited to the loop of TAR. Cdk9 92

93 phosphorylates Spt5, NELF-E and the RNAPII CTD. This causes the dissociation of NELF from TAR, and converts Spt5 into an elongation factor. It may then be the case that NELF-E could re-associate with TAR to help regulation at additional downstream pause sites as the transcription complex travels further down the genome. (D) In the absence of NELF, data presented in this work suggests a partial activation of transcription. This is indicative of producing both abortive and full length transcripts. It may be that DSIF alone exerts enough of a repressive effect to inhibit production of at least some transcripts, and therefore results in incomplete mrnas. DSIF alone appears unable to solely repress all transcription. Without the additional level of repression imposed by NELF, some processive transcription does occur, resulting in production of complete, full length HIV transcripts. 93

94 Chapter 3: Dominant Negative Mutant Cyclin T1 Proteins Inhibit HIV Transcription by Specifically Degrading Tat 94

95 Summary The positive transcription elongation factor b (P-TEFb) is an essential cellular cofactor necessary for transcription of the Human Immunodeficiency Virus type 1 (HIV-1). The cyclin T1 (CycT1) subunit of P-TEFb associates with the viral Tat protein at the transactivation response element (TAR). This represents a critical and necessary step for the stimulation of transcriptional elongation. Given this, the potential exists that CycT1 may serve as a target for the development of anti-hiv therapeutics. To create effective inhibitors of HIV transcription, mutant CycT1 proteins were constructed based upon sequence similarities between CycT1 and other cyclin molecules, as well as the defined crystal structure of CycT1. One of these mutants, termed CycT1-U7, showed a potent dominant negative effect on Tat-dependent HIV transcription despite a remarkably low steady-state expression level. Surprisingly, the expression levels of Tat proteins coexpressed with CycT1-U7 were significantly lower than Tat co-expressed with wild type CycT1. However, these expression levels of CycT1-U7 and Tat were able to be restored by treatment with proteasome inhibitors. Concomitantly, the dominant negative effect of CycT1-U7 was abolished by these inhibitors. These results indicate CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription that could be potentially used in the design of an anti-viral therapy. Contents of this chapter were published by Jadlowsky et al. in Retrovirology 2008, 5:6 [119]. 95

96 Introduction Transcription of Human Immunodeficiency Virus type 1 (HIV-1) is a highly and specifically regulated process in which several host cellular co-factors and the viral transactivator protein Tat are involved [20, 28]. Tat stimulates elongation of transcription with the aid of the positive transcription elongation factor b (P-TEFb), a heterodimeric complex comprised of cyclin T1 (CycT1) and cyclin dependent kinase 9 (Cdk9). Tat and CycT1 bind to the transactivation response element (TAR), an RNA stem loop structure located at the 5'-end (+1 to +59) of all viral transcripts [41, 120, 121]. This interaction results in recruitment of Cdk9 and the subsequent stimulation of its kinase activity by Tat [17]. Among three distinct P-TEFb complexes (CycT1/Cdk9, CycT2/Cdk9, and CycK/Cdk9), only the CycT1/Cdk9 complex can support Tat transactivation [38, 42, 43]. The interaction between Tat, TAR and CycT1 has been extensively studied [28, 41, 43, ]. Tat binds to the bulge region (+23 to +25) of TAR and the CycT1 subunit of P-TEFb through its central arginine-rich motif (ARM; a.a ) and its N- terminal activation domain (a.a. 1-48), respectively. CycT1, in turn, is believed to bind to the central loop (+30 to +35) of TAR through its Tat-TAR recognition motif (TRM; a.a ) in the presence of Tat [20, 28]. Human CycT1 is comprised of 726 amino acids and contains a cyclin box repeat domain (from positions 31 to 250), a coiled-coil sequence (from positions 379 to 530), and a PEST sequence (from positions 709 to 726). The N-terminal cyclin boxes are important for binding and activation of Cdk9. Residues from positions 251 to 272 are essential for the zinc ion-mediated binding between Tat and TAR [121]. This region also interacts with the HEXIM1 protein and 7SK small nuclear 96

97 RNA, the negative regulators of the kinase activity in P-TEFb [46, 47, 50, 123, 124]. The C-terminal region (a.a ) of CycT1 is dispensable for Tat transactivation since the N-terminal cyclin repeats (a.a ) and TRM (a.a ) of CycT1 interact with Cdk9, Tat and TAR [38-41, 120, 121]. Recently, we have determined the crystal structure of the N-terminal region (a.a ) of human CycT1 [125] and its interacting dimeric Cyclin T-binding domain in HEXIM1 [126]. Since P-TEFb is the essential cellular host co-factor of the viral Tat protein, this interaction serves as a potential target for anti-hiv therapeutics. Several approaches have been taken to block HIV transcription by targeting P-TEFb. First, mutant Cdk9 proteins defective in kinase activity have been shown to inhibit HIV transcription in cell culture systems [127]. A number of small compounds which inhibit Cdk9 activities or disrupt the Tat/TAR/P-TEFb interaction have also been tested [ ]. Another approach by Napolitano et al. aimed to inactivate Cdk9 by an oligomerization chain reaction [136]. Additionally, our group has constructed chimeric proteins containing wild type (wt) CycT1 and mutant Cdk9 which inhibited HIV replication up to 90% [137]. Moreover, several CycT1-binding proteins and their truncation mutants have been used as inhibitors of Tat transactivation [ ]. Finally, Bai et al. demonstrated that intrabodies against CycT1 inhibited Tat stimulated transactivation [141]. It is important to note, however, that because P-TEFb is involved in the transcription of many cellular genes [35], it is critical to exclusively block HIV-specific pathways in order to develop safe and effective anti-hiv therapies 97

98 In this study, we sought to construct dominant negative CycT1 mutant proteins capable of blocking HIV transcription. A sequence alignment between the cyclin proteins CycT1, T2 and K revealed ten very well-conserved regions essential to forming the alpha-helical cyclin box repeat domain. We introduced random mutations in the nine most conserved amino acid clusters in these regions and tested the resulting mutant CycT1 proteins for their ability to block HIV transcription. One of the mutant proteins, termed CycT1-U7, showed a potent, yet specific, dominant negative effect on HIV transcription, although the steady-state expression level of CycT1-U7 was surprisingly low. Western blot analysis revealed the expression levels of the Tat proteins coexpressed with CycT1-U7 were also significantly lower than those co-expressed with wt CycT1. Proteasome inhibitor treatment restored expression of CycT1-U7 and Tat proteins. As a consequence, these inhibitors diminished the dominant negative effect elicited by over-expression of CycT1-U7. Our results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription, which may potentially be further utilized in the development of safe and effective anti-hiv therapies. 98

