(HBV) Infection of the Liver Ahmed Mohamed Abdel-B Diab Purdue University

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January 2016 The oler of PLK1 in Hepatitis B Virus (HBV) infection of the liver Ahmed Mohamed Abdel-B Diab Purdue University

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This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated 12/26/2015

PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance

This is to certify that the thesis/dissertation prepared

By Ahmed Mohamed Abdel-B Diab

Entitled The role of PLK1 in Hepatitis B Virus (HBV) Infection of the Liver

For the degree of Doctor of Philosophy

Is approved by the final examining committee:

Ourania Andrisani Andy W. Tao Co-chair Fabien Zoulim

Co-chair Robert Geahlen

Xiaoqi Liu

To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material.

Approved by Major Professor(s): Ourania Andrisani

Laurie Jaeger 10/6/2016 Approved by: Head of the Departmental Graduate Program Date

THE ROLE OF PLK1 IN HEPATITIS B VIRUS (HBV) INFECTION OF THE LIVER

A Dissertation

Submitted to the Faculty

Purdue University

Ahmed M Diab

In Partial Fulfillment of the

Requirements for the Degree

Doctor of Philosophy

December 2016

Purdue University

West Lafayette, Indiana

ACKNOWLEDGEMENTS

I would like to begin by extending my gratitude to my committee members Robert

Geahlen, Andy W. Tao and Xiaoqi Liu as well as committee co-chairs Ourania Andrisani and Fabien Zoulim for accepting to read, evaluate and thoughtfully critique my work. I also thank them for their confidence, continuous support and mentorship throughout my stay at Purdue.

I would like to express my sincerest gratitude to my mentor and adviser Ourania

Andrisani for her exceptional mentorship and the tremendous support she provided throughout the years. Her dedication to her work was inspiration, attention to detail and perseverance was inspirational. I appreciate her relentless encouragement, being readily available for scientific, intellectual and emotional support as well as her honest and constructive advice.

I would like also to thank Fabien Zoulim and David Durantel for cordially granting me the opportunity to work with them and for their continuous professional and scientific support. My stay at INSERM Lyon was extremely beneficial for my scientific and personal development and it would have not been possible without their support. I will be utterly grateful for the expertise and experience I gained under their mentorship.

iii

I owe very special thanks to Ronald Hullinger for his outstanding mentorship, continuous support and the intellectual encounters. His wittiness, hospitability and exceptional culinary skills made my stay at Purdue memorable.

I would like also thank my colleagues at the Andrisani lab, Saravana Kumar Mani, Hao

Zhang, Huitao Fan, Zhibin Cui, Dante Johnson, Ellen Weigel, Zuo Ding and Li Ma. I would like to thank Adrien Foca, Nathalie Isorce, Dulce Alfaiate, Pascal Jalaguier, Suzanne

Faure-Dupuy, Maëlle Locatelli, Valentina D’Arienzo and Olivier Hantz at INSERM Lyon. I would like to particularly thanks Adrien Foca for his tremendous help to complete my work on the PLK1 project in Lyon. It has been a pleasure to work with him.

I am also obligated to thank my colleagues at Purdue Wen-Hung Want, I-Hsuan (Blair)

Chen, Meng-Ju Wu, Anton Iliuk, Mariya Krisenko, Lama Alabdi and Cindy Xing for their help. I particularly thank I-Hsuan chen for her help with the mass spectrometry work.

I thank François-Loïc Cosset and Floriane Fusil for the work on the humanized-liver mice and their help extending my findings to in vivo models.

Finally, I would like to thank my friends at Purdue who made Lafayette a second home and my family for their belief in me and their continuous encouragement.

