REGULATION OF CELLULAR GLUCOSE METABOLISM BY HIV-1 INFECTION

A Dissertation Submitted to the Temple University Graduate Board

In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY

By SATARUPA SEN MAY 2014

Examining Committee Members:

SHOHREH AMINI, Advisory Chair, BIOLOGY PRASUN K. DATTA, NEUROSCIENCE KAMEL KHALILI, NEUROSCIENCE ANTONIO GIORDANO, BIOLOGY MICHAEL NONNEMACHER, External Member, DREXEL UNIVERSITY

ABSTRACT

REGULATION OF CELLULAR GLUCOSE METABOLISM BY HIV-1 INFECTION SATARUPA SEN DOCTOR OF PHILOSOPHY TEMPLE UNIVERSITY 2014 DOCTORAL ADVISORAL COMMITTEE CHAIR: DR. SHOHREH AMINI, PH.D.

Regulation of Glucose metabolism is known to play an important role in pathogenesis of many diseases. Primarily because deregulation of this metabolic pathway can lead to either apoptosis or extended life span of the cells involved. Viruses are parasitic in nature, they utilize the host cellular pathways to support their own progeny; hence it is expected that viruses would regulate the central glucose metabolism of infected host cells. Human immunodeficiency virus type 1 (HIV-1) causes acquired immune deficiency syndrome, and it uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+

T cells induces G2 arrest and cell death within 2–3 days, HIV-1 infection of macrophages results in longer survival of infected cells and low constitutive viral production, generating viral reservoirs. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of infected cells by altering the enzymatic activity and protein expression of various glycolytic components. The data suggests that the two HIV-

1 target cell types exhibit very different metabolic outcomes. During viral replication in monocyte/macrophage lineage cells we observe increase in glycolytic protein expression and the same proteins show no modulation in T-cell lines post viral replication. Similar differential regulation is observed in case of enzymatic activity of glycolytic enzymes as well. We also conducted proteomic studies in collaboration with the proteomics core.

HIV-1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Vpr ii is known to cause cell cycle block in infected cell and bring about cell death. However, macrophages are resistant to cell death and are viral reservoir, even Vpr over expression does not cause apoptosis in these cell types. The goal of the study was to use a stable- isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry- based proteomics approach to characterize the Vpr response in macrophages. More than

600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which

136 were significantly altered upon Vpr overexpression in macrophages. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. We observed that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr- hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase

(HK), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase muscle type 2

(PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. We then focused on infected monocyte macrophages to identify if glycolytic components such as HK and G6PD were regulated by HIV-1 infection/replication. We report that Hexokinase-1 (HK-1) enzyme expression increases post infection of PBMCs where as the enzymatic activity of HK decreases. Similar effect is seen with HIV-1 replication in latently infected monocyte cell lines U1. The G6PD enzyme activity and expression both increases in infected PBMCs and in U1 cells post induction of viral replication with PMA. We also found that HK-1 translocate to the mitochondria of U1 cells post induction of HIV-1. It is known that the product of HK activity, Glucose 6-phosphate (G6P) releases HKI from the outer leaflet of mitochondria.

Hence we conclude that the viral infection decreases HK activity to have less G6P

iii produced in cell and increases G6PD enzyme activity ensuring the remaining G6P is quickly used up, supporting the adherence of outer mitochondrial membrane bound HK1.

This sequence of cellular events ensures longer survival of infected cells supporting the viral progeny to propagate in the cell. We further show that suppressing the Pentose phosphate pathway (PPP) by blocking G6PD activity is not only detrimental to the survival of the infected cells it also suppresses viral replication and promoter level transactivation of the viral LTR. Next we sought to identify if glycolytic enzyme PKM2, that is also known to play a nonmetabolic dual role as a protein kinase regulating gene transcription has any effect on the transcription of HIV-LTR. Our study demonstrates upregulation of pyruvate kinase isoform M2 (PKM2) expression in whole cell extracts and nuclear extracts of HIV-1JRFL infected PBMCs and during reactivation of HIV-1 in chronically infected U1 cells. We then focused on understanding the potential role of

PKM2 on HIV-1 LTR transactivation. Our studies demonstrate that over expression of

PKM2 leads to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -120 bp to -

80 bp to be essential for PKM2 mediated transactivation. This region contains the NFkB

DNA binding site and mutation of NFkB binding site attenuated PKM2 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis confirmed interaction of PKM2 with HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional co-activator of HIV-1 LTR. Hence it opens up another possible target to curb HIV-1 replication at transcriptional level. This study sheds light on the regulation of glycolytic pathway of host cells by HIV-1 infection and its consequences for the virus, opening up

iv new avenues to target viral replication and identify glycolytic markers of HIV-1 pathogenesis.

v DEDICATION

This thesis is dedicated to my grand parents

Mr. Prasanta K Sen, Mrs. Supriti Sen

&

Mr. Jagadindra Narayan Charaborty, Mrs. Bani Chakraborty

vi ACKNOWLEDGEMENT

This thesis is the culmination of years of hard work not only by myself but many people who have influenced my life and my choices. It is by the grace of the almighty and support of my family that I was able to achieve this feat. I would like to thank Drs.

Shohreh Amini and Prasun Datta for being wonderful mentors. I would like to express my gratitude towards my parents who have always supported me to fulfill my dreams and aspirations even if it was beyond their means. It was their dedication towards the well being of our family and society at large, which inspired me to take up a career path that gives me the opportunity to contribute towards the well being of millions of people all around the world living with or susceptible to AIDS in my small capacity. I would like to thank all my teachers in my school and college back home who played a big role in instilling the love for science in me. Also would like to thank my cousins, Sourish for making me realizing my priorities in life and Subhrangshu for setting up an excellent example to follow. I am forever great full to my little cousins Atmadeep and

Chandramouli for bringing so much joy to our lives and ease the stress of PhD. I am thankful to all my relatives back home for taking care of my parents, while I have been thousands of miles away and my inlaws for their love and affection. I would like to thank my family in the United States who have taken care of me all these years. A special mention to Hassen Wollebo, Rafau Kaminiski and Inna Rom for being my lifeline in graduate school. I am also grateful for the love and support I have received from my friends here at Temple University and back home. Last but not the least my wonderful husband Utsav for his continued love and support despite my absolute disregard for most worldly matter at home.

vii

TABLE OF CONTENTS

Page

ABSTRACT ...... ii

DEDICATION ...... vi

ACKNOWLEDGEMENT ...... vii

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER

1. INTRODUCTION ...... 1

AIDS ...... 1

Clinical progression of HIV-1 ...... 3

HAND/HAD ...... 4

Current treatment ...... 5

HIV-1 ...... 6

HIV-1 lifecycle ...... 11

Glycolysis ...... 13

Link between viruses & glucose metabolism ...... 14

HIV-1 infection & glucose metabolism ...... 16

Hypothesis & specific aims ...... 21

2. DIFFERENTIAL REGULATION OF HOST GLYCOLYTIC PATHWAY

BY HIV-1 INFECTION & EFFECT OF VPR ON MACROPHAGE

PROTEOME ...... 23

viii Introduction ...... 23

Materials & methods ...... 26

Results ...... 34

Discussion ...... 43

3. ANTIAPOPTOTIC ROLE OF GLYCOLYTIC COMPONENTS ARE

REGULATED BY HIV-1 INFECTION IN HOST CELL ...... 47

Introduction ...... 47

Materials & methods ...... 49

Results ...... 52

Discussion ...... 61

4. GLYCOLYTIC ENZYME PKM2 PLAYS NONMETABOLIC ROLE IN

REGULATION OF HIV-LTR TRANSCRIPTION ...... 66

Introduction ...... 66

Materials & methods ...... 68

Results ...... 71

Discussion ...... 77

5. CONCLUSION, SIGNIFICANCE & FUTURE DIRECTION ...... 80

BIBLIOGRAPHY ...... 83

ix LIST OF TABLES

Table Page

1. Macrophage proteins within metabolic pathways altered in response to HIV-1 Vpr……………………………………………………………………….38

x LIST OF FIGURES

Figure Page

1. HIV-1 virion schematic………………………………………………………… 7

2. HIV-1 life cycle………………………………………………………………… 11

3. Glycolytic pathway……………………………………………………………... 13

4. 10nm/ml PMA treatment efficiently induces HIV-1 viral production in U1 and J.Lat cells in a time dependent manner………………………………... 34

5. Induction of viral replication in U1 cells leads to changes in expression and activity of glycolytic enzymes……………………………………………... 35

6. Induction of viral replication in J.Lat cells has different effect on glycolytic enzyme expressions and enzyme activity………………………………………. 36

7. Experimental strategy for SILAC based proteomics…………………………… 37

8. Categorization of molecular function of differentially expressed proteins in Vpr transduced macrophages…………………………………………………37

9. Effect of Vpr over expression on macrophages glycolytic enzyme expression... 39

10. HIV-1 induces HK-1 expression in monocytes/macrophages………………….52

11. HIV-1 induces G6PD expression and activity…………………………………..53

12. HIV-1 increases mitochondria bound HK-1 levels in U1 cells………………… 54

13. Clotrimazole treatment removes mitochondria bound HK and caused mitochondrial de polarization…………………………………………………... 55

14. PPP blocker reagent induces apoptosis in virus replicating cells by inhibiting HK-1 mitochondrial translocation and blocking G6PD enzyme activity………. 56

15. DHEA-S treatment lead to decrease in HIV-1 replication and suppression of HIV-LTR promoter activity by blocking p65 nuclear translocation………… 57

16. PKM2 protein expression increases with HIV-1 replication in both whole cell and nuclear fraction………………………………………………….71

17. PKM2 protein over expression induces HIV-LTR activity…………………….. 72

xi 18. PKM2 dependent HIV-LTR transcriptional activation involves the p65-binding site of LTR………………………………………………………...73

19. PKM2 binds to HIV-LTR………………………………………………………. 74

xii CHAPTER 1

INTRODUCTION

*Text and figures used in this chapter were reproduced with permission

AIDS:

On June 5, 1981, the Centers for Disease Control and Prevention (CDC) in its Morbidity and Mortality Weekly Report (MMWR), described pneumonia in 5 homosexual men in

Los Angeles, California, USA, the very first documentation of what became known as acquired immunodeficiency syndrome (AIDS). In June same year, the MMWR reported additional diagnoses of P. carinii pneumonia, other opportunistic infections (OIs), and

Kaposi sarcoma (KS) in homosexual men from New York City and California (1). By the year-end there was report of total 270 such cases in gay men and 121 of those individual were dead (2). On September 24 1982, CDC introduces the term “AIDS” (acquired immune deficiency syndrome) for the first time, and its first case definition of AIDS was:

“a disease at least moderately predictive of a defect in cell-mediated immunity, occurring in a person with no known case for diminished resistance to that disease.” Later on cases of AIDS in infants receiving blood transfusion, female sexual partner of male with AIDS starts getting reported. In 1983 professor Robert Gallo of NIH and Professor Luc

Montagnier, of the Pasteur Institute in France, reports the discovery of a virus that could be the causative agent of AIDS. And finally comes to picture the retrovirus known as

Human immune deficiency virus (HIV) which gave rise to history’s worst epidemic with

25 million people dead and 33.4 million people currently leaving with HIV/AIDS.

Continues epidemiological studies found that HIV type 1, group M (HIV-1), the

1 predominant cause of the AIDS epidemic, had actually evolved from a virus that crossed the species barrier from chimpanzees to humans. The geographic range of the chimpanzee host, Pan troglodytes troglodytes’ habitat suggest that this cross-species transmission took place in central Africa early in the 20th century (3) . However the exact circumstances of cross-species transmission are uncertain. Presumably human exposure of simian viruses took place through hunting and related activities. Over time, the virus evolved to adept the human host and began to spread from person to person, the capacity of viruses in general to mutate indeed came handy in this scenario. Serum specimens collected from patient as early as the year 1959 was diagnosed for HIV-1 infection in retrospect (4) . HIV-2, a second type of HIV rarely found outside western Africa was originated in sooty mangabeys (5) . HIV-1 and HIV-2 are both considered causative agents of AIDS however there are some dissimilarity in between the immunological outcomes of the two infections. HIV-2 leads to a reduced immune activation generally low or even undetectable level of plasma viremia possibly leading to the generally reduced person-to-person transmission of this infection. Also the CD4+T cell counts are normal in HIV-2 infected individual for long time and the disease progression is comparatively slower. However once the disease kicks in, all pathological symptoms of the disease resulted by HIV-1 and HIV-2 infection is practically similar and so is the mortality risk (6, 7) .

2 Clinical progression of HIV-1

Acute Primary Infection- Once in the body, the virus infects a large number of CD4+

T cells and replicates rapidly. During this acute phase of infection, the blood has a high

HIV viral load that spread throughout the body, seeding primarily in the lymphoid organs such as the thymus, spleen, and lymph nodes. During this phase, the virus may integrate irreversibly in the cell’s genetic material. In the acute phase of infection, up to 70 percent of HIV-infected people suffer flu-like symptoms.

The Immune reaction to infection- 2 to 4 weeks after exposure to the virus, the immune system fights back with CD8+ T cells and B-cell-produced antibodies. HIV levels in the blood are significantly reduced at this point and at the same time, CD4+ T cell counts rebound.

Clinical Latency- During this phase, a person infected with HIV may remain free of

HIV-related symptoms for extended period of time from months to several years.