99 Results Construction and screening of CycT1 mutants. CycT1 is a member of the C-type cyclin family [44]. Its N-terminal 250 amino acids form two cyclin repeat boxes which are essential for the interaction with, and the activation of, Cdk9. Recently, we have determined the three dimensional crystal structure of CycT1 [125]. The cyclin boxes consist of two repeats, each containing five α-helices (Fig. 3.1A and B). Sequence alignment of three P-TEFb-forming cyclins T1, T2, and K from different species revealed that the secondary structure elements are well conserved among these cyclins, indicating that they play important roles in P-TEFb functions (Fig. 3.1B). Based on this secondary structure alignment, we selected the nine most conserved amino acid clusters in the cyclin box domain of CycT1 and introduced random mutations into a C-terminal truncation mutant of CycT1 (CycT1(1-280)). This truncation is sufficient to support Tat transactivation as described before [38, 120, 121] (Fig. 3.1C and Table 3.1). Mutations were introduced by oligonucleotides containing degenerate nucleotides corresponding to each conserved region. In total, 115 CycT1 mutants were constructed and tested for their activities on Tat transactivation by co-transfecting murine NIH 3T3 cells with an HIV LTR-Luciferase (Luc) reporter gene and Tat (Table 3.1). Since murine endogenous CycT1 (mcyct1) cannot support Tat transactivation, Tat activated the LTRdriven Luc expression only by approximately 10-fold (Fig. 3.2A, lane 2). Overexpression of the wt human CycT1 further activated the gene expression up to 70-fold (Fig. 3.2A, lanes 3 and 4). The luciferase activities obtained by over-expressing any of 99

100 the pool of mutant CycT1 proteins ranged from five to 70-fold. Fifteen mutants showed an equal or a higher activity than the wt, 45 mutants showed modest (50-100% of wt) activity and 55 had less than 50% of the activity of wt CycT1 in these cells (summarized in Table 3.1). These 55 mutants were further sequenced and tested for their dominant negative effect on HIV transcription by co-transfecting HeLa cells stably expressing the HIV-Luc reporter gene (HeLa/HR-Luc cells) with Tat (Fig. 3.2B and data not shown). An N-terminal CycT1 mutant exhibited the strongest dominant negative effect on Tat transactivation by promoting the degradation of Tat proteins. Of the 55 clones tested for their ability to block Tat transactivation in HeLa cells, one mutant containing four amino acid substitutions and one deletion in the second helix H2 of the N-terminal cyclin box repeat (residues HRFYM at a.a. position to IIWE; Fig. 3.1B), termed CycT1-U7, showed the strongest dominant negative effect (>90% inhibition) on HIV transcription in HeLa/HR-Luc cells (Fig. 3.2B, lanes 3 to 5). At least four other mutant CycT1 proteins constructed using the same oligonucleotides showed potent dominant negative effects on HIV transcription (60-90%, data not shown). Overexpression of CycT1-U7 affected neither the basal HIV transcription nor CMV-Luc reporter gene expression (Fig. 3.2C). Next, HeLa/HR-Luc cells stably expressing CycT1-U7 were created by infecting with a second lentiviral vector. Tat transactivation in these cells was scored by transfecting an increasing amount of Tat and measuring LTR-driven luciferase activity. In cells expressing CycT1-U7, Tat exhibited a significantly lower activity compared to the cells stably carrying the empty phr lentiviral vector (Fig. 3.2D). Western blot analysis revealed that the steady-state expression level 100

101 of CycT1-U7 is much lower than wt CycT1 (1-280) although equal amounts of plasmid were transfected (Fig. 3.3A, lanes 2 and 3 in the top panel). Interestingly, the expression level of Tat was also much lower in CycT1-U7-expressing cells than in wt CycT1 (1-280) expressing cells, (Fig. 3.3A, lane 2 and 3). In contrast, the expression levels of the endogenous CycT1 and Cdk9 in the presence of CycT1-U7 remained unchanged (Fig. 3.3A). These results suggested that Tat transactivation in CycT1-U7 expressing cells is kept at a low level because the steady state Tat expression is diminished in these cells. Since CycT1-U7 retains the wild type sequence of Tat-TAR recognition motif (Fig. 3.1C), we hypothesized that CycT1-U7 forms a complex with Tat, and this complex is rapidly degraded in cells. Expression of CycT1-U7 and Tat can be rescued by proteasome inhibitors. To further prove our hypothesis that CycT1-U7, together with Tat, is rapidly transferred to proteasomal degradation pathways, cells expressing Tat and either wt CycT1 (1-280) or mutant CycT1-U7 were incubated with the proteasome inhibitors, MG- 132 (50μM) or Epoxomicin (50μM) for 1, 3, and 5 hours prior to cell lysis. MG-132 showed a strong cytopathic effect when incubated for 5 hours (data not shown). The expression of both CycT1-U7 and Tat was partially restored in the presence of MG-132 (Fig. 3.3B, lanes 2 and 3 compared with lane 1), and much more efficiently restored in the presence of Epoxomicin (Fig. 3.3B, lanes 5 to 7 compared with lane 4). In contrast, the expression of wt CycT1 (1-280) and Tat remained virtually unchanged in the presence of these inhibitors (lanes 9 and 10 compared with lane 8). 101

102 The restoration of the CycT1-U7 and Tat expression by Epoxomicin was also observed at the cellular level by an indirect immuno-fluorescence (IF) assay (Fig. 3.4A). HA-tagged wt and mutant CycT1 and myc-tagged Tat proteins were co-expressed in HeLa/HR-Luc cells. Twenty-four hours after transfection, cells were untreated or treated with 25μM Epoxomicin for 3 hours. HA-CycT1 proteins were probed with mouse anti- HA and Cy2- conjugated anti-mouse IgG, and myc-tat proteins were probed with Texas Red-labeled anti-myc antibody. As shown in Fig. 3.4A, the expression of CycT1-U7 and Tat was maintained at low levels without Epoxomicin treatment. These protein levels became elevated when the cells were treated with Epoxomicin. The wt CycT1 and Tat proteins co-expressed with wt CycT1 were detected in the presence or absence of Epoxomicin. Finally, the inhibitory effect by CycT-U7 was diminished in transient (Fig. 3.4B) and stable (Fig. 3.4C) expression systems when the cells were incubated with 25μM Epoxomicin for 6 to 18 hours. Since it has been demonstrated that CycT1 is ubiquitinated in cells [142], we sought to examine whether CycT1-U7 is ubiquitinated by co-immunoprecipitation analysis (Fig. 3.5A). Ubiquitinated CycT1-U7 proteins were detected in HeLa/CycT1-U7 cells treated with 50μM Epoxomicin for 60 min (Fig. 3.5A, lane 2). Also, in this condition, the interaction between CycT1-U7 and Tat was detected by co-immunoprecipitation (Fig. 3.5B, lane 4). These results suggest that CycT1-U7 inhibits Tat-transactivation by rapidly recruiting Tat into an ubiquitin-dependent proteasomal degradation pathway. 102