TABLE OF CONTENTS

Page

LIST OF TABLES ...... ix LIST OF FIGURES ...... x LIST OF ABBREVIATIONS ...... xii CHAPTER 1. INTRODUCTION ...... 1 1.1 Hepatitis B...... 1 1.1.1 Pathogenesis ...... 1 1.1.2 Carcinogenesis ...... 2 1.1.3 Treatment and prevention...... 3 1.2 Hepatitis B virus ...... 3 1.2.1 Genome organization and structure ...... 3 1.2.2 HBV Life cycle ...... 4 1.3 References: ...... 5 1.4 Figures: ...... 7 CHAPTER 2. HEPATITIS B VIRUS (HBV) CORE ANTIGEN (HBC): FUNCTION IN VIRUS BIOSYNTHESIS AND HOST GENE REGULATION ...... 9 2.1 Introduction ...... 9 2.2 HBc structure ...... 10 2.3 HBc, a nucleic acid chaperone ...... 12 2.4 HBc localization ...... 13 2.5 HBc phosphorylation ...... 16 2.6 HBc and interaction with host factors ...... 19

Page

2.7 HBc as a regulator of transcription ...... 22 2.8 Unanswered questions and future directions ...... 24 2.9 References ...... 26 2.10 Figures...... 39 CHAPTER 3. MATERIALS AND METHODS ...... 40 3.1 Cellular models ...... 40 3.1.1 HepaRG cell lines...... 40 3.1.1.1 HepaRG cell culture: ...... 40 3.1.1.2 HepaRG-TR-PLK1 cell lines: ...... 41 3.1.1.3 Infection in HepaRG cells and Primary Human Hepatocytes ...... 41 3.1.2 HepG2-NTCP cell line and infection ...... 42 3.2 Production of HBV inoculum in HepG2.2.15 cells: ...... 43 3.2.1 Materials ...... 43 3.2.2 Protocol ...... 43 3.3 Tables ...... 46 3.4 References: ...... 47 CHAPTER 4. POLO-LIKE-KINASE 1 IS A PROVIRAL HOST-FACTOR FOR HEPATITIS B VIRUS REPLICATION: THERAPUTIC IMPLICATIONS ...... 48 4.1 Abstract...... 48 4.2 Introduction ...... 49 4.3 Results ...... 52 4.3.1 PLK1 is activated by HBV infection in non-dividing/differentiated hepatocytes ...... 52 4.3.2 PLK1 inhibitors, including BI 2536, suppress HBV DNA accumulation in persistently HBV-infected hepatocytes ...... 53 4.3.3 Loss-of-function of PLK1 suppresses HBV DNA accumulation in infected hepatocytes ...... 54

Page

4.3.4 A modest gain-of-function of PLK1 is associated with an increased HBV replication ...... 55 4.3.5 Reduction of HBV viremia in liver-humanized infected FRG mice treated with BI 2536 ...... 55 4.3.6 Effect of the combination of BI 2536 with approved or investigational anti- HBV molecules ...... 56 4.3.7 HBc is a substrate for PLK1 in vitro ...... 57 4.3.8 S/A substitution of in vitro PLK1 phosphorylation sites diminish HBV replication ...... 58 4.4 Discussion ...... 59 4.5 Materials and Methods ...... 62 4.5.1 Chemicals, antibodies, and others reagents ...... 62 4.5.2 HepaRG cells and primary human hepatocytes & infection by HBV ...... 63 4.5.3 HBV infection and analysis of viral parameters during replication ...... 63 4.5.4 Transfections, plasmids and siRNAs ...... 64 4.5.5 Humanized FRG mouse model and HBV infection ...... 64 4.5.6 In vitro PLK1 kinase assays ...... 65 4.5.7 Trans-complementation assay ...... 66 4.5.8 Statistical analysis ...... 66 4.6 References ...... 67 4.7 Acknowledgments ...... 71 4.8 Figure legends ...... 72 Figure 1: HBV infection activates PLK1...... 72 Figure 2: Effect of BI 2536 on HBV replication in non-dividing and non-transformed dHepaRG...... 73 Figure 3: Effect of BI 2536 on HBV replication in non-dividing and non-transformed PHH...... 73