Progression to AIDS- The immune system finally deteriorates to the point that the human body is unable to fight off other infections. The HIV viral load in the blood dramatically increases while the number of CD4+ T cells drops to dangerously low levels. An HIV-infected person is diagnosed with AIDS when he or she has one or more opportunistic infections, such as pneumonia or tuberculosis, and has fewer than 200

CD4+ T cells per cubic millimeter of blood (8) .

3 HAND/HAD:

The syndrome of cognitive and motor dysfunction observed after infection withHIV-1 has been designated HIV associated dementia (HAD) or HIV associated neurodegenerative disorder (HAND). Although antiretroviral therapy (ART) has resulted in a decrease in the incidence of HAND, it does not provide complete protection from

HAND. The prevalence of the dementia may increase as people live longer with AIDS.

Currently there is no specific treatment for HAND, mainly because of an incomplete understanding of how HIV infection causes neuronal injury and apoptosis. The principal pathway for HIV entry into the central nervous system (CNS) is through infected monocytes. The predominant pathogenesis of HAD is believed to involve activation of monocytic cells (macrophages and microglia) and their subsequent release of toxins that lead to neuronal and astrocytic dysfunction. Macrophages and microglia can be activated by HIV infection itself, by interaction with viral proteins, or by immune stimulation due to concurrent infection. It is possible that direct effects of viral proteins on neurons may also contribute to neurodegeneration, although neurons do not get infected by HIV it suffers from a by standard’s effect (9) .

4 Current treatments:

There's no cure for HIV/AIDS, but a variety of drugs can be used in combination to control the virus. Each of the classes of anti-HIV drugs blocks the virus in different ways.

Hence to avid generating drug resistant viral strain of HIV at least three drugs from two different classes are used in combination. The classes of anti-HIV drugs include:

Non-nucleoside reverse transcriptase inhibitors (NNRTIs): NNRTIs disable viral reverse transcriptase hindering HIV-1 replication. Examples include efavirenz (Sustiva), etravirine (Intelence) and nevirapine (Viramune).

Nucleoside reverse transcriptase inhibitors (NRTIs): NRTIs also blocks reverese transcriptase activity. Abacavir (Ziagen), and the combination drugs emtricitabine and tenofovir (Truvada), and lamivudine and zidovudine (Combivir).

Protease inhibitors (PIs): PIs disable protease, another protein that HIV needs to replicate. Examples include atazanavir (Reyataz), darunavir (Prezista), fosamprenavir

(Lexiva) and ritonavir (Norvir).

Entry or fusion inhibitors: These drugs block HIV's entry into CD4 cells. Examples include enfuvirtide (Fuzeon) and maraviroc (Selzentry).

Integrase inhibitors: Raltegravir (Isentress) works by disabling integrase, a protein that

HIV uses to insert its genetic material into CD4 cells.

5 HIV-1:

Human immunodeficiency virus type 1 (HIV-1) belongs to a large group of enveloped, single stranded, positive polarity RNA viruses called Retroviridae. All retroviruses have common viral structure organized by three poly-protein genes known as group specific antigen (gag), polymerase (pol) and envelope (env), a life cycle involving reverse transcription and integration of the viral genome into the host genetic material and the ability to mutate rapidly (10) . A subfamily within Retroviridae is the lentiviruses, which carry in addition to gag, pol and env genes combination of regulatory and accessory genes, making viral replication more complex. These viruses can latently infect host cells for long period of time. Neurological and immunological diseases follow viral reactivation. HIV-1 is a lentivirus, which targets and kills CD4 T lymphocytes and macrophages resulting in an impaired immune system, leaving the body susceptible to a wide range of bacterial, viral, fungal and protozoan pathogens etc. opportunistic infections. Since its discovery in the early 1980s, HIV-1 has been subject of intense studies leading to development of a number of antiviral drugs targeting different steps of the virus life cycle. Using highly active antiretroviral therapy (HAART), a cocktail of several drugs, AIDS became a manageable chronic illness. Unfortunately, the high frequency with which HIV-1 mutates to drug resistance presses need for new antiviral drugs targeting different steps of the virus life cycle.

6 HIV-1 virion structure:

Figure.1: HIV-1 virion schematic from NIAID.

The structure of HIV follows the typical pattern of the retrovirus family; HIV is spherical in shape and has a diameter of 1/10,000 of a millimeter. The viral envelope is composed of lipid bilayer essentially acquired during budding of the viral progeny from the membrane of host cell. Host-cell proteins, such as the major histocompatibility complex

(MHC) antigens and actin, remain embedded within the viral envelope, along with the viral envelope protein (Env). Each viral envelope subunit is made up of two non- covalently linked membrane proteins: glycoprotein (gp) 120, the outer envelope protein, and gp41, the trans membrane protein that anchors the glycoprotein complex to the surface of the virion. The Env copies protrude through the surface of the viral particle giving it an appearance of spikes (8) . 7 The viral core - HIV has three structural genes (gag, pol, and env) that contain information needed to make structural proteins for new virus particles. The bullet-shaped core or capsid within the viral envelop protects the two single strands of HIV RNA, each of which has a complete copy of the virus's genes, the capsid is made up of 2,000 copies of the viral protein, p24. Protein products of the gag poly-protein gene provide integrity and shape of a HIV virion. The env gene, codes for a protein called gp160 that is broken down by a viral enzyme to form gp120 and gp41, the components of the env protein and pol codes for catalytic proteins such as protease, reverse transcriptase (RT) and integrase

(IN) required for successful replication of the virus in host. HIV has six regulatory genes

(tat, rev, nef, vpu, vif, and vpr) containing information needed to produce proteins that control the ability of HIV to infect, replicate or cause disease.

Tat (transactivator of transcription) is a multifunctional early regulatory protein with variable strength of 86 -101 aa, that contributes to several pathological symptoms of

HIV-1 infection as well as playing a critical role in virus replication. It is a robust transactivating protein that induces a variety of effects by altering the expression levels of cellular and viral genes. Tat binds to a transactivation response (TAR) element at the 5’ end of nascent viral transcripts significantly boosting transcription of full-length viral mRNAs. Another interesting role of Tat is its interaction with several chromatin modifying complexes and histone modifying enzymes to relieve the latent virus LTR

(11) . Rev (regulator of expression of virion proteins) is responsible for the nuclear export of intron-containing HIV-1 RNA. It is a virally encoded sequence-specific RNA- binding protein. Rev shuttles between the nucleus and the cytoplasm and harbors both a nuclear localization signal and a nuclear export signal. These essential peptide motifs

8 have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export (12) .

Nef (negative factor) is a 27 KD myristoylated protein that is associated with the cytoplasmic face of cellular membranes. It is one of the first viral proteins to be expressed post infection, Nef has been established as a critical determinant of pathogenicity it was observed that humans or rhesus macaques infected with HIV-1 or

SIV (simian immunodeficiency virus) strains lacking intact Nef genes lead to long-term survival of the infected hosts (13) , Indicating that Nef may help dictate pathogenic outcome in natural infections of different species (14, 15) . Endocytosis of CD4, the primary entry receptor for the HIV-1 virus, from the surface of infected cells is accelerated in response to Nef. Which happens through the interaction of Nef with the cytoplasmic tail of CD4, the recruitment of AP2 (clathrin adaptor protein complex 2), and internalization through clathrin coated pits and subsequent transport to endosomes for lysozomal degradation. Nef diverts host MHC-I molecules to the trans-Golgi network- associated endosomal compartments by an endocytic pathway to finally target them for degradation (15) .

Vpu (viral protein u) is a small integral transmembrane protein, which is co translationally inserted into membranes of infected cells. Vpu consists of an N-terminal hydrophobic domain that function as membrane anchor and a hydrophilic cytoplasmic domain (16) . Vpu has two primary biological activities, the degradation of CD4 in the endoplasmic reticulum and the augmentation of virus secretion from the plasma membrane. In addition, expression of Vpu has been associated with a reduction in syncytia formation of infected cells, which may be a consequence of the reduced

9 presence of viral Env protein at the cell surface due to the more efficient shedding of viral particles in the presence of Vpu. Vpu has also been credited with a role in degradation of

CD4 (14, 16-19) .

Vif (viral infectivity factor) of HIV-1 is 192 amino acid cytoplasmic protein with essential role in replication in primary T cells and during natural infection. Though certain cultured cell lines are able to support growth of vif-deficient viruses, cell fusion experiments indicated that such cells lack expression of inhibitory factor(s) that naturally block viral replication when Vif is absent. The human gene APOBEC3G (A3G) was identified as being fully sufficient to prevent productive infection in the absence of Vif

(14) . Hence the primary contribution of Vif seems to be towards rendering protection to the virus against cellular restriction factor APOBEC3G.

Vpr (viral protein R) is a small 96-amino acid multifunctional protein. Vpr is important for targeting pre-integration complex to nucleus, it also is essential for HIV-1 infection of macrophages since virus deficient in Vpr is less efficient in replication in macrophages.

HIV-1 LTR activation by Vpr results in increased viral replication. Vpr-mediated transcriptional induction of HIV-1 involves interaction between Vpr with specific sequences that span the C/EBP and adjacent NFκB sites of HIV-1 LTR, and transcription factor, Sp1. Vpr induces apoptosis in several cell types, including lymphocytes, monocytes, astrocytes, and neurons (20, 21) .

The ends of each strand of HIV RNA contain an RNA sequence called the long terminal repeat (LTR). Regions in the LTR act as switches to control production of new viruses and can be triggered by proteins from either HIV or the host cell. HIV’s core also includes a protein called p7, the HIV nucleocapsid protein (22) .

10 HIV-1 life cycle:

Figure.2: HIV-1 life cycle Reproduced with permission from Furtado et al; N Engl J Med 1999; 340:1614-22.Copyright Massachusetts Medical Society.

Infectious virus initially binds to cellular receptors on the surface of susceptible cells via envelope glycoprotein, gp120 (Fig.2), primarily the CD4 receptor present on helper T- lymphocytes, macrophages and activated peripheral blood mononuclear cells (PBMC).

Additionally two co-receptors: a-chemokine receptor CXCR4 (T-cell lines) and b- chemokine receptor CCR5 (macrophages) are also utilized by the virus. HIV-1 strains

11 using the CCR5 and are known as R5 strains, and the once that use CXCR4 as a co- receptor are known as X4 strains, and there are some more strains that can use either co- receptor are known as R5X4 strains. The interaction of gp120 with the receptor and co- receptor results in a fusion of the viral and cellular lipid membranes and the viral core

(nucleocapsid) is released in to the cytoplasm. Post release of the nucleocapsid the reverse transcription complex is developed, which contains the MA, Vpr, RT and IN proteins and associates rapidly with microtubules. The single-stranded RNA genome of the virus is then copied into a double-stranded linear DNA molecule by viral enzyme reverse transcriptase (RT) using as a primer tRNAlys-3. Then the DNA enters the nucleus as a nucleic acid-protein complex known as the pre integration complex and is incorporated into the cell’s genome by the action of second viral enzyme, integrase (IN).

The covalently integrated form of viral DNA, which is defined as the provirus, serves as the template for viral transcription. Retroviral RNAs are synthesized, processed by capping, polyadenylation, splicing etc. and then transported to the cytoplasm, where they are translated to produce viral proteins. The proteins that form the viral core initially assemble into immature nucleocapsids together with two copies of full-length viral RNA.

As these structures bud through the plasma membrane, they become encapsulated by a layer of membrane that also harbors the viral envelope glycoproteins. During the budding process, a third viral enzyme, the protease, cleaves the core proteins into their final forms this final step primes the viral particles for the next round of infection and is termed maturation (12, 23, 24) .

12 Glycolysis

Figure.3: Glycolytic pathway modified with permission from Vander Heiden et al. Science. 2009, 324(5930): 1029–1033

Glycolysis is the anaerobic central energy metabolism pathway that converts glucose to pyruvate. The term “glycolysis” itself means sugar splitting, as that is exactly what happens during this pathway. Glycolysis can be divided in to two phases: energy investment and energy pay off. During the energy investment cycle the cell uses up ATP that is generated back in the energy pay off phase by substrate level phosphorylation and

NAD+ is reduced to NADH by electrons released from oxidation of glucose. Two important products of this pathway are high energy ATP and NADH (reduced nicotinamide adenine dinucleotide). It is a sequence of ten reactions that occur in the cytosol with ten intermediate metabolites. This pathway also generates various by

13 products during the glucose metabolism process. The end product of glycolysis is the raw material for the next step in respiration, the Krebs cycle. 2 NADH or NADre are produced along with 4 ATP molecules. Among the nine enzymes of glycolysis three are known as the regulatory enzymes of the pathway, which are Hexokinase (HK),

Phosphofrukto kinase -1 (PFK-1) and phosphoglycerate kinase (PG). The rate-limiting step in glycolysis is the reaction catalyzed by PFK-1.

Link between viruses and glucose metabolism of the host:

The Warburg effect, which is the induction of aerobic glycolysis, is common in cancer pathology and recently it was observed in various viral pathogenesis such as Kaposi’s sarcoma herpes virus (KSHV) (25) , feline leukemia virus (26) , Cytomegalovirus,

Herpes simplex virus (27) , Mayaro virus (28, 29) and Hepatitis C virus (30) etc.