103 Discussion Although P-TEFb is a potential target for the development of novel anti-hiv therapies, it had been extremely difficult to construct dominant negative CycT1 mutants capable of blocking HIV transcription [137]. This is presumably due to the high stability and the complex regulatory mechanism of the endogenous P-TEFb complex. In the present study, we constructed and evaluated a novel class of CycT1 mutant proteins (CycT1-U7) that explicitly block HIV transcription by promoting a rapid and specific degradation of Tat proteins co-expressing CycT1-U7. Resulting from a functional screen of 115 randomized mutant proteins, sequence analysis of CycT1-U7 showed five mutations including one amino acid deletion in the second helix of the cyclin box repeat fold (Fig. 3.1B). We have previously demonstrated that a CycT1 variant lacking this region (CycT1 ( )) is also unstable in cells [143]. This particular mutant exhibited a potent dominant negative effect on HIV transcription, potentially by a similar mechanism (data not shown). Therefore, H2 of CycT1 appears very important for maintaining the structural stability of CycT1 and the interface in between the two repeats. In addition, a residue directly preceding the first helix of the cyclin box repeat that varies between human and equine CycT1 has been previously shown to account for differences in the recognition of Tat/TAR complexes from HIV and EIAV [144]. Together, these data highlight the importance of the integrity of the first cyclin box repeat for the interaction with Tat. This region also appears to be essential for the interaction with Cdk9 [125, 137]. Interestingly, CycT1-U7 does not promote degradation of endogenous Cdk9. On the other hand, this mutant does bear the wild type sequence of Tat/TAR recognition motif (a.a ). Indeed, the complex between CycT1-U7 and Tat was 103

104 detected when the cells were treated with proteasome inhibitors (Fig. 3.5). The mutant CycT1-U7 proteins can form a complex with Tat and this complex becomes quickly degraded due to the instability of CycT1-U7. Therefore, we conclude that CycT1-U7 exhibits a strong dominant negative effect on Tat transactivation by specifically degrading the co-expressed Tat protein, without disturbing the endogenous P-TEFb complex (Fig. 3.6). It has been demonstrated that CycT1 interacts with other cellular transcription factors through its N-terminal cyclin box regions [145, 146]. It is of importance to examine whether CycT-U7 can also inhibit cellular transcription mediated by these factors using a similar pathway. Additionally, the TRM region of CycT1 also interacts with HEXIM1, the endogenous inhibitory protein of P-TEFb which interacts with this region [46], and it is possible that CycT1-U7 affects P-TEFb activity by reducing HEXIM1 levels. More detailed studies are required to assess the effect of CycT1-U7 on cellular transcription. Our results indicate that CycT1-U7/Tat is recruited to the ubiquitin-dependent degradation pathway. CycT1 seems to be ubiquitinated not only on its C-terminal PEST region (a.a ) but also at other regions [142]. It is to be noted that wt CycT1 (1-280) is resistant to degradation (Fig. 3.3). Although we have not identified the potential ubiquitination site(s) of CycT1-U7 in this study, it is possible that the cyclin box structure stabilizes the protein by preventing ubiquitination. Conformational changes induced by post-translational modifications such as phosphorylation may expose any additional ubiquitination sites in this region, which would represent a novel pathway to regulate P- 104

105 TEFb function. Constructing CycT1 mutants based on C-terminal truncated forms of wt CycT1 (CycT1(1-280)) is particularly beneficial in terms of HIV transcription. CycT1 (1-280) has been demonstrated to be sufficient for supporting Tat-transactivation [38, 120, 121]. In addition, Tat competes with HEXIM1 to increase active P-TEFb complexes [50, 124, 126]. CycT1 (1-280) can therefore bypass the 7SK/HEXIM-mediated complex regulatory pathway and be exclusively directed towards Tat-dependent transactivation, making CycT1 (1-280) proteins highly specific for Tat. Since the mechanism by which CycT1-U7 inhibits HIV transcription seems not to be through blocking the normal function of P-TEFb, but rather through a gain-offunction pathway, it represents a novel class of inhibitory molecules. Moreover, since the steady-state expression of CycT-U7 is very low, it may be an excellent candidate for gene therapy because the mutant proteins would not persist for a prolonged period of time, thereby avoiding induction of unwanted immune responses. Additionally, these proteins would work only when Tat is actively expressed in cells. HIV utilizes the cellular transcriptional machinery for its own replication. Therefore, it is important to inhibit this step without disturbing normal cellular functions. Since CycT1 interacts with Tat and TAR, it can be an excellent target to develop asafe and effective anti-hiv therapy. Here we present an example of a dominant negative CycT1 molecule which specifically blocks HIV transcription by causing rapid degradation of Tat. Studying the precise mechanism by which this mutant CycT1 protein inhibits HIV transcription could unveil novel regulatory pathways of the HIV life cycle 105

106 and therefore provide reliable clues for designing anti-hiv agents. 106

107 Materials and Methods Materials. HeLa, 293T or NIH 3T3 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) including 10% fetal bovine serum at 37 C with 5% CO 2. HeLa cells stably carrying an HIV-LTR-driven luciferase reporter gene (HeLa-HR-Luc cells) were established using phr lentiviral vector expressing the luciferase gene under the control of the HIV LTR, as described previously [72, 147]. Anti-myc, anti-ha, anti- CycT1, anti-cdk9, and anti-ub antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin antibody was purchased from Cell Signalling Technology (Danvers, MA). Anti-Tubulin was purchased from Sigma Aldrich (St. Louis, MO). Proteasome inhibitors, MG-132 and Epoxomicin were purchased from EMD Bioscience (San Diego, CA) and Alexis (San Diego, CA), respectively. Construction of CycT1 mutants. A structure based sequence alignment resulting from the crystal structure of the cyclin box repeat of human CycT1 [125] revealed highly conserved α-helical structures in the P-TEFb-forming cyclins T1, T2 and K (Fig. 1B). Based on this alignment, we selected the nine most conserved regions in the cyclin box repeat domain of CycT1 and introduced random mutations into a C-terminal truncation mutant of CycT1 (1-280) by using oligonucleotides that contain degenerated nucleotides at positions corresponding to each conserved helix and the Transformer Site Directed Mutagenesis Kit (Clontech) (Fig. 3.1 and Table 3.1). The resulting 115 CycT1 mutants were tested for their ability to support Tat transactivation in murine cells as described previously [120]. The CycT1 mutants that failed to activate HIV-transcription in murine 107