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Figure 4: Effect of PLK1 knockdown on HBV replication in dHepaRG and PHH...... 74 Figure 5: Effect of BI 2536 on HBV replication in liver humanized HBV-infected FRG mice...... 74 Figure 6: Combination treatment of BI 2536 and tenofovir, IFN-a, or Bay41-4109. 75 Figure 7: HBc is a phosphorylation substrate of PLK1 in vitro...... 75 4.9 Supplementary figure legends...... 76 Supplementary Figure 1: Dose response effect of competitive and non-competitive PLK1 inhibitors on HBV replication in non-dividing hepatocytes...... 76 Supplementary Figure 2: Comparison of batches of BI 2536...... 76 Supplementary Figure 3: Effect of BI 2536 on establishment of HBV infection in dHepaRG...... 76 Supplementary Figure 4: Limited over-expression of PLK1 leads to an increase of HBV replication in dHepaRG...... 77 Supplementary Figure 5: Effect of IFN-α and entecavir on HBV replication in liver humanized HBV-infected FRG mice...... 77 4.10 Figures...... 79 CHAPTER 5. HOST CELL INTERACTOME OF HBV CORE PROTEIN REVEALS A NOVEL LINK TO PARASPECKLE COMPONENTS ...... 90 5.1 Introduction ...... 90 5.2 Results ...... 93 5.2.1 Identification of HBc interacting proteins by AP-MS ...... 93 5.2.2 An overlap between interactomes of HBV core protein and HIV-1 Rev protein ……………………………………………………………………………………………………………….94 5.2.3 STRING network analysis reveals paraspeckle proteins as HBc interacting partners ……………………………………………………………………………………………………………….94 5.2.4 HBc associates with lncRNAs NEAT1 and NEAT2 ...... 95 5.2.5 HBc binds lncRNAs NEAT1 and NEAT2 during HBV replication & infection 96

viii

Page

5.2.6 Phosphorylation status of HBc regulates interaction with lncRNAs...... 96 5.3 Discussion ...... 97 5.4 Materials and Methods ...... 99 5.4.1 Chemicals, antibodies, and others reagents ...... 99 5.4.2 Cell culture ...... 100 5.4.3 Affinity purification and Protein Digestion ...... 100 5.4.4 LC-MS/MS Analysis ...... 101 5.4.5 Data analysis ...... 101 5.4.6 Bioinformatics ...... 102 5.4.7 Ribonucleoprotein Immunoprecipitation (RNP-IP) ...... 102 5.5 References ...... 104 5.6 Figure Legends ...... 111 5.7 Figures...... 113 5.1 Tables ...... 119 CHAPTER 6. CONCLUSION & FUTURE DIRECTIONS ...... 154 6.1 References ...... 157 VITA……………………………………………………………………………………………………………………………158

LIST OF TABLES

Table ...... Page 3.1 Calculating MOI 100 for a single well in a 12 well plate ...... 46 5.1 NEAT1 and NEAT2 interacting proteins that are part of HBc interactome ...... 119 Appendix Table 5.2 HBc interactome raw table ...... 120

LIST OF FIGURES

Figure ...... Page 1.1 HBV life cycle ...... 8 2.1 Subcellular Localization motifs in HBc CTD ...... 39 2.2 Schematic of the HBc CTD ...... 39 4.1 Activation of PLK1 upon HBV infection ...... 79 4.1S Dose response effects of PLK1 inhibitors on HBV replication ...... 80 4.2 Effect of BI 2536 on HBV replication in non dividing and non transformed hepatocytes ...... 81 4.3 Effect of BI 2536 on HBV replication in non dividing and non transformed PHH ...... 82 4.3S Effect of BI 2536 on the establishment of HBV infection in HepaRG cells ...... 83 4.4S Expression of PLK1 in HepaRG cells leads to an increase in HBV replication ...... 83 4.5 Effect of BI 2536 on HBV replication in liver-humanized HBV infected mice ...... 84 4.5S : Effect of IFN-a and entecavir on HBV replication in liver-humanised HBV-infected FRG mice ...... 85 4.6S Monitoring of Weight, human serum albumin and ALAT during B1 2536 treatment in liver-humanised HBV-infected FRG mice ...... 86 4.6 Combination between BI 2536 and tenofovir, IFN-a, or Bay41-4109 (CpAM) ...... 87 4.7 HBc is a phosphorylation substrate for PLK1 in vitro ...... 88 4.7S Trans-complementation assay for Plk1 phosphorylation sites ...... 89 5.1 Interactome analysis identifies known and novel HBc interacting partners ...... 113 5.2 Overlap between HIV-1 Rev and HBc interactomes ...... 113 5.3 Global map of HBc interactome ...... 114 5.4 HBc binds lncRNAs NEAT1 and NEAT2 ...... 115