During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production and decreases oxygen consumption, i.e., the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis of KSHV-infected endothelial cells but uninfected cells remain unaffected. Therefore, similar to cancer cells, the Warburg effect is associated with maintaining KSHV latently infected cells (25) . Two related herpes viruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) infection of fibroblast and epithelial host cells was determined using mass spectrometry and both the viruses led to divergent effects on the host cell (27) . Another study revealed that in cultured Vero cells infected with the alpha virus Mayaro showed 2 fold increase in glucose consumption and increased lactate production showing overall increase of glycolytic flux (28) . Hepatitis C virus (HCV) infection affects the

14 mitochondrial-respiratory chain hence inducing oxidative stress. Using cell lines inducibly expressing different HCV constructs (30) ; it was shown that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation. This study shows that HCV protein expression activates hypoxia-inducible factor 1α (HIF-1α) by normoxic stabilization of its subunit. Subsequently, expression of HIF-1α controlled genes including the ones coding for glycolytic enzymes were significantly up regulated.

Stabilization of HIF-1α was also found in liver biopsy specimens from patients with chronic hepatitis C (30) . Similarly another aspect of utilizing glucose metabolism by virus is seen in case of human T-cell leukemia virus (HTLV) (31) as the receptor binding domains of both HTLV-1 and -2 envelope glycoproteins can interact with glut1 receptor and uses it for infecting target cells. Abrogating Glut1 expression can inhibit

HTLV-1 envelope-driven infection. During this interaction of the virus with glut-1 receptor glucose transport was inhibited and starvation induced glut1 expression also increased HTLV-1 infection. These observations suggest that studying the less explored facet of host viral interaction in terms of glucose metabolic pathway can lead to more therapeutic targets for intervention.

15 HIV-1 infection and glucose metabolism:

HIV-1 infection causes modulation of many host cellular pathways. The switch from oxidative phosphorylation to glycolysis may benefit enveloped virus production by increasing biomass including nucleotides, fatty acid, and lipid (25) . Enveloped viruses require increased lipid production to keep up with the needs of producing viral progeny by budding (27) . If HIV-1 infection causes increased metabolic demand in host cell to support its own replication, it can alter the glucose metabolic pathway in the host cell. It has been shown that GLUT3 expression is increased during HIV infection and glucose transport activity is also increased (32) . In a recent study, it was shown that the metabolite profile of HIV-1 infected CD4+T-cells and macrophages are different than those of the uninfected healthy cell (33) . HIV-1 infected CD4+ T-cells have a higher rate of glucose uptake and the pool size of glycolytic intermediates is also increased. In contrast, HIV-1 producing macrophages had a substantial reduction in glucose uptake, also a reduction in the pool size of glycolytic metabolites was observed. One important aspect of this study is that the two cell types studied here have very different characteristics when infected with HIV-1. HIV-1 infection of CD4+ T-cells kills the host cells within a period of 2-3 days post-infection but in the case of macrophages, cells do not die soon after infection but rather they act as reservoir for the virus in the body of the host. As mentioned before, survival of a cell is connected to glucose metabolism; hence this difference in metabolite profile in these two cell lines after HIV-1 infection could have some interpretation in terms of deciding the fate of these cells which in turn will have an effect on viral replication.

16 Influence of HIV-1 proteins on glycolytic pathway: Gp120 and glycolysis: It has been reported that gp120 treatment induces about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels (34, 35) . The experiments were performed on mixed cultures of brain cells and also in neuroblastoma culture where only neuronal cells were present. The gp120 induced toxic effect on neuronal culture was reduced if cells were supplied with a high concentration of glucose (20), suggesting that gp120 neurotoxicity may result from the impairment of neuronal metabolism.

Vpr and glycolysis: Recently we have characterized the Vpr response of macrophages using stable-isotope labeling by amino acids in cell culture (SILAC) coupled with a mass spectrometry-based proteomics approach (36) . Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with recombinant adenoviral vector harboring HIV-1 Vpr. More than 1000 proteins were quantified in SILAC coupled with

LC-MS/MS approach. The proteomic data showed significant changes in the glycolytic pathway of U937 cells upon Vpr overexpression. The most prominent effect observed was on the pentose phosphate pathway and pyruvate metabolism. As discussed before,

Vpr protein can also contribute to the regulation of HIF-1α hence Vpr may also regulate

HIF-1α mediated glycolytic regulation. In a recent study, extracellular Vpr was found to alter the glycolytic pathway through impairment of GAPDH activity, resulting in decreased levels of ATP (37) . Decreased GAPDH activity can be attributed to the translocation of GAPDH to the nucleus of the cell, which is observed in apoptotic cells hence making it unavailable in the cytoplasm as a glycolytic enzyme. Involvement of

GAPDH in apoptosis was first demonstrated in cultured cerebellar granule cells and cortical neurons undergoing spontaneous apoptosis (38) . GAPDH was overexpressed

17 prior to apoptosis and an antisense oligonucleotide against it could decrease apoptosis percentage (39) . These observations point to a role for Vpr in altering astrocyte metabolism and indirectly affecting neuronal survival in the onset of HAND through regulation of GAPDH.

Anti apoptotic switches stemming from glycolytic pathway: relevance to HIV-1:

In an event of HIV-1 infection T-cells die within 3-4 days-post infection but monocyte/macrophages can survive for a much longer period after being infected. Hence the question arises regarding the survival mechanism of monocyte/ macrophages after

HIV-1 infection. It is imperative for the virus to make sure that the host cell sustains long enough post infection to support the replication of viral progeny. Apart from the role as a glycolytic enzyme to phosphorylate glucose, the protective effects of hexokinase against cell death have long been observed in tumor cells. In addition, hexokinase II, the second isoform of hexokinase, is up regulated in rapidly growing tumor and the type I and II isoforms of hexokinase have N-terminal sequences that target them to bind the outer mitochondrial membrane (OMM), and both proteins can associate with voltage dependent anion channel (VDAC), which is a major protein on the outer mitochondria membrane behaving as a diffusion pore facilitating exchange of molecules between mitochondria and cytoplasm of cell (37-39). There are a few mechanisms that could regulate the anti-apoptotic effect of hexokinase through VDAC binding; in one mechanism hexokinase binding to VDAC prevents cytochrome c release by closing the mitochondrial pore (37). In a second mechanism, hexokinase directly prevents Bax translocation to the mitochondria and subsequent permeabilization, also Cyclophilin D can play a role in regulating this interaction and lastly increase in mitochondrial

18 cholesterol content can enhance this interaction (40). As a common pathology of viral infection severe oxidative stress comes into play. In order to survive, a cell must find a way to counter excess of ROIs. The product of pentose phosphate pathway (PPP)

NADPH can counter ROS. Also an increase in the PPP shunt will ensure on increase in production of several useful intermediate metabolites such a ribulose-5-phosphate, which can contribute to DNA repair. PPP activation will ensure that cells are protected from apoptosis induced by ROIs and DNA repair inhibition. Therefore it can be expected that there should be a noticeable increase in the PPP shunt in HIV-1 infected monocyte/macrophages as they survive under the stress of undergoing HIV-1 latent or rampant productive infection. This characteristic can be used to target those infected cells that have increased dependency on the PPP to support cellular survival by countering

ROIs produced by viral replication, as blocking this pathway will not harm uninfected cells as much as infected cells harboring viral progeny. These observations suggest that viral utilization of the glycolytic pathway is a very potent target for antiviral therapy.

Glycolytic markers of viral infections can be very helpful in development of novel therapeutics to counter both productive infection and viral latency.

Viruses and host regulatory networks have always shared an important relationship. Due to the dependency of viral lifecycle in host cell, host viral interactions play an important role in deciding the fate of the infected cell as well as sustaining the propagation of viral progeny. The effect of viral infection on the host glycolytic pathway can be very beneficial for supporting the viral progeny by utilizing the various intermediates of this pathway such as the nucleotides and NADH produced during the PPP and by utilizing the anti apoptotic and pro- apoptotic activities of glycolytic components such as HK, Taldo

19 and GAPDH. Hence it is clear that a reciprocal interaction between HIV-1 and host glycolytic pathway can be beneficial for the virus. It is imperative to identify the precise effects of HIV-1 infection on the host glycolytic pathway. The close relationship between cell survival and glycolytic pathway makes it an important pathway to study in latent reservoir cells of the virus. From all the observations regarding the possible modulation of the glycolytic pathway of cell by HIV-1 infection, it is safe to say that in latently infected cell’s cellular glycolytic pathway must get modified accordingly to support the survival of these cells. All these possibilities suggest that detailed study of the HIV-1 influence of the host glucose metabolism network can prove to be immensely useful in terms of a new target area for anti-viral treatment.

20 Hypothesis:

HIV-1 infection of activated CD4+T cells and monocyte/macrophages can alter the glucose metabolism pathway of the host cell, which affects the survival of the host cell and supports viral replication. Interfering with these modulations can lead to possible perturbations of viral replication and pathogenesis.

Specific aim 1: To identify change in the glycolytic pathway of HIV-1 target cells.

Sub Aims:

I. To identify changes in the expression and activity of principal glycolytic enzymes with

HIV-1 infection in two different host cell types.

II. To detect changes in the metabolite pools of the glycolytic pathway as an effect of important HIV-1 viral protein Vpr in longer surviving monocyte cell line.

Specific aim 2:

To investigate the possible effect(s) of the changes in the glycolytic pathway of host cells in regulation of host cell survival and apoptosis.

Sub aims:

I. To decide if glycolytic enzyme hexokinase is regulated by HIV-1 infection and has any role in deciding the fate of the infected cells.

II. To observe if interfering with the viral regulation of glycolysis lead to inhibition viral replication.

21 Specific aim 3: To examine the effect of regulation of the glycolytic pathway on the viral replication.

Sub Aims:

I. To identify the effect of PKM2 over expression on HIV-1 LTR.

II. To determine potential molecular interaction between the HIV-LTR and glycolytic pathway component(s) such as PKM2.

22 CHAPTER 2

DIFFERENTIAL REGULATION OF HOST GLYCOLYTIC PATHWAY BY

HIV-1 INFECTION & EFFECT OF VPR ON MACROPHAGE PROTEOM

*Some text and figures used in this chapter were previously published in PLoS

One and are reproduced with permission under the Creative Commons CC BY 3.0.

Introduction:

HIV-1 uniquely infects both activated CD4+ T cells and terminally differentiated macrophages during the course of HIV-1 pathogenesis. While HIV-1 infection of CD4+

T cells induces G2 arrest and cell death within 2–3 days by the effect of HIV-1 viral protein Vpr (40) , HIV-1 infection of macrophages leads to long-lived survival along with low constitutive viral production hence generating viral reservoirs (41) , these reservoirs are huge hindrance for successful HIV-1 eradication and plays a major role in

HIV-1 associated neurodegenerative disease in the CNS (42) . These differences suggest there is something fundamentally very different in terms of effect of HIV-1 infection on these two different host cell type. As discussed before cellular glycolytic pathway and cellular apoptosis are very closely related and are often regulated by one another. Hence it is becoming increasingly apparent that upon viral infection changes occur in the metabolic profile of the cell, which is critically important for viral replication and survival or apoptosis of host. In a recent study it was shown that the metabolite profiles of HIV-1 infected CD4+T cells and macrophages are different than that of the uninfected healthy cells (33) . HIV-1 infected CD4+T cells had higher glucose uptake and pool size of glycolytic intermediates were also increased; whereas HIV-1 infected macrophages

23 had substantial reduction in glucose uptake and subsequent reduction of the pool size of glycolytic metabolites. This difference in metabolite profile in these two cell lines after

HIV-1 infection could have some interpretation in terms of deciding the fate of these cells which in turn could have an effect on viral replication. Analysis of expression and activity of major glycolytic pathway enzymes was conducted using western blotting and enzymatic activity assay. And the comparison showed the differential regulation of host glycolytic pathway by HIV-1 infection in T-cell lineage cells vs. macrophage lineage cells.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a small 96-amino acid multifunctional protein (21, 40, 43) . Vpr is essential for HIV-1 infection of macrophages since virus deficient in Vpr is less efficient in replication in macrophages

(44) . Furthermore, extracellular Vpr can rescue replication of Vpr-deficient HIV strains in macrophages (45) . HIV-1 LTR activation by Vpr results in increased viral replication

(46) . Vpr induces apoptosis in several cell types, including lymphocytes, monocytes, astrocytes, and neurons (20, 47) . However, HIV-1 infected macrophages are resistant to apoptosis (48) . These observations suggest that Vpr modulates macrophage proteome to promote viral replication and induce anti-apoptotic pathways. This acquired anti- apoptotic phenotype may promote reservoir formation in this cell type. Therefore, analysis of the macrophage proteome in Vpr expressing macrophages can help to better understand mechanisms involved in HIV-1 replication and survival.

A variety of stable-isotope labeling strategies, such as isotope-coded affinity tag (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ) and stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry (MS)-based

24 proteomics allows reliable identification and quantitative analysis of multiple proteins in complex samples (20) . SILAC, as a metabolic labeling method was used, since it is simple, efficient, and allows for almost complete heavy isotope incorporation in cells . To explore novel mechanisms underlying Vpr-mediated modulation of macrophage proteome, LC-MS/MS, along with SILAC was employed to assess quantitatively Vpr- induced perturbation of protein expression in U937 derived macrophages. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS measurement, among which

136 were significantly altered upon Vpr overexpression in macrophages showing Vpr- induced up-regulation of enzymes in the pyruvate metabolism, pentose phosphate pathway.

25 Materials and methods:

Cells: The cells used in these studies are U937, Jurkat, U1, and J.Lat.