108 cells were sequenced and further tested for their ability to block Tat transactivation in HeLa cells as described previously [137]. The mutant CycT1 (termed CycT1-U7) that exhibited the strongest inhibitory effect on Tat-dependent HIV transcription was used in this study. Sequences of the mutagenic oligonucleotides are shown in Table 3.2. Generation of stable cell lines. CycT-U7 was subcloned downstream of a CMV promoter in a modified phr -SIN lentiviral vector [147, 148]. The VSV-G pseudotyped lentiviruses were produced by co-transfection with packaging plasmids (pmd.g and p8.9i,[118]), and used to infect HeLa cells and HeLa-HR Luc cells. Transfection and reporter assays. HeLa or NIH 3T3 cells were transfected with 0.5μg of pef-cyct1 (wt or mutant constructs) and an HIV-Luciferase reporter construct, in the presence or absence of ptat (0.01μg) using Lipofectamine 2000 according to the manufacturer s instructions (Invitrogen). Twenty- four hours after transfection, cells were harvested and lysed. The protein concentrations of the cell lysates were determined by Protein Assay kit (BioRad). Luciferase activities in the cell lysates were measured as described previously [72]. Ubiquitination assays. HeLa cells stably expressing myc-epitope tagged mutant CycT1 proteins were expressed and, when indicated, treated with 50 μm Epoxomicin for 1 hour. Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (50 mm Tris-HCl, 0.15 M NaCl, 1 mm EDTA, 1% Sodium deoxycholate, 1% NP-40, 0.1% SDS, 1mM DTT [ph 7.4]) in the presence of protease inhibitors. After pre-clearing with protein-g 108

109 sepharose coupled with normal mouse IgG, cell lysates were incubated with 0.5 μg of monoclonal antibody against c-myc (F-7; Santa Cruz Biotechnology) overnight at 4 C. After the cell lysates were allowed to bind to the antibody, reaction mixtures were incubated with protein- G sepharose beads (Roche) for 1 hour at 4 C. The beads were washed extensively with RIPA buffer and the proteins remaining on the beads were eluted by incubation with SDS loading buffer (50 mm Tris-HCl, 2% SDS, 10% glycerol, 2 mm EDTA, 0.1 M DTT and 0.01% bromophenol blue, ph 6.8) and subjected to SDS- PAGE, followed by Western blotting with anti-ub antibody (Santa Cruz biotechnology). Proteasome inhibitor treatment. 293T cells (2x10 5 ) were transfected with 1 μg of plasmids encoding HA-tagged wt CycT1 (1-280) or CycT1-U7 in the presence or absence of the plasmid encoding HA-tagged HIV-1 Tat (0.2 μg) using calcium phosphate. Twenty-four hours post-transfection, cells were treated with MG-132 (50 μm), Epoxomicin (50 μm) or DMSO (solvent control) at 37 C for 1, 3 and 5 hours. Cells were then harvested and lysed in RIPA buffer. The protein concentrations in the cell lysates were determined by Protein Assay Kits (BioRad, Palo Alto, CA). The same amounts of cellular proteins (20 μg) were separated by SDS-PAGE followed by Western blot analysis to detect the HA-epitope tagged CycT1 proteins and myc-epitope tagged Tat proteins. Immunofluorescence (IF) assay. HA-tagged wt and mutant CycT1 and myc-tagged Tat 109

110 proteins were co-expressed in HeLa/HR-Luc cells using Lipofectin (Invitrogen). Twenty-four hours after transfection, cells were untreated or treated with 25 μm Epoxomicin for 3 hours. Cells were fixed with 4% formaldehyde and blocked with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 1% sodium azide and 10% normal donkey serum. After washing with PBS, HA-CycT1 and were probed with mouse anti-ha monoclonal antibody (Santa Cruz) and Cy2-conjugated donkey antimouse IgG (Jackson ImmunoResearch). Myc-Tat proteins were probed with anti-myc monoclonal antibody (Santa Cruz) pre-labeled with Zennon Texas Red anti-mouse IgG1 (Invitrogen). Nuclei were stained with Hoechst (Sigma). Fixed cell images were captured on a Deltavision DV-RT (Applied Precision, Inc. Issaquah, WA.) microscopy system using the Deltavision Softworx program. Acknowledgements This work was supported by NIH funded grant R21 AI62516 and the Cell and Molecular Biology training grant (5TG32 GM ) from the NIH, American Foundation for AIDS Research (AmFAR) RGGN, the Deutsche Forschungsgemeinschaft (GE 976/5), Japan Human Science Program, and the Center for AIDS Research (CFAR) at Case Western Reserve University. 110

111 Figure 3.1. Construction of mutant CycT1 proteins. A. Structure of the cyclin box repeat domain (1-281) of CycT1. Two repeats of five α-helices each form the conserved cyclin box (blue). Flanking N- and C-terminal helices, which are important for the specificity of cyclins, are depicted in yellow and red, respectively. B. Schematic representation of C-terminally truncated wt CycT1 and the dominant negative CycT1-U7 mutant used in this study. Secondary structure of conserved α-helices (dotted regions in cyclin box 1 and hatched regions in cyclin box 2) together with two helices at N- and C- terminal (gray) locate in the N-terminal cyclin boxes in CycT1. Random mutations were introduced into the nine most conserved regions (shown by thin lines) in the cyclin box 111

112 domain of a C-terminal truncation mutant of CycT1 (CycT1(1-280)). - in the CycT1- U7 sequence represents a deletion site. The truncated wt and mutant CycT1 employed in this study are also shown. C. A schematic representation of the full-length Cyclin T1. Amino acid motifs such as cyclin boxes, Tat-TAR recognition motif (TRM), coiledcoiled region, and PEST sequence are depicted. 112

113 Table 3.1 Overview of CycT1 mutants used in this study 113

114 Figure 3.2. N-terminal mutant CycT1 proteins (CycT1-U7) exhibit a strong dominant negative effect on HIV transactivation. A. CycT1-U7 cannot support Tat transactivation in murine cells. NIH 3T3 cells were transfected with HIV-Luc reporter gene in the presence (lane 2-6) or absence (lane 1) of Tat (0.1 μg) with or without increasing amounts (0.2 and 0.5 μg) of wt human CycT (lanes 3 and 4) or CycT1- U7 (lanes 5 and 6). Twenty-four hours after transfection, luciferase activities were measured as described before. B. CycT1-U7 shows strong dominant negative effects on Tat-transactivation. Increasing amounts of CycT1-U7 (0.2, 0.4 and 0.6 μg) were transfected in HeLa/pHR-Luc cells in the presence of Tat (0.02 μg). Luciferase activities were measured as described above. C. CycT1-U7 was unable to inhibit basal HIV transcription and CMV-driven transcription. The plasmid (0.6 μg) encoding CycT1-U7 114

115 (gray bars) or an empty vector (black bars) was co-transfected in HeLa cells with HIV- LTR-Luciferase or CMV-Luciferase reporter plasmid (0.05 μg) in the absence of Tat. Luciferase activity was measured as described above. D. Tat has lower activity on HIV- LTR in cells stably expressing CycT1-U7. Increasing amounts of Tat were transfected in Hela/pHR-Luc cells stably carrying a lentiviral vector encoding no protein (empty vector; gray diamonds) or CycT1-U7 proteins (black triangle). Luciferase activities were measured as described in the Materials and Methods section. Error bars represent the standard deviation of triplicate measurements. Data are representative of four independent assays. 115