Figure Page 5.5 HBc binds lncRNAs NEAT1 and NEAT2 in HBV replication and infection ...... 116 5.6 HBV infection does not induce NEAT1 and NEAT2 ...... 117 5.7 HBc mutants bind lncRNAs NEAT1 and NEAT2 ...... 118

xii

LIST OF ABBREVIATIONS

HBV-Hepatitis B Virus

HBs-HBV S antigen

HCC-Hepatocellular Carcinoma

HBc-HBV core antigen

NTCP-sodium taurocholate cotransporting polypeptide cccDNA-covalently closed circular DNA pgRNA-pregenomic RNA

HBx-HBV X protein

CTD-C-terminal domain rcDNA-relaxed circular DNA

SRPK1-Serine/Arginine Protein Kinase 1

NLS-nuclear localization sequence

CRS-cytoplasmic retention signals

HSV-1-herpes simplex virus type 1

EBV-Epstein Barr virus

PKC-protein kinase C

PLK1-Polo-like-kinase1

xiii lncRNA-long non-coding RNAs

PHH-Primary Human Hepatocytes dHepaRG-differentiated HepaRG cells

WHO-World Health Organization

CHB-Chronic Hepatitis B

CDK2-Cyclin Dependent Kinase 2

SP-Serine/proline

DP-Serine/ aspartic acid

AP-Serine/alanine

S/T-serine/threonine

SUZ12-Suppressor of Zeste 12

ZNF198-Zinc Finger, MYM-Type2 p.i.-post-infection d.p.i- days post infection

HBs-HBV S antigen

HBe-HBV E Antigen

EC50 -Effective Concentration 50%

CC50 -Cytotoxic concentration 50% siRNA-small interfering ribonucleic acid

HCV- Hepatitis C virus

HepaRG-TR-PLK1CA -HepaRG- tetracycline regulated-PLK1 constitutively active vge- virus genome equivalents

xiv

DMSO- Dimethylsufoxide qPCR- quantitative Polymerase Chain Reaction

RTqPCR- Reverse transcription quantitative Polymerase Chain Reaction

ETV- entecavir

IFN-α- Interferon –α

FRG- Fah-/-/Rag2-/-/Il2rg-/-

CpAM- Core protein Allosteric Modulators

A- alanine

D- aspartic acid

WCE- Whole Cell Extract

MAPK- Mitogen Activated Protein Kinase

HTA- Host Targeting Agent

DAA- Direct Acting Agent

RNAi- Ribonucleic Acid interference

PIV5- parainfluenza virus 5

NS5A- Nonstructural

ABSTRACT

Diab Ahmed M. Ph.D., Purdue University, December 2016. Molecular Regulatory Mechanisms In Hepatitis B Virus Pathogenesis. Major Professor: Ourania Andrisani

With 250 million reported chronic infections globally, Hepatitis B Virus (HBV) is a major human health issue, linked to increased risk for development of hepatocellular carcinoma (HCC). Current treatments to control chronic HBV infection remain ineffective. New and effective therapies that target the persisting viral molecules are needed in order to clear infection.

My research aims to understand the role of hepatitis B virus (HBV) in inducing HCC and identify novel host molecules that are targeted by HBV. Understanding how HBV modifies and manipulates host cellular pathways is critical for the development of mechanism based therapeutics.