U937: The U-937 cell line was derived from malignant cells obtained from the pleural effusion of a patient with histolytic lymphoma. Distributed by ATCC (CRL 1593.2).

U937 cells mature and differentiate in response to a number of soluble stimuli, adopting the morphology and characteristics of mature macrophages. U937 cells are of the myeloid lineage and so secrete a large number of cytokines and chemokine either constitutively (e.g. IL-1 and GM-CSF) or in response to soluble stimuli such as phorbol myristic acid (PMA).

Jurkat: Jurkat cells are an immortalized line of human T lymphocyte cells that are used to study the expression of various chemokine receptors susceptible to viral entry, particularly HIV.

U1: The U1 cells were a gift from Dr. Jay Rappaport. In 1987 by Folk TM et al (49) ,

Infection of U937 cell with HIV-1 viral strain LAV created U1 cell line. Limiting dilution technique cloned the cells that survived the infection cycle and acute replicative phase. A panel of clone was obtained and investigated further to identify HIV-1 expression pattern. The clones that were expressing low HIV levels were readily induced to produce high viral titer by treatment with PMA. Hence U1 cell containing two HIV-1 proviruses works as a monocytic latency model of HIV-1.

J.Lat: To create J.Lat cells, Jurkat cells were infected with viral particle containing HIV- based retroviral vector containing the Tat and GFP open reading frames both under the control of the HIV promoter in the 5′ long terminal repeat (LTR). Differential fluorescence-activated cell sorting (FACS) based on GFP expression was used to isolate

26 GFP-negative cells by FACS 4 days after infection. This population presumably harbored both uninfected cells and cells with transcriptionally silenced proviruses. To activate HIV expression, this population was treated with TPA or tumor necrosis factor α (TNF-α) and purified GFP-positive cells by FACS. Following standard serial dilution methods followed by FACS separation the cell population harboring latently infected provirus was created and we used the J.Lat clone 6.3 for our experiments.

Cell culture technique: The above-mentioned cells were all suspension cells so the following method was employed to culture them. The frozen cell containing vial was thawed by gentle agitation in a 37°C water bath. Vial was removed from the water bath as soon as the contents were thawed, and decontaminated by dipping in or spraying with

70% ethanol. The vial contents were transferred to a centrifuge tube containing 9.0 mL complete culture medium and spun at approximately 125x g for 5 to 7 minutes to obtain cell pellet. Cell pellets were re suspended with RPMI media supplemented with 10%

FBS, penicillin streptomycin antibiotic and dispense into a 25 cm2 or a 75 cm2 culture flask. The culture was incubated at 37°C in a 5% CO2 in air atmosphere maintaining incubator.

HIV-1 replication in U1: To induce HIV-1 replication the U1 cells were treated with

10nm/mL PMA over-night and the media was replaced with fresh RPMI supplemented with 10% FBS.

HIV-1 replication in J.Lat 6.3: To induce HIV-1 replication the U1 cells were treated with 10nm/mL PMA over-night and the media was replaced with fresh RPMI supplemented with 10% FBS.

27 p24 ELISA: The HIV-1 p24 antigen capture assay for the detection of Human

Immunodeficiency Virus Type 1 (HIV-1) p24 in tissue culture media. Advanced bioscience laboratory catalogue # 5421 was done according to manufactures protocol.

HK Enzyme activity assay: Cells were lysed using the following buffer: 50 mM potassium phos-phate, 2 mM dithiothreitol (DTT), 2 mM EDTA, and 20 mM sodium fluoride. Approximately 10 µl of freshly lysed cell supernatant was added to 1,000 µl of

100 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 10 mM ATP, 10 mM MgCl2, 2 mM glucose,

0.1 mM NADP, and 0.1 U/ml of G6PD (Sigma-Aldrich). HK activity was determined by following the G6P-dependent conversion of NADP to NADPH spectrophotometrically at

340 nm at 37°C.

G6PD enzyme activity assay: Cells were lysed using 0.05 M Tris-HCL pH 7.5 buffer with 5mm MgCL2 and 0.1% saponin. Briefly, the substrate mixtures containing 2.5 mm

G6P, 0.2 mm NADP in H2O, and the WST-8/1-methoxy PMS the hydrogen of NADPH produced by G6PD enzyme activity converts WST-8 to WST-8 formazan in the presence of a hydrogen carrier, 1-methoxy PMS and gives the dark yellow to orange color as a indication of G6PD activity increasing with darker color visible in naked eye. It is possible to express G6PD activity as increase of NADPH concentration by reading absorbance at 460 nm after incubation for 30 or 60 min.

PKM2 activity assay: The Pyruvate Kinase Activity Assay Kit from Bio vision (#K709-

100) was used as per manufacturer’s protocol; the kit provides a simple and direct procedure for measuring pyruvate kinase activity. Pyruvate concentration is determined by a coupled enzyme assay, which results in a colorimetric (570 nm) product, proportional to the pyruvate present.

28 Western blot: Protein samples (50µg) were separated by gradient SDS-PAGE and then transferred to a nitrocellulose membrane in a blotting chamber (BioRad) at 100V for 30 min. The membrane was blocked with 5% powdered milk in Tris-buffer saline solution

(pH 7.6) containing 0.05% Tween-20 (TBS/T) then probed with antibodies that had been diluted 1:500 (anti-HK-1, HK-2, G6PD, and Grb2 antibodies from Cell signaling).

Membranes were incubated with primary antibodies overnight at 4C, washed, and then incubated with appropriate secondary antibodies conjugated with Licor IR dyes at room temperature for 1h. The membranes were then analyzed with digital imaging in Licor odyssey machine.

13 15 13 Chemicals and Antibodies: Heavy lysine and arginine ([ C6, N2]-L-lysine and [ C6]-

L-arginine) were obtained from Cambridge Isotope (Andover, MA) and light amino acids

(L-lysine and L-arginine) were obtained from Sigma-Aldrich (St. Louis, MO). All components of cell culture media were obtained from Life Technologies (CA) and protease inhibitor cocktail was obtained from Sigma-Aldrich (St, Louis, MO). SILAC

DMEM Media was obtained from Pierce Biotechnology and the dialyzed FBS was purchased from HyClone (Logan, UT). Trypsin was purchased from Promega (Madison,

WI). All the chemicals were HPLC-grade unless specifically mentioned. The antibodies against HK-1, HK-2, G6PD, PKM2 and Grb2 were obtained from Cell Signaling

Technology (Danvers, MA); HIF-1 alpha antibody was obtained from BD Biosciences

(San Jose, CA).

Construction of Recombinant Adenoviruses: To construct recombinant adenoviral vector harboring HIV-1 Vpr, Vpr cDNA from the dual-tropic (CCR5 and CXCR4) strain of HIV-1 89.6 [25] was used. Vpr cDNA (288 bp) was excised from pcDNA3-Vpr and

29 cloned into the EcoRI and NheI sites of the adenovirus-shuttle plasmid pDC515 under the control of the murine cytomegalovirus promoter (purchased from Microbix Inc., Ontario,

Canada). Adeno-Vpr recombinant shuttle containing Vpr sequence (pDC515-Vpr) was transfected into HEK-293 cells with pBHGfrt (del) E1, 3FLP, and a plasmid that provides adenovirus type5 genome deleted in E1 and E3 genes. Plaques of recombinant adenovirus arising as a result of frt/FLP recombination were isolated, grown, and purified by cesium chloride density equilibrium banding. Empty shuttle plasmid, pDC515, was used to construct control adenoviral vector (Adeno-null, a virus without a transgene).

Differentiation of U937 Cells into Macrophages and Culture in SILAC Media: SILAC

DMEM media was supplemented with 10% dialyzed fetal bovine serum, 1%

13 streptomycin/penicillin. The medium was then divided and supplemented with C6 L-

13 15 arginine and C6, N2-L-lysine or normal L-arginine and L-lysine, to produce heavy or light SILAC medium, respectively. U937 cells were treated with 100 ng/ml of phorbal myristate acetate (PMA) for 3 hrs in complete RPMI medium at 37°C and then washed with 1XPBS and cultured for an additional 24 h in complete RPMI medium at 37°C. For

SILAC experiments, the PMA differentiated cells were then grown in parallel in either light or heavy media for 5 days, with media replacement every 24 h.

Transduction of SILAC Labeled Cells with Adenoviral Constructs: The PMA differentiated cells (macrophages) grown in SILAC media were then transduced with adenoviral stock corresponding to a multiplicity of infection (MOI) of five plaque- forming units per cell. The heavy labeled cells were transduced with Adeno-Vpr, while the light-labeled cells were transduced with Adeno-Null. The cells were harvested at 72 h post infection.

30 Preparation of Protein Samples, 1-D SDS-PAGE Separation and In-gel Trypsin

Digestion: Proteins were processed for gel electrophoresis-liquid chromatography-mass spectroscopy (GeLC-MS/MS) proteomics analysis. Total cell proteins were extracted from cells transduced with Adeno-Vpr and control cells using RIPA buffer (25 mM

Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS).

Protein quantification was performed using the method of Bradford (Bio-Rad Protein

Assay). 60 µg of total proteins (30 µg “heavy” and 30 µg “light”) were diluted with

Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol. The mixture was heated for 5 min at 90°C and loaded onto 10% polyacrylamide gel. 1-D SDS-PAGE separation was performed using a mini Protean II system (BioRad) at 200 V for 45 min.

Bands were visualized with Simply Blue Safe Stain and lanes were sliced into 11 sections, which were diced into ~ 1×1 mm. After distaining with 50% v/v Acetonitrile

(ACN) in 25 mM ammonium bicarbonate buffer (bicarbonate buffer), proteins within gel pieces were reduced with 10 mM DTT in bicarbonate buffer and alkylated by incubation with 50 mM iodoacetamide in bicarbonate buffer. After gel dehydration with 100% ACN, the gel pieces were covered with approximately 40 µL of 12.5 µg/mL trypsin in bicarbonate buffer. In gel digestion was done at 37°C for 12 h, trypsin was inactivated with formic acid at 2% final volume and peptides were extracted and clean-up using C18

Tip column (ZipTips®) as previously described in (50) .

GeLC-MS/MS and Data Analysis: Peptides were dried in a vacuum centrifuge then resuspended in 30 µL of 0.1% v/v TFA/H2O. Peptide samples were loaded onto 2 µg capacity peptide traps (CapTrap; Michrom Bio-resources) and separated using a C18 capillary column (15 cm 75 mm, Agilent) with an Agilent 1100 LC pump delivering

31 mobile phase at 300 nL/min. Gradient elution using mobile phases A (1% ACN/0.1% formic acid, balance H2O) and B (80% ACN/0.1% formic acid, balance H2O) was as follows (percentages for B, balance A): linear from 0 to 15% at 10 min, linear to 60% at

60 min, linear to 100% at 65 min. The nano ESI MS/MS was performed using a HCT

Ultra ion trap mass spectrometer (Bruker). ESI was delivered using distal-coating spray

Silica tip (id 20 µm, tip inner id 10 µm, New Objective, Ringoes, NJ). Mass spectra were acquired in positive ion mode, capillary voltage at −1200 V and active ion charge control trap scanning from 300 to 1500 m/z; Using an automatic switching between MS and

MS/MS modes, MS/MS fragmentation was performed on the two most abundant ions on each spectrum using collision-induced dissociation with active exclusion (excluded after two spectra, and released after 2 min). The complete system was fully controlled by

HyStar 3.2 software.

Mass spectra data processing was performed using Mascot Distiller (Version 2.4.3.3) with search and quantitation toolbox options. The generated de-isotoped peak list was submitted to an in-house Mascot server 2.4.0 for searching against the SwissProt database version 2013_01 (538849 sequences; 191337357 residues). Mascot search parameters were set as follows: species, Homo sapiens (20,233 sequences); enzyme, trypsin with maximal 2 missed cleavage; fixed modification: cysteine carbamidomethylation; variable modifications: methionine oxidation, Gln->pyro-Glu (N-term Q), Glu->pyro-Glu (N-term

E), Label: 13C (6) 15N(2) (K), Label: 13C(6) (R); 0.90 Da mass tolerance for precursor peptide ions; and 0.6 Da for MS/MS fragment ions. SILAC quantitation was performed in Mascot Distiller using SILAC K+8 R+6 quantitation method; SILAC ratios for heavy and light peptide pairs were calculated by Simpson’s integration method, minimum 1

32 peptide with unique sequence and 0.05 of significant threshold. The following criteria were used to evaluate protein identification: one or more unique peptides with ion score

≥45 and two or more unique peptides with ion score ≥30 (p≤0.05 threshold); proteins identified were extracted using MS Data Miner (MDM) [26]. Quantified proteins with

≥1.5 and ≤0.7 fold change were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis (IPA) software

(www.ingenuity.com) for bioinformatics analysis.

Statistical analysis: The Statistics used for IPA analysis can be found at

(http://www.ingenuity.com/index/html). For western blot analysis Student’s t-test was used for statistical analysis and p*≤*0.05 was considered statistically significant.