116 Figure 3.3 CycT1-U7 promotes the degradation of Tat. A. Western blotts depict the steady-state expression of Tat proteins co-expressed with wt or mutant CycT1. HAepitope tagged Tat (lanes 1-3) and HA-tagged CycT (wt: lane 2) or HA-CycT1- U7 (lane 3) were co-expressed in 293T cells. Twenty-four hours after transfection, cells were lysed with RIPA buffer (25 mm Hepes-KOH, 150 mm KCl, 1 mm EDTA, 1% Triton X100, 0.1% NP-40, ph 7.4), and soluble proteins were separated by 12% SDS- PAGE. The ectopically expressed CycT1 and Tat proteins were detected by anti-ha antibody. The endogenous proteins (CycT1, Cdk9 and Tubulin) were also detected by Western blotting. B. The expression of CycT1-U7 and Tat was restored by proteasome inhibitors. 293T cells were transfected with HA-tagged wt CycT1 (1-280) (lanes 8 to 10) or HA-CycT1-U7 (lanes 1 to 7) and HA-Tat as described above. Twenty-four hours after 116

117 transfection, cells were treated with DMSO (lanes 1, 4 and 8), MG-132 (50 μm: lanes 2, 3 and 9) or Epoxomicin (50 μm: lanes 4 to 7 and 10) for 1 (lanes 2 and 5), 3 (lanes 3, 6, 9 and 10) and 5 hours (lane 7). Cells were then lysed in RIPA buffer and subjected to SDS-PAGE. The ectopically expressed CycT1 and Tat proteins were detected by anti- HA antibodies. 117

118 Figure 3.4. Epoxomicin restores CycT1-U7 and Tat expression. A. HA-tagged wt and mutant CycT1 and myc-tagged Tat proteins were co-expressed in HeLa/HR-Luc cells. Twenty-four hours after transfection, cells were untreated or treated with 25 µm Epoxomicin for 3 hours. HA-CycT1 proteins were visualized with mouse anti-ha antibody and Cy2-conjugated donkey anti-mouse IgG. Myc-Tat proteins were seen with Texas Red-conjugated anti-myc antibody. Nuclei were stained with Hoechst. B. The inhibitory effect by CycT1-U7 was diminished by Epoxomicin. HeLa/HR-Luc cells were transfected with CycT1-U7 expression plasmids (0.5 μg) or empty plasmids (0.5 μg) in the presence of Tat. Cells were treated with DMSO (-) or 25 μm Epoxomicin for 6 hours and 18 hours as indicated, and the Luc activities were measured. The results were presented as relative luciferase values obtained with CycT1-U7 divided by the values 118

119 with the empty vector at each time point. C. Increasing amounts of Tat were transfected in Hela/pHR-Luc cells stably expressing CycT1-U7 proteins. 24 hours after transfection, cells were untreated (open circles) or incubated (closed circles) with Epoxomicin (25 μm) for 6 hours prior to luciferase assay. Data are expressed as fold activation relative to the value obtained with untreated cells. Error bars represent the standard deviation of triplicate measurements. Data are representative of three independent assays. 119

120 Figure 3.5. CycT1-U7 is ubiquitinated. A. HeLa cells stably expressing myc-cyct1- U7 proteins (HeLa /myc-cyct1-u7) were treated (lanes 1 and 2) or untreated (lane 3) with Epoxomicin (50 μm) for 30 min prior to cell lysis. The Myc-CycT1-U7 proteins were immunoprecipitated with anti-myc antibody followed by Western blot analysis with anti-ub antibody to detect ubiquitinated Myc-CycT1-U7 proteins (upper panel). Normal mouse IgG (migg) was used as a negative control for immunoprecipitation (lane 1). The expression of the Myc-CycT1-U7 proteins in 10% of the input samples was also detected by Western blot analysis using anti-myc antibody (lower panel). B. CycT1-U7 binds Tat. HeLa/myc-CycT1-U7 cells were transfected with HA-Tat. The cells were treated (lanes 1, 3 and 4) or untreated (lanes 2, 4 and 5) with Epoxomicin (50 μm) for 30 min prior to cell lysis. The Myc-CycT1-U7 proteins were immunoprecipitated with anti-myc antibody (lanes 4 and 6) followed by Western blot analysis with anti-ha antibody to detect Tat 120

121 proteins. Normal rabbit IgG (rigg) was used as a negative control (lanes 3 and 5). Tat proteins in the input samples (10%) are also shown (lanes 1 and 2). 121

122 Figure 3.6. Proposed model for the mechanism of dominant negative effect elicited by CycT1-U7. Wild type CycT1 forms a complex with Cdk9 as an active P-TEFb, and interacts with Tat and TAR RNA. Alternatively, CycT1-U7 associates with Tat but the CycT-U7/Tat complex is immediately degraded via an ubiquitin-dependent proteasomal pathway. This degradation can be prevented by proteasome inhibitors. 122

123 Name Sequence Position (a.a.) Mut 1 GTCTCACAATTGNNNATCAACNNNGCTATAGTANNNATGCATCGATTC Mut 2 CACTGCTATAGTATACATGNNNNNNNNNNNNNNNATYCAGTCCTTCACAC Mut 3 GCTCCAGCAGCCNNNNNNNNNNNNGCTAAAGTGGAGGAGCAGCCC Mut 4 GCTAAAGTGGAGGAGNNNNNNNNNNNNNNNTTGGAACATGTCATCAAGGTAGC Mut 5 CAAAAAATTGGAACATNNNNNNNNNNNNGCANNNNNNTGTCTCCATCCTC Mut 6 TTGCAACAAGTTCAANNNNNNNNNNNNNNNNNNAGCATAATTTTGCAGTT Mut 7 CTGGTCATTTTAAGNNNNNNNNNNNNNNNNNNTTAGGCTTTGAACTAAC Mut 8 ATTTTGCAGACTTTAGGCNNNNNNNNNNNNATTGATCACCCACATACTC Mut 9 CTTTAGGCTTTGAACTANNNNNNNNNNNNNNNCATGTAGTAAAGTGCAC Table 3.2 Sequences of mutagenic oligonucleotides. 123

124 Chapter 4: Dominant Negative Mutant Cyclin T1 Proteins that Inhibit HIV Transcription by Forming a Kinase Inactive Complex with Tat 124

125 Summary Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virusspecific interaction, CycT1 may potentially serve as a target for the development of anti- HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies. Contents of this chapter were published by Jadlowsky et al. in J. Gen. Virol [149]. 125