Here, I provide a review for the HBV core protein (HBc), its function and the host molecules it usurps during infection and pathogenesis. I also identify Polo-like-kinase 1

(PLK1) as a proviral factor in HBV pathogenesis. I demonstrate by loss of function as well as gain of function approaches that PLK1 inhibition suppresses viral replication both in vitro and in vivo. I also show that HBc is a phosphorylation substrate for PLK1 in vitro and I mapped the PLK1 phosphorylation sites to HBc residues S168, S176 and S178.

Finally, I used a global proteomics approach to study human HBc interactome identifying

xvi potential interacting partners with interesting links to viral pathogenesis. Collectively, my research expands our understanding of the role of HBc in HBV pathogenesis and the ensuing transformation of the liver.

CHAPTER 1. INTRODUCTION

1.1 Hepatitis B

The seminal discovery of the Hepatitis B virus by the Nobel Prize winner Baruch

Blumberg in 1965 marks the birth of the field of HBV virology 1. We have since witnessed major breakthroughs in the study of HBV epidemiology, diagnosis, pathogenesis, oncogenicity and treatment. These efforts have culminated in the development of the HBV vaccine in 1969 by Blumberg and his colleagues which granted them a well-deserved Nobel Prize in medicine. Soon afterwards, Dane and his colleagues obtained the first electron microscope images of Dane particles, a 42 nm infectious viral particles with a distinct inner core 2. Yet with over 5 decades of breakthroughs in HBV research, chronic HBV infection claims over 600,000 death annually from the ensuing cirrhosis or hepatocellular carcinoma 3.

1.1.1 Pathogenesis

Despite these original discoveries, the mechanisms for establishment and maintenance of HBV infection remain poorly understood. HBV is one of the most efficacious human pathogens having infected over 2 billion people 4. This is due to the unique genomic organization of HBV which allows the virus to ‘disguise’ as a minichromosome in the nuclei of infected hepatocytes. This allows HBV to evade the

2 host’s innate immune response 4. Additionally, the ability of HBV to hijack the cellular transcription machinery to replicate efficiently enables the virus to sustain chronic infection. HBV is not directly cytopathic and the ensuing liver damage is a side effect of the repeated-yet inefficient- attempts of the host’s immunity to suppress the chronic infection 5. Based on clinical markers such as serum levels of HBV DNA and HBsAg, chronic carriers of HBV are classified into different phases. While the natural history varies among chronic carriers, the different stages/phases of infection usually start with an immune-tolerant phase, followed by an immune-reactive phase, immune-control and finally immune-escape 5. These phases do not necessarily occur sequentially. Factors such as patient’s age, gender and immune status collectively dictate if infection would be cleared or if it will chronically persist leading to liver fibrosis, cirrhosis and eventually hepatocellular carcinoma (HCC).

1.1.2 Carcinogenesis

HCC is a major disease ranking 5th among males and 7th among females overall cancer 6. Chronic HBV infection has been long described as a major oncogenic agent in liver cancer pathogenesis. While the oncogenic role of HBV remains unclear, the combination of chronic liver inflammation, apoptosis and regeneration, the accumulation of genetic and epigenetic alterations is thought to induce transformation

7,8. Additionally, HBV DNA integrations into host DNA and the oncogenic co-factor X protein provide more direct effectors of transformation 7. While the vaccine against HBV is efficient, an estimated 250 million people are chronically infected with HBV -according

3 to the World Health Organization, with the risk of developing HCC. New and effective therapies targeting chronic HBV infection are needed.

1.1.3 Treatment and prevention

Current anti-HBV therapies are not sufficient to control chronic infection thus the risk for cancer development remains high. Successful and sustained suppression of viral replication is essential in order to prevent disease progression and prolong patient survival 9. The last decade has witnessed major advances in HBV therapy with the introduction of nucleoside analogs as well as interferon-alpha as anti-HBV therapeutics.

Limitations to the available treatments arise from the side effects associated with prolonged interferon treatment and the resistance developed against nucleoside analogs in long-term therapy 10,11. Such limitations drive the ongoing research efforts to develop novel therapeutics with more sustained suppression of viral replication and enhanced viral clearance. Understanding the molecular mechanisms of HBV replication is therefore essential for successful treatment. The core protein stands as an important target for the next generation of antivirals as it plays multiple roles in the viral life cycle.