33 Results:

Figure.4: 10nm/ml PMA treatment efficiently induces HIV-1 viral production in U1 and J.Lat cells in a time dependent manner. (A) p24, of U1 cells treated with PMA (10nm/ml) collected at 24,48,72h .72hour post induction being the highest 8929+/-652 ng/ml. (B) p24, of J.Lat cells collected in three different time points. 72 hour post induction being the highest 10021+/-321 ng/ml. Values represent means ± S.E of three experiments. *indicate p value<0.05,

34

Figure.5: Induction of viral replication in U1 cells leads to changes in expression and activity of glycolytic enzymes. (A) Western blot analysis of HK1, HK2, G6PD and PKM2 in U1 cells control untreated and induced with PMA. (B) Densitometry analysis of the western blots. (C) HK ,(D) G6PD, (E) PKM2 Enzymatic activity assay control and induced with PMA measured in calorimetric method. Values represent # means ± S.E of three experiments. *indicate p value<0.05, indicate p value>0.05 or

35 Fig.6: Induction of viral replication in J.Lat cells has different effect on glycolytic enzyme expressions and enzyme activity: (A) western blot analysis of HK1, HK2, PKM2 and G6PD in J.Lat cell line control untreated and induced with PMA. (B) Densitometry analysis of the western blots. (C) HK, (D) G6PD and (E) PKM2 enzymatic activity assay in J.Lat cells control and induced with PMA measured in calorimetric method. Values represent means ± S.E of three experiments. *indicate p value<0.05, #indicate p value>0.05 or not significant. 36 Fig.7: Experimental strategy for SILAC based proteomics: PMA differentiated U937 cells cultured in light or heavy media and then transduced with Adeno-Null or Adeno-Vpr virus, respectively. Protein lysates were prepared and mixed in 1:1 ratio. Sample complexity was reduced prior to LC-MS/MS analysis by fractionation at the protein level by SDS-PAGE. Expression levels of selected proteins were validated by western blot analysis.doi:10.1371/journal.pone.0068376.g001

Fig.8: Categorization of molecular function of differentially expressed proteins in Vpr transduced macrophages: The pie graph demonstrates that among the 136 differentially expressed proteins majority of them cluster in the metabolic pathways and metabolism of proteins.doi: 10.1371/journal.pone.0068376.g002

37 Table 1. Macrophage proteins within metabolic pathways altered in response to HIV-1 Vpr. doi:10.1371/journal.pone.0068376.t001

38

Fig.9: Effect of Vpr over expression on macrophages glycolytic enzyme expression: (A) Western blot analysis of protein lysates prepared from macrophages derived from U973 cells transduced with Adeno-Null or Adeno-Vpr virus. Molecular weight of respective protein is shown in kDa. (B) Densitometric analyses of the representative proteins were done after normalization to Grb2 levels. Values represent means ± S.E of three experiments. *indicate p value<0.05, #indicate p value>0.05 or not significant. doi: 10.1371/journal.pone.0068376.g005

39 Fig.4: PMA treatment induces HIV-1 replication in U1 and J.Lat 6.3 cell line in a time dependent manner: 10nm/ml of PMA was used to induce HIV-1 replication in chronically infected U1 cell and J.Lat 6.3 cell line. The p24 ELISA data collected from the supernatant showed that the amount of HIV-1 p24 in creases with time and at 72 hours post induction shows a reasonably high p24 count 8929+/-652 ng/ml for U1 cells and 10021+/-321 ng/ml (Fig. 4). Hence in the following experiments cell lysates from U1 and J.Lat 6.3 at 72hpi will be used.

Fig.5: Induction of viral replication in U1 cells leads to changes in expression and activity of glycolytic enzymes: U1 cell were treated with 10nm/ml of PMA over night and the media was replaced on the following day with fresh media. Cells were harvested and analyzed with western blot for protein expression and enzymatic assay for enzyme activity. The data shows that the expression level of HK-1, G6PD, and PKM2 increased,

HK2 expression level did not change much in response to HIV-1 replication induced by

PMA (Fig.5A, 5B). The enzymatic activity assay data showed that despite of increase in

HK-1 expression and unchanged HK-2 expression the enzymatic activity of HK-1 decreased with viral replication in U1 cells. However the enzymatic activity of G6PD increased with HIV-1 replication. The PKM2 enzyme activity also decreased with HIV-1 replication in U1 cells (Fig.5C, D, E).

Fig.6: Induction of viral replication in J.Lat 6.3 cells has different effect on glycolytic enzyme expressions and enzyme activity: J.Lat 6.3 cells were treated similarly as U1 cells and were harvested to be analyzed by western blotting and enzymatic activity assay.

In J.Lat cell line the induction of HIV-1 replication did not bring about any significant changes in terms of expression of HK-1, HK-2, G6PD and PKM2 (Fig.6A, 6B). Only the

40 HK enzymatic activity increased in J.Lat cells with induction of HIV-1 replication. G6PD and PKM2 enzymatic activity did not show significant difference between induced and uninduced cells (Fig.6C, D, E).

Fig.7: Proteome of Vpr Transduced Macrophages: In this study, PMA differentiated

U937 macrophages were cultured in both light and heavy media. The light labeled cells were transduced with Adeno-Null virus and the heavy labeled cells were transduced with

Adeno-Vpr. Cell pellets prepared 72 h post transduction were lysed, and the lysates were combined and subsequently fractionated by SDS-PAGE. After in-gel digestion, the proteins were identified and quantified by LC-MS/MS (Fig7). This analysis was performed once and a total of 614 proteins were identified and quantified. For quantitative analysis of differences between paired experimental samples a ratio of ≥1.5 or ≤0.7 was chosen as threshold for screening significantly changed proteins. Using this criterion a total of 136 proteins that displayed significant changes in Vpr expressing macrophages were identified, among which 67 and 69 were up- and down- regulated.

Fig.8: Functional Characterization of Identified Proteins and Bioinformatics Analysis:

The 136 differentially expressed proteins identified in response to Vpr over expression were used, to perform a biological function, pathway and network analysis using the

Ingenuity Pathway Analysis (IPA) software. According to the molecular function analysis (Fig.8), most of the proteins were related with metabolic pathways (39%), protein metabolism (17%), cell cycle regulation (15%), signal transduction (12%), phagosomal activity (10%), membrane trafficking (10%), gene expression (8%), RNA metabolism (8%), cell cycle (6%), extracellular matrix organization (6%), signal

41 transduction (5%), cell adhesion molecules (4%) and unclassified (22%). The list of the proteins that were altered and are related with metabolic pathway is listed in Table 1.

Fig.9: Validation of Protein Identification and Quantification: The functional characterization and bioinformatics analysis revealed that the pathways that were significantly altered involved glycolysis, mitochondrial dysfunction, and HIF-1α signaling. Hence the relative abundance of some of the proteins in these pathways was validated by western blot analysis. The western blot analysis demonstrated that the changes in the ratios of representative proteins (HK-1, HK-2, PKM2, G6PD), between adeno-null transduced and adeno-Vpr transduced macrophages (Figure 9A and B) are consistent with that derived from SILAC studies. There was a significant increase in HIF-

1 alpha levels in Vpr transduced macrophages.

42 Discussion:

Based on our preliminary findings and literature reviews we hypothesized that HIV-1 infection regulates the cellular glucose metabolism pathway of host. The possible changes in the pathway by the viral infection could either be a cellular response to viral infection or result of viral regulation of the pathway to support it’s own progeny. Due to the divergent nature of cell death and survival in T-cell lineage cells and Monocyte macrophage lineage cells once infected by HIV-1, we identified two chronically infected cell lines U1 and J.Lat 6.3 as our study model. As U1 cell line represents the monocyte/macrophage lineage cells and J.Lat 6.3 cell line represents the T-cell lineage target cells for HIV-1. The experiments analyzing the state of various glycolytic pathway components in terms of protein expression and enzymatic activity showed that HIV-1 infection lead to significant changes of the glycolytic pathway components in U1 cells.

The effect on J.Lat 6.3 cells were not so significant in terms of protein expression of glycolytic components but there was increase in hexokinase enzyme activity in J.Lat 6.3 cells post induction of viral replication. Which is exactly opposite of what was seen in U1 cells. U1 cells showed significant increase in the protein expression of HK1, G6PD and

PKM2. We followed up with these observations in our later chapters as all three enzymatic proteins have significant role to play in terms of cellular survival, proliferation and transcriptional regulation of target genes. The difference between the metabolic profile of U1 and J.Lat could be consistent with their cellular fate post infection. HK1 could play a role in supporting mitochondrial integrity hence supporting survival of infected U1 cells so that in turn the virus has a shelter for its replication to take place.

G6PD plays a supporting role in the same account to HK-1 and apart from that up

43 regulated G6PD activity means increased PPP flux which produces various cellular macromolecules (51, 52) for the virus to use for construction of its own progeny viral particles. We conducted the Vpr proteomic studies in the U937 cell since these cells are proven to not be resistant to apoptosis (48) . In recent times proteomics has contributed significantly in HIV research to investigate not only HIV pathogenesis but also for identification of potential biomarkers (53-55) . In this study, we utilized the high- throughput quantitative proteomic approach using SILAC to obtain information about the macrophage proteome in the context of Vpr-host interaction since Vpr from strain 89.6 demonstrates PP2A-dependent apoptosis in CD4+ T cells and Vpr gene polymorphism is known to influence clinical outcomes (56, 57) . A total of 136 different proteins were identified as having altered abundances in Vpr transduced macrophages, including those involved in pyruvate metabolism, pentose phosphate pathway, mitochondrial dysfunction, oxidative stress, HIF-1 alpha signaling, and cell cycle: G2/M DNA damage checkpoint regulation. Our data are in concordance with the transcriptome analysis that also demonstrated the interference of HIV-1 in host energy metabolism pathways (58,

59) . In the Vpr study in macrophages we observed trend similar to U1 in increased expression of HK-1, G6PD, PKM2 proteins. In addition to all of these enzymes the total expression of HIF-1α also increased in tune with our earlier observation (60, 61) . HIF-

1α up regulation is significant because it is one of the major regulators of the glycolytic pathway. GLUT-1, HK, G6PD, GAPDH and many other glycolytic enzymes are regulated by HIF-1α protein (62) ; most of these glycolytic components have a hypoxia responsive element present in the promoter. HIV-1 has been shown to up regulate expression of HIF-1α (60) . Hence HIV-1 can regulate glycolysis via the Vpr-HIF1α axis

44 to create an environment that is not only advantageous for viral replication and biogenesis, but also for long-term survival of infected macrophages. HK is the rate- limiting enzyme that converts glucose in to glucose-6-phosphate (G6P), while G6PD a member of the pentose phosphate pathway (PPP) is involved in the conversion to G6P to

6PG (63) . Furthermore, up regulation of hexokinase expression and shunting of glucose through the pentose phosphate pathway (PPP) creates a restrictive environment for cytochrome c-mediated apoptosis as a result of increased translocation of HK to outer mitochondrial membrane (OMM), and generation of NADPH, respectively (64) , (65) .

Cellular redox status is maintained by scavenging ROS by glutathione whose synthesis is regulated by NADPH (65) . In addition, up regulation of PPP and activation of G6PD also promotes nucleotide biosynthesis that provides the nucleotide pool (66) required for sustained HIV-1 replication. All these proteins were seen to be up regulated with HIV-1 replication in U1 cell lines but not in J.Lat 6.3 cell lines indicating the virus utilizes the host glycolytic pathway in monocyte/macrophage lineage cells to avoid apoptosis of host, facilitating viral replication and latency in these cells but no such changes are observed in

T-Cell undergoing rapid productive infection and death.

Interestingly Among the proteins whose expression is down regulated in Vpr transduced macrophages are Glutamate dehydrogenase 2 (GLUD2), Adenylate kinase 2 (AK2) and

Transketolase (TKT). GLUD2 is a mitochondrial enzyme involved in glutamate metabolism it catalyzes the reversible oxidative deamination of glutamate to alpha- ketoglutarate (67) . Dysregulation of glutamate metabolism in macrophages therefore can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of

NeuroAIDS. AK2 is a mitochondrial enzyme that regulates adenine nucleotide

45 interconversion (68) . AK2 is known to mediate mitochondrial apoptosis through the formation of AK2-FADD-caspase-10 (AFAC10) complex (69) . Both these observations suggest that Vpr induces changes in macrophage proteome is creating an anti-apoptotic environment in these cells to support its survival and in turn provide room for viral replication. Our studies demonstrate how HIV-1 replication can regulate the host glucose metabolism pathway by modulating its components and also single viral protein has comparable impact and underscores the role of Vpr in modulating changes at the transcriptome and proteome level in HIV-1 infected host. Following chapter will further investigate the role of the identified proteins, specifically HK and G6PD in anti-apoptotic pathways in macrophages and PKM2 in HIV-1 replication.

46 CHAPTER 3

ANTIAPOPTOTIC ROLE OF GLYCOLYTIC COMPONENTS ARE

REGULATED BY HIV-1 INFECTION IN HOST CELL

Introduction

The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-

6-P) by hexokinases (HKs), a family with 4 isoforms (70) . It has been observed that G-

6-P is a potent inhibitor of HK in physiological conditions. Among all the isoforms of

HKs the HK1 and HK2 have the capacity to bind to the mitochondria of the cell (71) .

Expression of HK1 in brain cells and red blood cells makes it a more potent subject for our study in the scenario of HIV-1 infection, as association of HIV-1 to the infection of macrophages in the central nervous system is a widely studied area. The infected macrophages also act as reservoir of HIV-1 in the host system (72) . This reservoir scenario is now one of the most studied areas in the HIV-1 field and rightfully so as the inability to remove these reservoirs from the host system makes cure of HIV-1 an impossible task. In order to come up with a strategy to get rid of the macrophage reservoir from the host system it is important to understand how they avoid apoptosis despite of the increased stress from the viral persistence. According to various studies in field of tumor biology Hexokinase is already known to play a role in cell survival by helping to sustain mitochondrial membrane integrity. Hexokinase binding to VDAC suppresses the release of inter-membrane space proteins and inhibits apoptosis, thereby contributing to the survival of tumor cells (71) .