126 Introduction The transcription of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process in which several host cellular co-factors and the viral transactivator protein, Tat, are involved [20, 28]. Tat stimulates the elongation of transcription with the aid of the positive transcription elongation factor b (P-TEFb). The active form of P-TEFb is a heterodimer comprised of cyclin T1 (CycT1) and cyclindependent kinase 9 (Cdk9) [146]. Tat and CycT1 bind to the transactivation response element (TAR), an RNA stem loop structure located at the 5 end (+1 to +59) of all HIV transcripts [41, 120, 121]. This interaction results in the recruitment of Cdk9 and the subsequent stimulation of its kinase activity by Tat [17]. Among 726 amino acids, the first 272 amino acids of CycT1 that form the cyclin box repeats are sufficient for HIV transcription, since these residues are responsible for the interactions with Tat, TAR and Cdk9 [38-41, 120, 121]. Since P-TEFb is the essential host cellular co-factor for Tat, it serves as a potential target for anti-hiv therapeutics. Several approaches have been taken to block HIV transcription by targeting P-TEFb [131], which include the use of small compounds or mutant proteins that inhibit Cdk9 kinase activity [127, 130, 132, 137] or disrupt the interaction between Tat, TAR and CycT1 [128, 129, 133, 134], using intrabodies against CycT1 [141], or oligomerization chain reaction to inactivate Cdk9 [136]. It is important to note, however, that because P-TEFb is involved in the transcription of many cellular genes [35], it is critical to exclusively block HIV-specific pathway(s) in order to develop safe and effective anti-hiv therapies. 126

127 Previously, Diehl et al have demonstrated that a threonine residue (T156) at the C-terminal end of the linker region between two of the cyclin boxes of CycD1 is critical for nuclear import and activation of Cdk4 [150]. Mutation of this threonine to alanine resulted in a strong dominant negative blockage of nuclear import and phosphorylation of Cdk4 [150]. In CycT1, there are three threonines (T143, T149, and T155) that are very well conserved between CycT1 and T2, at the corresponding region (a linker region between two cyclin boxes). We therefore mutated each of these threonines to alanines either singly or in combination in the expression plasmid encoding the N-terminal 280 amino acids of CycT1, which are sufficient for supporting HIV-transactivation. Of these, the mutant CycT1 containing three threonine to alanine mutations (T143, 149, 155A) was able to inhibit HIV transcription. Further analysis revealed the mechanism behind this inhibition is a failure to stimulate the kinase activity of Cdk9 despite association among the mutant CycT1, Cdk9, and Tat. This dominant negative mutant may represent an unexplored class of specific inhibitors of HIV transcription, which could potentially aid in the development of new therapeutics to target HIV infection. Results A mutant CycT1 protein containing triple T to A mutations in the N-terminal region blocks HIV transactivation The constructs, (CycT1-280 (T143A), CycT1-280(T149A), CycT1-280 (T155A), CycT1-280 (T143, 149, 155A)) were first tested for their ability to support Tat- 127

128 transactivation (Fig.4.1a,b). The plasmids encoding wild type (wt) or mutant CycT1 were co-transfected with HIV-LTR-Luciferase (Luc) reporter plasmids and Tat in NIH 3T3 cells. As previously reported, Tat alone exhibited a modest transactivation of HIV- LTR Luc in these cells due to the inability of the murine endogenous CycT1 to interact with Tat/TAR [39, 41, 120] (Fig.4.1b, lane 2). CycT1 proteins containing a single mutation showed levels almost equal to the wild type level of Tat-transactivation (Fig.4.1b, lanes 4 to 6). In sharp contrast, the triple mutant CycT1-280 (T143, 149, 155A) was unable to support Tat-transactivation (Fig.4.1b, lane 7). All mutant proteins were expressed at similar levels as the wt CycT1, as assessed by Western blot analysis (Fig.4.1d for the expression of CycT1-280 (T143, 149, 155A)). Next, the dominant negative effect of CycT1-280 (T143, 149, 155A) was examined by transient over-expression in the presence of Tat in HeLa/HR-Luc cells that contain chromosomal HIV-LTR-Luc reporter genes introduced by lentiviral vector [147]. An increasing amount of CycT1-280 (T143, 149, 155A) proteins inhibited HIV-Luc reporter gene expression (up to ~85%) in the presence of Tat in HeLa cells (Fig.4.1c, lanes 3 to 5). Over-expression of CycT1-280 (T143, 149, 155A) proteins inhibited neither the basal HIV transcription nor the transcription from the CMV promoter, which indicates that the dominant negative effect of CycT1-280 (T143, 149, 155A) is also highly specific for Tat-transactivation (Fig.4.2). HeLa/HR-Luc cells stably expressing CycT1-280 (T143,149,155A) were also established using a phr- lentiviral vector and employed in titration of Tat activity. In these mutant-expressing cells, Tat showed a lower activity on HIV transcription than in HeLa/HR-Luc cells stably carrying the empty lentiviral vector (Fig.4.1e). Compared with the transient expression system, the inhibitory 128

129 effect of the mutant protein was somewhat less potent in the stably expressing cells. This is presumably because of a lower expression level of the mutant proteins introduced by a lentiviral vector. (Fig.4.1c and e) CycT1-280 (T143,149, 155A) proteins associate with Cdk9 and Tat as a kinasenegative complex Our previous studies indicated that mutant CycT1 proteins defective in interacting with Tat or Cdk9 showed only a small dominant negative effect on HIV transcription [137]. Therefore, we next tested whether this CycT1-280 (T143,149,155A) protein was able to interact efficiently with Tat or Cdk9. HA-tagged CycT1 proteins were coexpressed with myc-tagged Tat in 293T cells. The wt and mutant CycT1 proteins were immunoprecipitated with anti-ha antibodies. Tat and the endogenous Cdk9 proteins associated with HA-CycT1 were detected by Western blot analysis using anti-myc and anti-cdk9 antibodies, respectively. As shown in Fig. 4.3A, CycT1-280 (T143,149,155A) retained the same ability to interact with Cdk9 and Tat as wt CycT1 (Fig.4.3a, lanes 2 and 3). Recent work has demonstrated that approximately 50% of P-TEFb molecules in HeLa cells are transcriptionally inactive due to an association with cellular HEXIM1 proteins and 7SK snrna [51, 151]. Therefore, we subsequently examined whether the truncated wt and mutant CycT1 proteins can associate with HEXIM1 by coimmunoprecipitation. As shown in Fig.4.3b, neither wild type nor mutant CycT1 (1-280) proteins interacted with HEXIM1, whereas the full-length protein did associate with HEXIM1 in the presence of Tat. These results are despite the fact that it is the N- 129

