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Regulation of Cellular Glucose Metabolism by Hiv-1 Infection

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Regulation of Cellular Glucose Metabolism by Hiv-1 Infection 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
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