47 Here we report that in order to render protection to the infected monocyte cells the HK enzyme activity is cut down by HIV-1 viral replication and instead expressed HK protein is accumulated to the mitochondria of the cell supporting the mitochondrial integrity of the infected cell is the primary strategy applied by the virus to avoid apoptosis of its host cell hence supporting the viral progeny. Removal of this excess HK accumulation from mitochondria or inhibition of this mitochondrial interaction of HK can lead to apoptosis of infected cell. The PPP also plays an important role in supporting this regulation through G6PD enzymatic activity and production of cellular macromolecules. We also report that PPP blocker reagent DHEA-S can have inhibitory effect on the viral replication / reactivation in U1 cells by inhibiting the nuclear translocation of p65.

48 Material and methods:

Cells: PBMC cells were isolated from buffy coat using the standard ficoll gradient centrifugation method and cultured in RPMI media containing 10% FBS, 10mg/ml gentamicin and 5µg/ml PHA-P. The U1 cell line consists a chronically infected clone from the parent pro-monocyte cell line, U937. U1 cells contain two HIV-1 pro-viruses, which are non-replicating. U1 cells were maintained in RPMI medium supplemented with 10% FBS and gentamicin. The U937 cells were also cultured in RPMI media containing 10% FBS and 10mg/ml gentamicin.

HIV-1 infection: PBMCs were infected with HIV-1SF162 and infected cell population was harvested at Day 3 and Day 6 post infection. Supernatant from the infected cells were collected to analyze viral replication status by p24 ELISA.

HIV-1 replication in U1: To induce HIV-1 replication the U1 cells were treated with

10nm/mL PMA over-night and the media was replaced with fresh RPMI supplemented with 10% FBS. p24 ELISA: The HIV-1 p24 antigen capture assay for the detection of Human

Immunodeficiency Virus Type 1 (HIV-1) p24 in tissue culture media. Advanced bioscience laboratory catalogue # 5421 was done according to manufactures protocol.

HK Enzyme activity assay: Cells were lysed using the following buffer: 50 mM potassium phos-phate, 2 mM dithiothreitol (DTT), 2 mM EDTA, and 20 mM sodium fluoride. Approximately 10 µl of freshly lysed cell supernatant was added to 1,000 µl of

100 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 10 mM ATP, 10 mM MgCl2, 2 mM glucose,

0.1 mM NADP, and 0.1 U/ml of G6PD (Sigma-Aldrich). HK activity was determined by

49 following the G6P-dependent conversion of NADP to NADPH spectro-photometrically at 340 nm at 37°C.

G6PD enzyme activity assay: G6PD assay was done following manufacturer’s protocol

(Dojindo).

Isolation of mitochondria: Cytoplasmic and mitochondrial fractionation were done after collecting dry cell pellet from the U1 cells after due treatment with PMA and CTM; the protocol followed was found online published by Chandel Lab, Cells were scraped in 1 ml mitochondria isolation buffer and Spinned at 1,000 rpm, 10 min, to pellet nuclei. The supernatant is spinned 15,000 rpm, 20 min to pellet mitochondria the pellet is solubilized in 1X lysis buffer (from 10X lysis buffer + 1:1000 PMSF). The mitochondrial isolation buffer contains 250mM sucrose, 10mM Tris-HCl pH 7.4, 0.1mM EGTA.

Adeno viral transduction: Recombinant adenoviral vector harboring HIV-1 Vpr was constructed using Vpr - cDNA from the dual-tropic (CCR5 and CXCR4) strain of HIV-1

89.6, as described in previous publication (36) .The PMA differentiated cells

(macrophages) were transduced with adenoviral stock corresponding to a multiplicity of infection (MOI) of ten plaque-forming units per cell.

Western blot: Protein samples (50µg) were separated by gradient SDS-PAGE and then transferred to a nitrocellulose membrane in a blotting chamber (BioRad) at 100V for 30 min. The membrane was blocked with 5% powdered milk in Tris-buffer saline solution

(pH 7.6) containing 0.05% Tween-20 (TBS/T) then probed with antibodies that had been diluted 1:500 (anti-HK-1, HK-2, G6PD, and Grb2 antibodies from Cell signaling).

Membranes were incubated with primary antibodies overnight at 4C, washed, and then incubated with appropriate secondary antibodies conjugated with Licor IR dyes at room

50 temperature for 1h. The membranes were then analyzed with digital imaging in Licor oddessy machine.

Apoptosis analysis by propidium iodide staining: Post treatments the cells were harvested by centrifugation, washed with PBS and fixed in ice-cold ethanol (70% final concentration). After incubation for 24 hours at −20°C cells were washed with PBS, stained with propidium iodide 10 µg/ml in PBS containing 250 µg/ml RNasA and incubated at 37°C for 30 minutes in the dark before analysis by fluorescence-activated cell sorting (FACS). Percentage of apoptotic cell population in each experimental condition was analyzed with the Guava EasyCyte mini system and using the Guava

CytoSoft cell cycle program according to the manufacturer's instructions (Guava

Technologies, Hayward, CA).

JC-1staining for detection of mitochondrial depolarization: Post treatment cells were harvested by centrifugation and washed with PBS and stained with JC-1 dye following the manufacturers protocol (Invitrogen, Mito probe M34152). The percentage of depolarized mitochondria containing cells were determined with the Guava EasyCyte mini system (Guava Technologies, Hayward, CA) by analyzing the ratio of conversion from red to green fluorescence.

51 Results:

Fig.10: HIV-1 induces HK-1 expression in monocytes/macrophages: PBMCs were infected with HIV-1SF162 (A, B, C) HK-1, HK-2 protein expression in infected PBMCs 6dpi by western blot. U1 cells and media were harvested at 72h post PMA induction of viral replication (D, E, F) HK-1 protein expression was analyzed 72hpi in U1 cell lysates. (G) HK enzyme activity in PBMCs infected with HIV-1SF162 6dpi measured by OD was normalized by protein concentration (H) HK activity assay was performed on lysates from 72h post induction (hpi) in U1 cells and in U937 cells 72 hours post PMA treatment. * Indicates P value <0.05. # Indicates P value > 0.05.

52 Fig.11: HIV-1 induces G6PD expression and activity (A, B) G6PD protein expression in infected PBMCs was analyzed at the 6dpi by western blot. U1 cells and media were harvested at 72 h post PMA induction of viral replication. (C, D) G6PD protein expression in U1 cells 72hpi was analyzed with western blot. (E) G6PD enzyme activity was measured in HIV-1SF162 infected PBMCs 6dpi. (F) G6PD enzyme activity in U1 cells 72hpi and in U937 cells 72hour post PMA treatment. * indicates P value <0.05. # indicates P value > 0.05.

53 Fig.12: HIV-1 increases mitochondria bound HK-1 levels in U1 cells : U1 cells were harvested 72hr post PMA induction and were subjected to cytoplasmic-mitochondrial fractionation. (A, B) The lysates were analyzed by western blots for the expression of HK1. VDAC01 was used as control for purity and protein loading of mitochondrial fraction & tubulin as cytoplasmic control. (C, D) U937 cells were treated with PMA and fractionated similarly as a control for PMA effect on the mitochondrial expression of HK-1. (E) PI staining shows apoptotic cell percentage among U1 cells treated/untreated with CTM for 8 hours post 72h. (F, G) Differentiated U937 transduced with Adeno Vpr were fractionated into cytoplasm and mitochondrial fractions and the lysates were analyzed by western blot for the expression of HK1. COX IV was used as control for purity and protein loading of mitochondrial fraction & tubulin as cytoplasmic control. * Indicates P value <0.05. 54 Figure 4

Cytoplasm A. Mitochondria Clotrimazole Clotrimazole (microM) 0 10 25 (microM) 0 10 25

102 HK1 102 HK1

17 CoxIV 52 Tubulin

B.

C.

Control PMA PMA+25µM CTZ

52.48% 20.19% 25.14%

79.81% 74.86% 47.26%

Fig.13: Clotrimazole treatment removes mitochondria bound HK and caused mitochondrial de polarization: U1 cells treated with doses of CTM for 8 hours post 72hr of PMA treatment were subjected to cytoplasmic and mitochondria fractionation. (A, B) Dose dependent effect of CTM treatment on subcellular HK expression was analyzed in cytoplasmic and mitochondria fraction by western blot. COX IV was used to normalize mitochondrial fraction and tubulin was used to normalize the cytoplasmic fraction. (C) CTM mediated changes in mitochondrial membrane potential were assessed by the MitoProbe JC-1. Mitochondrial depolarization is indicated by the decrease in red fluorescence in conjunction with a gain in green fluorescence intensity. The changes in red fluorescence are shown in U1 cells treated with CTM 72hour post PMA treatment.

55

Fig.14: PPP blocker reagent induces apoptosis in virus replicating cells by inhibiting HK-1 mitochondrial translocation and blocking G6PD enzyme activity: (A) Effect of DHEA-S treatment on G6PD activity of U1 cell induced with PMA. (B, C) Effect of DHEA-S treatment on G6PD protein expression of U1 cells induced with PMA. U1 cells were treated with 100, 200, 300 mM of DHEA-S for 10 hours and followed by 10nm/ml PMA treatment or control DMSO treatment for 72 hours. (D) PI staining of the cells shows the percentage of apoptotic cells in each condition sample. (E, F) Effect of DHEA-S treatment on total HK-1 level in PMA induced U1 cells. U1 cells were treated with 300 mM of DHEA-S for 10 hours followed by 10nm/ml PMA treatment and subjected to cytoplasmic mitochondrial fractionation. (G, H) Effect of DHEA-S treatment on the mitochondrial HK expression of PMA induced U1 cells. (I) DHEA- S mediated mitochondrial depolarization is indicated by the decrease in red fluorescence in conjunction with a gain in green fluorescence intensity. * Indicates P value <0.05 56 Fig.15: DHEA-S treatment lead to decrease in HIV-1 replication and suppression of HIV-LTR promoter activity by blocking p65 nuclear translocation: U1 cells were treated with 100, 200, 300 mM of DHEA-S for 10 hours and followed by 10nm/ml PMA treatment or control DMSO treatment for 72 hours. (A) p24 ELISA assay showing the amount of viral replication in the different condition post induction with PMA. HIV- LTR transfected in U937 cells were treated with DHEA-S and/or PMA (B) Luciferase assay showing effect of DHEA-S treatment on HIV-LTR activity. (C) Effect of 10 hour DHEA-S treatment on PMA induced nuclear translocation of p65 in U1 cells with Lamin as nuclear loading control. * Indicates P value <0.05.

57

Fig.10: HIV-1 induces HK-1 expression in monocytes/macrophages:

To explore whether HIV-1 infection of monocytes/macrophages cell line U1 modulates expression of the enzymes involved in glucose metabolism, we assessed the levels and activity of HK that predominantly channels phosphorylated glucose in to catabolic pathways to generate ATP, and Gluose-6-phosphate dehydrogenase (G6PD), the first rate limiting enzyme of the pentose phosphate pathway (PPP), a key enzyme in the generation of NADPH and ribulose 5-P (the pre cursor of ribose 5-P, i.e. a critical building block of nucleic acids). Lysates from 6dpi mock infected and HIV-1 infected PBMCs (p24 levels

70.9±10.3 ng/ml) were analyzed for HK enzyme activity and western blot. The data shows that the HK-1 enzyme expression increased with infection but the HK-2 did not increased significantly (A, B, C). HK enzyme activity in infected PBMCs decreases (G).

A similar pattern of HK-1 upregulation was also observed in U1 cells following reactivation of HIV-1 replication upon PMA stimulation (p24 level 8653±652) but no change in HK-2 expression was observed (D, E, F). Total HK enzymatic activity was significantly decreased (H). To further ascertain that PMA treatment of U1 cells did not inhibit total HK activity, U937 (the parent pro-monocytic cell line of U1) cells were treated with PMA in a similar fashion and HK enzyme assay was performed. PMA treatment of U937 cells on the contrary led no significant change in HK enzymatic activity.

Fig.11: HIV-1 induces G6PD expression and activity: To evaluate the effects of HIV-1 expression on G6PD expression and enzymatic activity cell lysates were prepared from

HIV-1 infected PBMCs and PMA activated U1 cells. Assessment of G6PD enzymatic

58 activity and expression demonstrated significant increase both in activity and expressions in PBMCs (A, B, E) and in U1 cells (C, D, F).