130 terminal region which contains the HEXIM1-binding domain [123]. This is presumably because Tat can compete with HEXIM1 for CycT1 binding, as has recently been demonstrated [50, 124]. Indeed, the interaction between CycT1-280 and HEXIM1 was observed in the absence of Tat, and co-expression of Tat diminished this interaction (Fig.4.4). Interestingly, when the mutant CycT1-280 (T143,149,155A) proteins were coexpressed with Tat, the amount of the endogenous CycT1 associated with Tat was significantly decreased as examined by co-immunoprecipitation assays (Fig.4.3c, WB: α- CycT1 and α-ha, lanes 1 and 2), indicating that the mutant CycT1 can efficiently form a complex with Tat by competing with the endogenous CycT1. These results indicate that use of the truncated CycT1 is advantageous since these proteins are not segregated by HEXIM1/7SK and therefore remain as a heterodimer with Cdk9, which can preferentially interact with Tat [50, 124, 126]. Additionally, the Tat-associated kinase assays revealed that the kinase complex associated with the mutant CycT1 (T143, 149, 155A) had a lower CTD-kinase activity (Fig. 4.3c, lower panel, lane 2) although a similar amount of the endogenous Cdk9 associated with Tat (Fig.4.3c, WB: anti-cdk9, lanes 1 and 2). These results indicate that the mutant CycT1 (T143, 149, 155A) proteins can interact with Tat and Cdk9 just as wt, without HEXIM1-interaction, and block HIV-transcription by forming a kinase-inactive complex. A computer simulation of the predicted conformational change of the cyclin boxes by these mutations was performed using 3D-Jigsaw program (Fig.4.5). A shift of the region between Glu 95 and Glu 102, which is important for the interaction with Cdk9, is observed in the mutant CycT1 (Fig 4.5). This prediction indicates that these mutations 130

131 may have an impact on the conformation of the Cdk9-binding domain of CycT1 necessary for stimulation of Cdk9 kinase activity. Discussion Construction of mutant cyclin proteins that show dominant negative effects against endogenous Cyclin/Cdk complexes has been quite challenging. This is presumably because the precise mechanism by which these cyclins activate the kinase activity of their corresponding Cdks is not fully understood. In order to clarify the structure-function relationship of CycT, which is critical for development of effective dominant negative CycT1 mutants, we have previously determined the crystal structure of the cyclin box region of CycT1 [125]. The resultant three-dimensional structure indicated that CycT1 shares structural similarities to other cyclins [125]. These results suggest that certain functional motifs are well-conserved among cyclin molecules, although there is wide sequence diversity among them. Constructing CycT1 mutants based on C-terminal truncated forms of wt CycT1 (CycT1(1-280)) is particularly beneficial in terms of HIV transcription. Previously CycT1(1-280) was demonstrated to be sufficient for supporting Tat-transactivation [38, 39, 120]. In this study, we identify a dominant negative CycT1 mutant, CycT1-280 (T143,149,155A) that exhibits a relatively modest inhibitory effect on HIV transcription. This CycT1 mutant was constructed based on a previous report of a dominant negative Cyclin D1 (CycD1) mutant that blocks Cdk4 [150]. Diehl et al., demonstrated that mutant CycD1 proteins containing a threonine-to-alanine mutation at position

132 (T156A), blocked Cdk4 kinase activity by preventing Cdk4 from entering into nucleus [150]. In CycT1, there are three threonines (T143, T149, and T155) that are well conserved between CycT1 and T2, at the corresponding region (a linker region between two cyclin boxes). None of the single threonine-to-alanine mutations resulted in alteration of the ability of CycT1 to support Tat-transactivation (Fig.4.1b). However, a triple mutant CycT1-280 (T143,149,155A) failed to activate HIV transcription in murine cells and exhibited a potent dominant negative effect on HIV transcription in human cells (Fig.4.1b and c). Unlike the CycD1 mutant, we did not observe a clear difference in the cellular localization between wt CycT1(1-280) and CycT1-280 (T143,149,155A) (Data not shown). Moreover, CycT1-280 (T143,149,155A) interacted with Tat and Cdk9 in a manner identical to wt (Fig.4.3a). Interestingly, Tat preferentially interacted with the mutant protein over the endogenous full-length proteins (Fig.4.3c). This, presumably, is because the truncated CycT1 proteins do not interact with HEXIM1 (Fig.4.4) and therefore, remain as a heterodimer with Cdk9, the form that can readily interact with Tat. Since CycT1 (1-280) proteins do not interact with 7SK snrna and HEXIM1 proteins in the presence of Tat, they can bypass the 7SK/HEXIM-mediated complex regulatory pathway and be exclusively directed towards Tat-dependent transactivation. This makes CycT1 (1-280) proteins highly specific to Tat. Indeed, the full-length version of these mutant CycT1 proteins exhibited less potent dominant negative effect on HIV transcription (data not shown), which may potentially provide important insights into the mechanism of 7SK/HEXIM1-mediated P-TEFb regulation. The Tat-associated kinase (TAK) activity associated with CycT

133 (T143,149,155A) was lower than TAK activity associated with wt-cyct1 (Fig.4.3c). Therefore, it is possible that CycT1-280 (T143,149,155A) cannot activate Cdk9 kinase activity to the level achieved by wt CycT1. These results indicate that the mutant CycT1-280 (T143,149,155A) proteins can block HIV-transcription by forming a specific and stable, kinase inactive complex with Cdk9 and Tat. HIV utilizes cellular transcriptional machinery for its own replication. Therefore, it is important to specifically inhibit this step without disturbing cellular functions. Given the interaction of CycT1 with Tat and TAR, it holds promise as an excellent target for the development of safe and effective anti-hiv therapies. Here we present the first example of a dominant negative CycT1 molecule which specifically blocks HIV transcription. Studying the precise mechanism by which certain mutant CycT1 proteins can inhibit HIV transcription may unveil novel regulatory pathway(s) of the HIV life cycle and therefore provide valuable insight for designing anti-hiv agents. 133

134 Materials and Methods Materials. HeLa, 293T or NIH 3T3 cells were maintained in Dulbecco s Modified Eagle s Medium (DMEM) supplemented with 10% fetal bovine serum at 37 C with 5% CO 2. HeLa cells stably carrying an HIV-LTR driven luciferase reporter gene (HeLa- LTR-Luc cells) were established using phr lentiviral vector as described previously [118]. Anti-myc, anti-cdk9, and anti-ha antibodies (Santa Cruz Biotechnology) were used in Western blots and immunoprecipitations. Anti-FLAG antibody was purchased from Sigma-Aldrich. The FLAG-tagged HEXIM1 expression plasmid was a generous gift from Monica Montano. Construction of CycT1 Mutants. Based on the previous identification of a dominant negative cyclin molecule [150], the conserved regions between CycT1 and T2, and the 3- dimensional crystal structure of the cyclin box region of CycT1, specific point mutations were introduced by Quick Change Site-Directed Mutagenesis Kit (Stratagene) using oligonucleotide primers containing mis-match nucleotides at the position of desired point mutations. Transfection and Reporter Assays. NIH 3T3 cells were transfected with 0.5 μg of wild type or mutant CycT1 and an HIV-Luciferase reporter construct, in the presence or absence of 0.1 μg Tat using Lipofectamine 2000 according to the manufacturer s instructions (Invitrogen). HeLa, or HeLa-LTR-Luc cells were transfected in a similar manner with indicated amounts of CycT1, Tat, or FLAG- tagged HEXIM1. Twenty-four hours post-transfection, cells were harvested and lysed with Passive Lysis Buffer 134