1.2 Hepatitis B virus

1.2.1 Genome organization and structure

Like other hepadnavirus viruses, HBV is a small encapsidated DNA containing virus with a 3.2 kb partially double stranded genome. HBV has a relatively small genome with only 4 open reading frames that encode 7 viral proteins. This simplicity of the virion is argued to correlate with complexity of the functions of the different viral proteins,

4 several of which have been shown to have pleiotropic roles in the life cycle of the virus

12,13. The compact nature of HBV also dictates that HBV would adapt to its host’s environment by hijacking or mimicking host proteins in order for it to efficiently complete its life cycle as well as to evade the immune system. Such mechanisms of molecular mimicry and deregulation are common to other viruses 5,14.

The compact genome of HBV displays a certain degree of complexity with 4 overlapping open reading frames, a poly-adynelation signal, an encapsidation signal, 2 enhancers and 4 promoters all tightly regulating viral transcription and replication 15. Of the 4 open reading frames of HBV: one encodes the viral polymerase, the preS/S encodes the 3 different envelope proteins, the preC/C encodes the core (HBc) and e antigens, and finally the smallest one encodes the regulatory X protein 15.

1.2.2 HBV Life cycle

HBV is a hepatotropic virus that specifically infects liver cells 8. Entry into hepatocytes is mediated by the binding of the viral envelope proteins to the human sodium taurocholate cotransporting polypeptide (NTCP) receptor (Fig. 1) 16. In the cytoplasm of infected hepatocytes, nucleocapsids are released and the HBV genome is transferred to the nucleus. Once inside the host cell nucleus, the viral genome is converted into covalently closed circular DNA (cccDNA) which serves as a template for the transcription of viral RNAs 15. Inside the nucleus of infected cells, the cccDNA associates with nucleosomes and assumes chromatin structure 17. Reverse transcription from the cccDNA results in 4 viral transcripts all of which share a single polyadenylation

5 site. Pre-genomic RNA (pgRNA) is the largest RNA species transcribed from cccDNA. It encodes for all viral proteins by alternative splicing. pgRNA also serves as template for reverse transcription of the viral genome. The other viral transcripts encode three viral surface proteins, and the small regulatory X protein. HBV replicates through reverse transcription 12. Viral reverse transcription takes place in the cytoplasm within the viral capsids, a process mediated by the virus-encoded reverse transcriptase in the presence of the core protein 15.

1.3 References

1. Blumberg BS. The discovery of the hepatitis B virus and the invention of the

vaccine: a scientific memoir. J. Gastroenterol. Hepatol. 2002;17 Suppl:S502–3.

2. Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients with

Australia-antigen-associated hepatitis. Lancet (London, England) 1970;1:695–698.

3. Gerlich WH. Medical Virology of Hepatitis B: how it began and where we are now.

Virol. J. 2013;10:239.

4. Revill P, Yuan Z. New insights into how HBV manipulates the innate immune

response to establish acute and persistent infection. Antivir. Ther. 2013;18:1–15.

5. Dandri M, Locarnini S. New insight in the pathobiology of hepatitis B virus

infection. Gut 2012;61 Suppl 1:i6–17.

6. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular

carcinogenesis. Gastroenterology 2007;132:2557–76.

7. Fallot G, Neuveut C, Buendia M-A. Diverse roles of hepatitis B virus in liver cancer.

Curr. Opin. Virol. 2012;2:467–73.

8. Dandri M, Petersen J. Mechanism of Hepatitis B Virus Persistence in Hepatocytes

and Its Carcinogenic Potential. Clin. Infect. Dis. 2016;62 Suppl 4:S281–8.

9. Zoulim F. Hepatitis B virus resistance to antiviral drugs: where are we going? Liver

Int. 2011;31 Suppl 1:111–6.

10. Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues.

Gastroenterology 2009;137:1592–1593.

11. Durantel D, Zoulim F. New antiviral targets for innovative treatment concepts for

hepatitis B virus and hepatitis delta virus. J. Hepatol. 2016;64:S117–31.