Fig.12: HIV-1 increases mitochondria bound HK-1 levels in U1 cells: Since mitochondria bound hexokinase confers protection against cell death (71) we asked the question whether HIV-1 modulates translocation of HK from the cytoplasm to the mitochondria in monocyte/macrophages and thereby regulate survival of HIV-1 infected macrophages. To demonstrate such a phenomenon to be operative in the U1 model of

HIV-1 infection, U1 cells were induced with PMA and harvested after 72 hours when virus production peaks and were subjected to cytoplasmic and mitochondrial fractionation. HK-1 levels in these fractions were increased significantly compared to uninduced cells (A, B) Mitchondria bound HK level increased around 6±1.3 fold. To ascertain the effect of PMA treatment on the over expression of mitochondria HK a control experiment was performed with U937 cell line in the similar fashion as U1. The data shows that PMA treatment of U937 does not lead to increase in mitochondrial HK expression (C, D). VDAC01 was used as mitochondrial marker to show the purity of mitochondrial fraction. As a reciprocal proof of concept, U1 cells were induced by PMA and 72 hours post induction were treated with various CTZ doses or DMSO control for

10 hours, Clotrimazole (CTZ) is a drug that has been shown to cause detachment of mitochondria bound hexokinase. Post treatment the cells were fixed with 70% ethanol and stained with propidium iodide (PI) for apoptotic cell percentages. It was observed that treatment with CTZ without PMA induction of U1 cells leads to significantly higher amount of apoptosis in the cells than treatment of CTZ on PMA-induced cell (E). Ad-Vpr

59 transduced differentiated U937 cells were analyzed for mitochondrial HK expression and data shows that Vpr over expression in U937 lead to increased expression of HK-1 in mitochondrial fraction (F, G).

Fig.13: Clotrimazole treatment removes mitochondria bound HK and caused mitochondrial de polarization: Clotrimazole (CTZ) was used in the previous experiment to show that increase in mitochondria bound HK1 in HIV-1 replicating cells provides anti apoptotic support to the infected cell. Here we show how CTZ removes mitochondria bound HK-1 in U1 cells in a dose dependent manner (A, B). Also JC-1 dye stained U1 cells post CTZ treatment shows mitochondrial depolarization. (C).

Fig.14: PPP blocker reagent induces apoptosis in virus replicating cells by inhibiting

HK-1 mitochondrial translocation and blocking G6PD enzyme activity: U1 cells pretreated with 300 µM of DHEA-S for 10 hours was followed by PMA induction and cells were harvested to analyze enzymatic activity and expression of G6PD. The data shows that DHEA-S pre treatment lead to reduced G6PD enzymatic activity (A) but did not alter G6PD protein expression (B, C). Pretreatment of U1 cells with 100, 200, 300

µM DHEA-S for 10 hours was followed by PMA induction and cells and media harvested at 96 hour post induction showed does dependent increase in apoptosis significantly high only in the PMA induced cells but the cells that were only treated with

DHEA-S for 10 hours and were not induced by PMA for viral replication did not show any significant percentage of apoptotic cell death (D). U1 Cells treated with 300µM U1 were analyzed by western blotting to detect expression of HK-1 and the data showed

60 decrease in HK-1 expression in the cell lysates (E, F). U1 cells similarly treated were fractionated and level of mitochondrial HK-1 was analyzed by western blot the data shows that DHEA-S treated cells had less HK-1 expression in mitochondria and HK-1 expression was normalized with COX IV expression level (G, H). Similarly treated U1 cells were harvested for J.C-1 dye staining as described before and analyzed for mitochondrial depolarization, we found that DHEA-S treatment lead to increase in mitochondrial depolarization (I).

Fig.15: DHEA-S treatment lead to decrease in HIV-1 replication and suppression of

HIV-LTR promoter activity by blocking p65 nuclear translocation: Pretreatment of U1 cells with 100, 200, 300 µM DHEA-S followed by PMA induction and media collection at 72 hour post induction. The media harvested from the PMA induced DHEA-S pretreated U1 cells revealed significant decrease in HIV-1 viral replication by p24 ELISA assay (A). Interestingly the apoptotic cell death percentage between 0 µM and 100 µM

DHEA-S treated PMA-induced cell is not very significant (Fig.14D) however the decrease of viral replication between these two samples is significant. Nuclear fraction of cells treated with DHEA-S 300µM showed reduced nuclear translocation of p65 in response to PMA induction (B, C). U937 cells transfected with HIV-1 LTR Full length when pre treated with DHEA-S showed 6± 2.1 fold less transcriptional activation in response to PMA induction (D).

61 Discussion:

Enveloped viruses rely on cellular machinery and materials to support their own progeny, viruses are known to influence various cellular pathways to obtain genetic material, sustainable macromolecule such as structural proteins, and membrane. It has been shown in various other viruses such as Kaposi’s sarcoma herpes virus (25) , feline leukemia virus (26) , Cytomegalovirus (73) , Herpes virus (27) etc. Hence we decided to look in to the regulation of host cellular glycolytic pathway of HIV-1 infected cells, especially the cells that are known to be serving as viral reservoir in the host body. In our experiments we found that the expression of HK-1 protein, which is also the very first enzyme of the glycolysis pathway, increased with viral replication in U1 cells but the expression of other isoform of HK, the HK-2 protein did not show much significant increase hence we focused on the HK-1 isoform there on. The enzymatic activity of HK-

1 goes down with viral replication. Decreased enzyme activity might imply a decrease in glucose phosphorylation leading to less production of Glucose-6-phosphate. The increased HK-1 protein expression however brings up a question about the utility of this excess HK-1 protein in the infected cell. Since HK-1 is known to play a role in inhibiting apoptosis by binding to mitochondrial membrane and interacting with VDAC (71) . We looked at the subcellular localization of this increased HK-1 in PMA induced U1 cells. It turns out that the subcellular localization of HK-1 changes in U1 cells with viral replication and increased expression of HK-1 is seen in the mitochondrial fraction of cell hence hinting that the viral replication causes HK-1 over expression in mitochondrial membrane of cell to support the mitochondrial health. To find out if this increased expression indeed supports cell survival we treated cells with clotrimazole and the

62 treatment of un induced cells i.e. the ones that did not have increased expression of HK-1 in the mitochondria showed higher percentage of apoptosis post treatment hence proving that the increased HK-1 expression indeed confers protection to virus producing U1 cells.

Further noticed that under the same conditions both the protein expression and enzyme activity of G6PD increases which implies that under the conditions of viral replication in

U1 cells decreased HK-1 activity leads to less production of the metabolite G6P which safeguards the increased HK-1 expression in the mitochondrial membrane of U1 cells since cytosolic G6P can cause disassociation of mitochondria bound HK-1 (74) . The increased activity of G6PD enzyme however suggest a shift towards the pentose phosphate pathway in the infected cell which leads to increased production of various macromolecules such as nucleotides and as a by product of this pathway there is excess production of NADH that protect the cell against the excess ROS produced hence further supporting the health of the viral reservoir cell. More over treatment with DHEA-S the

PPP blocker not only reduces G6PD enzymatic activity it also reduces the amount of virus produced in U1 cells in response to PMA induction. The DHEA-S treated U1 cell once induced by PMA cannot increase HK-1 expression proving again that HK-1 over expression is result of HIV-1 replication so blocking the replication results in inhibition of HK-1 over expression as well. In order to find out if this effect of DHEA-S on viral replication is connected to HIV-1 promoter activity we treated U937 cells with DHEA-S followed by transfection of HIV-1 LTR and PMA induction 24hours post transfection.

We report that DHEA-S treated transfected cells failed to trans activate the LTR and in similarly treated U1 cells p65 nuclear translocation was blocked which could lead to the reduced LTR activity and inhibition of viral replication. All these observations proof the

63 important role HK, G6PD and PPP plays in supporting the sustenance of HIV-1 viral reservoir cells giving us a possible target for intervention and eradication of viral reservoir and replicating cells. However targeting these enzymes globally in host body will have adverse effects, as HK is the very first enzyme in the pathway and targeting HK will cause the entire pathway to collapse. Clotrimazole (CTZ) treatment has been shown to remove HK from the mitochondria of the cells (75) leading to cellular apoptosis.

However we found that CTZ is not selective about its HK removing actions and both un induced and induced U1 cells gets affected with the treatment the un induced once more than the induced once as they do not have the excess accumulation of HK in the mitochondria hence similar treatment of CTZ results in more apoptosis in un induced U1 cells. Hence we looked at the treatment with DHEA-S; DHEA-S has been shown to inhibit the mitochondria binding of HK-1 (76) . The cells such as the infected once supporting HIV-1 replication are heavily dependent on the mitochondria bound HK-1 according to our data; hence DHEA-S treatment should be detrimental to the infected cells as they will loose their important life support system but spare the once that does not depend on this translocation i,e the normal cells. Our data shows exactly that; DHEA-S treatment does not affect the un-induced cells where as it does affect the infected cells by increasing apoptosis in them. Uninduced cells don’t die as much as they don’t relay on solely HK over expression in mitochondria and do not have any lethal stress in their system. Hence DHEA-S treatment can directly target the cells that are producing virus and by destroying them it can stop viral production at the core by taking out its hubs.

Interestingly our data shows that the DHEA-S treatment can also block the viral replication as seen in the p24 level. And this inhibition is not a result of apoptosis of

64 those cells because when compared between the rate of apoptosis with 100 µM DHEA-S treatment with control, there isn’t a significant difference however the viral replication decreases with the same treatment significantly and continues to decrease in a dose dependent manner. It was found in studies in HIV-1 patients that the DHEA-S level significantly reduces in them and the reduction is higher in later stages of disease progression (77) . These observation suggests that HIV-1 virus infections evidently regulates the glycolytic pathway of host and in doing so it has the benefit of controlling the apoptosis of infected cells as these two pathways are interconnected. It is also clear from this study that mitochondria bound HK of infected cells and the dependence of infected cells on PPP can serve as a new target to eliminate virus replicating cells from host system and DHEA-S shows potential to achieve that.

65 CHAPTER 4

GLYCOLYTIC ENZYME PKM2 PLAYS NONMETABOLIC ROLE IN

REGULATION OF HIV-1 LTR TRANSCRIPTION

Introduction

Pyruvate kinase (PK) is an enzyme that catalyzes the final step of glycolysis, transfer of a phosphate group from phosphoenol pyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP. In proliferating cells, PKM2 plays a decisive role in shuttling glucose metabolites either to catabolic or anabolic pathways, controlling nutrient use and energy expenditure. This is particularly relevant to cancer cells, which often prefers the process of aerobic glycolysis. Pyruvate kinase deficiency, due to defects in pyruvate kinase expression or activity, is the second most common cause of hemolytic anemia (63, 78, 79) . PKM2 has long been known to exist in two conformations: a tetramer, which is enzymatically active as a pyruvate kinase, and a dimer, which is not

(80) . PKM2 is capable to nuclear localization in its dimeric form and may act as a transcription regulator, the tetramer form remains in the cytoplasm and carries out its duties as a metabolic enzyme (81-83) . Earlier studies have demonstrated up regulation of

PKM2 in HIV-1 infected macrophages and in HIV-1 infected human astrocytes treated with cocaine (84) ; however, its role in HIV-1 LTR activation remains unknown. Recent studies demonstrate that PKM2 has nonmetabolic functions and plays significant role in regulating gene transcription (62, 81) . PKM2 can function as a dual-specificity protein kinase by phosphorylating Stat3 at Y705 and histone H3 at T11 (81, 85) . Furthermore, phosphorylation of H3-T11 by PKM2 leads to the induction of Myc gene transcription

66 following dissociation of HDAC3 (85) . HDAC3 inhibition leads activation of latent

HIV-1 (86) . Our earlier observation in U1 cells led us to further investigate the role of

PKM2 in HIV-1 replication and primarily its possible effect on the HIV-1 LTR transcription since PKM2 has been shown to be involved in regulation of transcription of its target proteins. Here we report HIV-1 infection induces PKM2 nuclear translocation in infected cells and the nuclear PKM2 can interact with the HIV-LTR and increase its transcriptional activity. We also predict that the interaction site of PKM2 protein and

HIV-LTR over laps with the p65 and LTR interaction sites.

67 Materials and methods:

Cells: U1, U937 cells as previously described (Chapter 2). PBMCs isolated from buffy coat as previously described (Chapter 3). TZMBL cell was obtained from ATCC. The

U1, U937 and PBMCs were cultured as previously described. The TZMBL cells were cultured in DMEM media supplemented with 10% FBS and penicillin streptomycin.

Western blotting: Protein samples (50µg) were separated by gradient SDS-PAGE and then transferred to a nitrocellulose membrane in a blotting chamber (BioRad) at 100V for

30 min. The membrane was blocked with 5% powdered milk in Tris-buffer saline solution (pH 7.6) containing 0.05% Tween-20 (TBS/T) then probed with antibodies that had been diluted 1:500 (anti PKM2, tubulin, Grab2) antibodies from Cell signaling).

Membranes were incubated with primary antibodies overnight at 4C, washed, and then incubated with appropriate secondary antibodies conjugated with Licor IR dyes at room temperature for 1h. The membranes were then analyzed with digital imaging in Licor oddessy machine.

Enzymatic activity: The Pyruvate Kinase Activity Assay Kit from Bio vision (#K709-

100) was used as per manufacturer’s protocol; the kit provides a simple and direct procedure for measuring pyruvate kinase activity. Pyruvate concentration is determined by a coupled enzyme assay, which results in a colorimetric (570 nm) product, proportional to the pyruvate present.

Plasmids: PKM2 expression plasmid was cloned in pCDNA3 vector and provided by Dr.

Prasun Datta. HIV-LTR plasmid was previously constructed in our laboratory as described in (87) . CMV-p65 plasmid was previously constructed in our laboratory as described in (88) .

68 Transfection and Reporter assay: Cells were transfected with 0.1 µg of reporter plasmid

HIV LTR-Luc /full length or deletion mutant or co-transfected with 0.3/0.5 µg of PKM2 expression cDNA. The amount of DNA used for each transfection was normalized with pcDNA3 vector plasmid. Cell extracts were prepared 24 h after transfection, and

Luciferase assay was performed as per manufacturers protocol (Promega, Madison, WI).