135 (Promega). Protein concentrations of the cell lysates were determined by Protein Assay kit (BioRad). Luciferase activities in the cell lysates were measured as described previously [72]. Tat-associated kinase (TAK) assay. The kinase activity associated with Tat was measured by TAK assay as described before [152]. Briefly, myc-tagged Tat proteins were overexpressed in 293T cells in the presence or absence of HA-CycT1 (T143,149, 155A). Twenty-four hours after transfection, Tat proteins were immunoprecipitated from the cell lysates using anti-myc antibodies. The CTD kinase activity was measured by adding 5µCi γ-[ 32 P] ATP and purified GST-CTD (200ng) in kinase buffer (50mM Tris- HCl, 1mM MgCl 2, 150mM NaCl, 1mM DTT, ph 7.4) for 60 min at room temperature. The reaction mixtures were then subjected to 12% SDS-PAGE, followed by autoradiography to detect the phosphorylated GST-CTD. Co-immunoprecipitations. HA-epitope tagged wild-type and mutant CycT1 proteins were expressed in 293T cells in the presence of myc-epitope tagged Tat proteins, or FLAGtagged HEXIM1 proteins by transfection using calcium phosphate. Forty-eight hours after transfection, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50mM Tris-HCl, 0.15M NaCl, 1mM EDTA, 1% Sodium deoxycholate, 1% NP-40, 0.1% SDS, 1mM DTT [ph 7.4]) in the presence of protease inhibitors (Sigma). Cell lysates were incubated with 0.5 µg of monoclonal antibody against c-ha (F-7; Santa Cruz Biotechnology) overnight at 4 C. After the cell lysates were allowed to bind to the antibody, reaction mixtures were incubated with protein-a sepharose beads (Roche) for 1 135

136 hour at 4 C. Immunoprecipitated proteins were washed extensively with RIPA buffer and subjected to SDS-PAGE, followed by Western blotting with anti-cdk9, anti-myc (Santa Cruz Biotechnology), or anti-flag (Sigma-Aldrich). Acknowledgments This work was supported by NIH R21 AI62516 and the Cell and Molecular Biology training grant (5TG32 GM ) from the NIH, as well as American Foundation for AIDS Research (AmFAR) RGGN, Japan Human Sciences Program, and the Center for AIDS Research (CFAR) at Case Western Reserve University. 136

137 Figure 4.1. Mutant CycT1 proteins containing triple T-to-A mutations in the N- terminal region also block HIV transactivation. (a) A schematic representation of the mutant CycT1-280 (T143A), CycT1-280 (T149A), CycT1-280 (T155A) and CycT1-280 (T143,149,155A). (b) CycT1-280 (T143, 149, 155A) is unable to support Tattransactivation in murine cells. NIH 3T3 cells were transfected with HIV-Luc reporter gene in the presence (lanes 2 7) or absence (lane 1) of Tat (0.1 μg) and wt (lane 3) and mutant (lanes 4 7) human CycT1 (0.5 μg) as indicated. The result is shown as fold activation relative to the luciferase activity obtained without Tat. (c) CycT1-280 (T143, 149, 155A) has dominant negative effects on Tat-transactivation. Increasing amounts of 137

138 CycT1-280 (T143, 149, 155A; mutcyct1) (0, 0.2, 0.4 and 0.6 μg, lanes 2 5) were transfected in HeLa/pHR-Luc cells in the presence of Tat (0.02 μg). Data are representative of three independent assays. (d) CycT1-280 (T143,149,155A) expresses in HeLa cells at a similar level to wild-type, as determined by Western blot analysis using anti-ha antibodies. (e) Tat has lower activity in cells stably expressing CycT1-280 (T143,149,155A). Upper panel: an increasing amount of Tat-expression plasmid was transiently transfected into HeLa/HR-Luc cells stably carrying a lentiviral vector encoding no protein (empty vector: closed circles) or HA-CycT1-280 (T143,149,155A: open circles). Lower panel: HA-CycT1-280 (T143,149,155A) proteins are detected in the lysate of the stable cells ( mutcyct1 ), but not in the control cell lysates ( vector ), as seen by Western blot analysis using anti-ha antibody. 138

139 Figure 4.2. The dominant negative effect of CycT1-280 (T143,149,155A) is specific for Tat. CycT1-280 (T143,149,155A) was unable to inhibit basal HIV transcription and CMV-driven transcription in the absence of Tat. Plasmids (0.6 μg) encoding CycT1-280 (T143,149,155A) (hatched bars) or an empty vector (black bars) were co-transfected in HeLa cells expressing HIV-LTR-Luciferase or CMV-Luciferase reporter plasmid (0.05 μg). 139

140 Figure 4.3. CycT1-280 (T143, 149, 155A) proteins associate with Cdk9 and Tat as a kinase- negative complex. (a) HA-tagged wt (lane 2) and mutant CycT1-280 (T143, 149, 155A) proteins (lane 3) were overexpressed in 293T cells in the presence of myctagged Tat proteins, and immunoprecipitated from the cell using anti-ha antibodies. Tat and the endogenous Cdk9 associated with HA-CycT1 were detected by anti-myc and anti-cdk9 antibodies, respectively. (b) The truncated version of CycT1 (1 280) does not interact with HEXIM1 in the presence of Tat. HA-tagged full-length (1 726; lane 1) and truncated (1 280) CycT1 [wt, lane 2, and CycT1-280 (T143,149,155A), lane 3] proteins were over-expressed in 293T cells in the presence of FLAG-tagged HEXIM1 proteins and myc-tagged Tat proteins and immunoprecipitated from the cell lysates with anti-ha 140

141 antibodies. The HEXIM1 proteins associated with HA-CycT1 were detected by anti- FLAG antibodies. (c) Tat proteins associated with the mutant CycT1-280 (T143,149, 155A) proteins have a lower CTD-kinase activity. Myc-epitope tagged Tat proteins were overexpressed in 293T cells in the absence (lane 1) or presence (lane 2) of CycT1-280 (T143,149,155A), and immunoprecipitated with anti-myc antibodies. The endogenous CycT1 and Cdk9 proteins, and HA-CycT1 (1-280) associated with Tat were detected by Western blot analysis as indicated. The Tat-associated kinase (TAK) assays were performed as described previously [152]. 141

142 Figure 4.4. Tat can compete with HEXIM for binding to CycT Increasing amounts (0, 0.2 and 0.4 μg) of Myc-Tat were co-transfected with HA-CycT1-280 (0.5 μg) and Flag-HEXIM1 (0.3 μg) in HeLa cells. HA-CycT1-280 proteins were immunoprecipitated with anti-ha antibody. Tat and HEXIM1 proteins associated with CycT1-280 were detected by Western Blot analysis using anti-myc and anti-flag antibodies, respectively. 142

143 Figure 4.5. Predicted conformational changes in CycT1 (1-280). Three T-to-A mutations were introduced into the structural model of CycT1 previously demonstrated by Anand et al. (2007) using the 3D-Jigsaw program. The computer simulation indicated a shift in a region between Glu 95 and Glu 102 (arrows). 143