Cytoplasmic nuclear fractionation: Performed on cell pellets according to manufacturers protocol (Thermo scientific 78833).

HIV-1 infection: PBMCs were infected with HIV-1SF162 and infected cell population was harvested at Day 3 and Day 6 post infection. Supernatant from the infected cells were collected to analyze viral replication status by p24 ELISA.

Chromatin Immunoprecipitation (ChIP) Assay: TZMBL cells were grown overnight in

100-mm dishes to 60–70% confluency; cells were then treated with 10nm/ml PMA.

Plates were returned to the incubator for 4 hours. Cells were cross-linked with formaldehyde and harvested, and ChIP was performed. For these studies, only 5 × 106 cells were used per immune-precipitation reaction because the HIV-LTR present at a high copy number. The remainder of the procedure followed standard protocols for ChIP analysis, as recommended by the manufacturer (Upstate Biotechnology, Inc.). The resulting DNA was analyzed by PCR using the following primers:

TZMBL: FWD: 5’-TAGAGTGGAGGTTTGACAGCCG-3’ REV: 5’-GTACAGGCAAAAAGCAGCTGCT-3’

69 U1: FWD: 5’-TTAGCAGAACTACACACCAGGGCC-3’ REV: 5’-CCGAGAGCTCCCAGGCTCAGATCT-3’

Antibody used in the ChIP procedure was against PKM2 and p65 (purchased from Cell signaling) as well as rabbit anti-mouse IgG provided by Paul Pozniak. All ChIP assays were performed twice.

70 Results:

Fig.16: PKM2 protein expression increases with HIV-1 replication in both whole cell and nuclear fraction. (A, B) PKM2 protein expression increases in U1 cells as show before (Fig 4,Chapter 2). (C, D) Expression of nuclear PKM2 increases in U1 cells post induction of viral replication with PMA. (E, F) Expression of PKM2 increases in PBMCs infected with HIVSF162. Nuclear expression of PKM2 increases with HIV-1 infection in PBMCs. 71 A

B C LTR -456/+66 LTR-156/+66 10 14 12 8 10 6 8

4 6 4 TR Fold Change TR Fold change L

2 L 2 0 0 Control PKM2 Control PKM2

D E LTR-120/+66 16 14 12 10 8 6

TR Fold Change 4 L 2 0 Control PKM2

Fig.17: PKM2 protein over expression induces HIV-LTR activity: (A) Schematic representation of HIV-LTR deletion mutants. Mapping of HIV-LTR section most responsive to PKM2 over expression. Effect of PKM2 over expression in (B) LTR 456/+66. (C) LTR-156/+66, (D) LTR-120/+66, (E) LTR-80/+66

72

Fig.18. PKM2 dependent HIV-LTR transcriptional activation involves the p65- binding site of LTR: (A) HIV-LTR promoter activity under over expression of PKM2, p65, and PKM2-p65 co expression. (B) HIV-LTR mutant with deleted NFκB binding site promoter activity under PKM2, p65 and Tat over expression.

73 Fig.19. PKM2 binds to HIV-LTR (A) ChIP with PKM2 and p65 in TZMBL cells using HIV-LTR primer -201/+3. (B) ChIP with PKM2 and p65 in U1 cells using HIV-LTR primer -374/+43.

74 Fig. 16: PKM2 protein expression increases with HIV-1 replication in both whole cell and nuclear fraction: U1 cells were treated with PMA as described in Chapter 2, as seen before PMA induced HIV-1 replication increased whole cell PKM2 enzyme expression about 1.5 fold and the nuclear expression of PKM2 also increased at a similar amount.

However in PBMCs infected with HIV-1SF162. Showed about 2.66±0.85-fold increase in

PKM2 expression and Nuclear PKM2 expression increases by 3±0.4 fold.

Fig.17: PKM2 dependent HIV-LTR transcriptional activation involves the p65-binding site of LTR: U87MG cells were transfected with HIV-LTR full length (-476/+66) or deletion mutants LTR-156/+66, LTR-120/+66, LTR-80/+66 and PKM2 expression plasmid (300ng). The promoter activity was compared with basal level of activity of each promoter construct transfected with empty vector pCDNA3. (17.B) The data shows that

PKM2 over expression induces HIV-LTR-476/+66 promoter reporter activity by 6.4±2.3 folds, -156/+66 promoter activity increases by 10.7±1.6 fold and -120/+66 promoter activity by 11.3±2.6 folds. The -80/+66 Promoter activity increased only 1.2±0.5 fold.

Fig.18: PKM2 dependent HIV-LTR transcriptional activation involves the p65-binding site of LTR: U87MG cells were transfected with 50ng of LTR-120/+66, the minimal promoter required for PKM2 mediated LTR transactivation along with PKM2 (300ng), empty plasmid, p65 and combinations of 100ng- p65 with 300ng of PKM2. The total amount of DNA used in each transfection reaction was normalized by empty vector

DNA. The promoter activity was compared with basal level activity of promoter transfected with PcDNA3 alone. The data showed that (Fig.18, A) PKM2 lead to 8±1.9

75 fold activation and p65 lead to a 13.6+_2.7 fold activation of the LTR, when co transfection of 100 ng p65 and 300 ng of PKM2 reduced the fold activity to 8.1±2.3 fold.

Showing competitive effect on the promoter. Next the HIV-LTR promoter mutant with the deletion of the three NFκB binding sites at the LTR was transfected in to U87MG cells and those cells were then transfected with PKM2, p65, Tat101 expression plasmid and reporter luciferase assay was performed. The data showed that (B) neither PKM2 nor p65 could lead to the transactivation of HIV-1 LTR mutant, only Tat 101 lead to a

592±21 fold increase.

Fig 19. PKM2 protein binds to the HIV-LTR: TZMBL and U1 cells both carrying copy of HIV-1 LTR were used to confirm if there is any protein DNA interaction between the

HIV-1 LTR and PKM2. The data shows that in both cell line tested PKM2 protein does interact with HIV-LTR (19. A, C). p65 being a known LTR binding protein was used as a positive control for the assay. To ensure adequate amount to PKM2 presence in the nucleus of the cell PMA treatment was done in both the cell line 4hr (TZMBL) prior and

24hr (Prior) to harvesting. The western blotting data confirms the induction of nuclear

PKM2 with PMA treatment.

76 Discussion:

PKM2 adds a fascinating new twist in ever evolving metabolic story. The double-edged sword of metabolism and transcription regulation is what we pursued in this study. The observed increase in PKM2 protein expression in both U1 cells (induced) and PBMCs infected with HIV-1 leads to the obvious question of its attendant downstream effects in viral replication.. In the previous chapter we have seen how viral infection mediated changes in glycolytic pathway can be beneficial to the virus in terms of supporting the longevity of the infected macrophages. In this chapter however we focused our attention to elucidate the role of increased PKM2 protein expression on HIV-1 LTR activation.

The rationale for this aim is based on the recent demonstration that PKM2 regulates gene transcription by acting as a protein kinase (78, 82). In tune with our hypothesis we found that induction of viral replication increases nuclear levels of PKM2 in the infected cells.

In our subsequent studies we show that the PKM2 over expression lead to LTR transactivation. To delineate the region in the HIV-1 LTR that regulates PKM2 mediated transactivation we performed mapping experiments using deletion constructs of HIV-

LTR. Our results show that PKM2 over expression induces the -476/+66, the -156/+66, and the -120/+66 LTR-luciferase constructs. But the construct spanning -80/+66 did not show significant upregulation in response to PKM2 over expression. These observations, suggests that most likely the NFκB binding site is involved in the regulation of the LTR by PKM2 since the -80/+66 construct that lacks the NFκB binding sites but still has the

Sp1 binding sites did not show significant upregulation. The studies also suggest that p65 and PKM2 could be binding partners and have an additive effect on the LTR, or p65 and PKM2 could compete for the same binding site in the LTR and have a competitive

77 effect on the LTR when expressed together. To further ascertain the implication of this observation co-transfection of p65 and PKM2 was performed to assess effects on LTR transactivation. Co-expression of PKM2 and p65 decreased the LTR activity when compared to p65 alone illustrating competition for binding on LTR. We also used a LTR construct with deletion of the NFκB binding sites in a transfection experiment which showed that neither p65 nor PKM2 could trans-activate this construct indicating the importance of NFκB binding site in PKM2 mediated upregulation of HIV-1 LTR.

Finally, we ascertained by ChIP analysis the interaction of PKM2 with HIV-LTR in

TZMBL and U1 cells that contain copies of HIV-LTR integrated in their genome. PMA was used to induce nuclear translocation of PKM2. The results confirmed physical interaction of PKM2 and HIV-LTR in both cell lines. This is a significant observation since we report for the first time the interaction between a component of glycolytic pathway and HIV-LTR in regulation of HIV-1 transcription. Future studies need to be performed to decipher if the protein DNA interaction is dependent on any posttranslational modification of the PKM2. PKM2 is already known to be regulated by posttranslational modifications such as phosphorylation, acetylation, sumolyation etc.

(86-89) . Since p300/CBP enhances NF-κB transcriptional activity by acetylating both

NF-κB/p65 and surrounding histones (38, 90), future studies will help understand if posttranslational modification of the PKM2 protein effects NFkB’s interaction with HIV-

LTR.

78 CHAPTER 5

CONCLUSION SIGNIFICANCE AND FUTURE DIRECTIONS

The role of glucose metabolism has not been that widely studied in terms of HIV-1 infection and pathogenesis. In recent past there has been a few exception to that trend due to the appearance of aggravated metabolic disorder in patients living longer with AIDS in the era of antiretroviral therapy (91-93) Based on our earlier studies demonstrating involvement of HIF-1α (57) in the pathogenesis of NeuroAIDS we hypothesized that

Vpr in a HIF-1a dependent manner might be involved in glucose metabolism of HIV-1 infected macrophages. Our studies show that HIV-1 infection lead to significant changes in the glycolytic pathway of monocyte/ macrophage lineage infected cells by altering the enzymatic activity and protein expression of various glycolytic components. We have also performed proteomic studies and found that viral protein Vpr is directly involved in modulating changes in the expression of enzymes involved in glycolysis. Our studies focused on monocyte macrophages to elucidate if the modulation of certain glycolytic components such as HK and G6PD, that are also shown to be involved in regulating cell survival and apoptosis in other pathogenic conditions were regulated by HIV-1 infection/replication. We report that HK enzyme activity decreases with HIV-1 replication in PBMCs where as its expression increases. Similar effect is seen with induction of HIV-1 replication with PMA in latently infected monocyte cell line, U1.

Similar increase was also seen with the expression and activity of G6PD in U1 cells. We also found that HK-1 translocate to the mitochondria of U1 cells post induction of HIV-1 replication. It is known that Glucose 6-phosphate releases HK1 from the OMM. Hence,

79 we conclude that HIV-1 infection decreases HK activity to reduce G-6-P levels, but increase G6PD enzyme activity to ensure rapid consumption of G-6-P. This, phenomena supports the interaction of HK1 with OMM to ensure survival of infected cells. We further show that inhibition of the pentose phosphate pathway (PPP) has detrimental effect on both infected cell survival and viral replication. The observation that DHEA-S

(PPP blocker) inhibits viral replication is corroborated by our findings that DHEA-S inhibits LTR activity by inhibiting p65 activity (94). Earlier studies involving DHEA and

HIV-1 have shown down regulation of HIV-1 replication and reduced re-activation of latent HIV-1 reservoirs (95, 96) but our studies illustrate the possible mechanism in terms of reduced NFκB activation and blocking of PPP also inhibits macromolecule synthesis thereby modulating HIV-1 biogenesis. Identification of cellular proteins in addition to already known transcription factors such as NFkB, SP1, and CEBP that interact with the

HIV-1 LTR is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression in HIV-1JRFL infected PBMCs and during reactivation of

HIV-1 in chronically infected U1 cells. Furthermore, we observed that infection of

PBMCs with HIV-1 and reactivation of HIV-1 in U1 cells results in increased levels of nuclear PKM2. Hence we focused on understanding the potential role of PKM2 on HIV-1

LTR transactivation. Our studies demonstrate that over expression of PKM2 in U937 and in TZM-bl cells lead to transactivation of the HIV-1 LTR reporter construct. Using various deletions constructs of HIV-1 LTR, we mapped the region spanning between -

120 bp to -80 bp to be essential for PKM2 mediated transactivation. This region contains the NFkB DNA binding site and mutation of NFkB binding site attenuated PKM2

80 mediated transactivation of HIV-LTR. Chromatin immune-precipitation (ChIP) analysis in PMA activated TZM-bl cells and U1 cells demonstrated interaction of PKM2 with

HIV-1 LTR. Our studies suggest that PKM2 is a transcriptional co-activator of HIV-1

LTR, which illustrates yet another possible target to curb HIV-1 replication at the transcriptional level. This idea of establishing links between regulation of cellular glucose metabolism and HIV-1 infection opens up a new perspective for targeting the disease and possibly curbing HIV-1 replication at the promoter level utilizing metabolic therapeutic molecules. More studies need to be conducted to identify various epigenetic and posttranslational modification of the glycolytic enzymes post HIV-1 infection to conclude exactly how viral infection regulates these glycolytic components. Finally, various small molecules, biological inhibitor and their chemical analogues can be brought about to be a part of HIV-1 treatment regime once the exact functionality of each of the glycolytic components in terms of disease pathogenesis is established.

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