12. Locarnini S, Zoulim F. Molecular genetics of HBV infection. Antivir. Ther. 2010;15

Suppl 3:3–14.

13. Zlotnick A, Venkatakrishnan B, Tan Z, et al. Core protein: A pleiotropic keystone in

the HBV lifecycle. Antiviral Res. 2015;121:82–93.

14. Tindle RW. Immune evasion in human papillomavirus-associated cervical cancer.

Nat Rev Cancer 2002;2:59–64.

15. Fields BN, Knipe DM, Howley PM. Fields Virology, 5th Edition. (Knipe DM, Howley

PM, eds.). Lippincott Williams & Wilkins; 2007.

16. Yan H, Zhong G, Xu G, et al. Sodium taurocholate cotransporting polypeptide is a

functional receptor for human hepatitis B and D virus. Elife 2012;1:e00049.

17. Bock CT, Schwinn S, Locarnini S, et al. Structural organization of the hepatitis B

virus minichromosome. J. Mol. Biol. 2001;307:183–196.

18. Watashi K, Urban S, Li W, Wakita W. NTCP and Beyond: Opening the Door to

Unveil Hepatitis B Virus Entry. Int. J. Mol. Sci. 2014, 15(2), 2892-2905.

1.4 Figures:

Figure1: HBV life cycle, (Courtesy of Watashi et al., 2014) attachment to the NTCP receptor allows HBV entry into hepatocytes. Viral transcription takes place in the nucleus, new genomes are replicated in the cytoplasm inside viral capsids 18.

CHAPTER 2. HEPATITIS B VIRUS (HBV) CORE ANTIGEN (HBC): FUNCTION IN VIRUS BIOSYNTHESIS AND HOST GENE REGULATION

2.1 Introduction

Hepatitis B virus (HBV) is a major human pathogen that infects the liver, with at least 2 billion people infected today, according to World Health Organization 1. While

90-95% of infected adults clear the infection, the remaining develop chronic infection, which increases the risk for developing liver cancer 2. The reason that HBV infection persists in 5% of infected adults and over 80% of those infected at birth is still not understood 3. In the nuclei of infected hepatocytes, HBV DNA persists as a covalently closed circular DNA molecule (cccDNA) which assumes chromosome-like structure, serving as template for viral transcription. The HBV genome is small, 3.2 kb, expressing four overlapping RNAs, with open reading frames encoding viral polymerase (P), regulatory X protein (HBx), surface (S) and core (HBc) antigens. In this review, I focus our discussion on the function of HBc protein. HBc is viewed classically as a structural protein since it self-assembles to form the viral capsid. Significantly, accumulated evidence supports that HBc function extends beyond its structural role, having a role in nearly every step of HBV replication. Indeed, HBc has been implicated in subcellular trafficking, reverse transcription as well as epigenetic regulation of viral and host genomes. This renders HBc an excellent target for mechanism based antivirals 4,5. This

10 review aims to highlight how the unique structure as well as post-transcriptional modifications of HBc help explain its multilayer roles in HBV biosynthesis and host gene expression.

2.2 HBc structure

HBc is a 21 kDa protein with distinct N- and C-terminal domains connected by a hinge region 6,7. HBc assembles into dimers forming icosahedral capsid particles 8. Each capsid is composed of 120 HBc dimers, and interestingly capsid formation takes place in the absence of other viral proteins 8. The full-length protein consists of 183 amino acids.

The N- terminal domain of HBc, spanning amino acid residues 1-140, is the capsid assembly domain, and indeed truncated HBc 1-140 is sufficient for self-assembly into particles 8. On the other hand, the C-terminal domain (CTD) of HBc, spanning amino acids 141-183, is dispensable for viral assembly. Interestingly, capsids lacking the last 34 amino acids maintain assembly capacity in vitro but do not support genome replication

9, suggesting a role for the CTD in this process. Of the 34 amino acid residues that comprise the CTD, 14 are arginines and at least 7 other residues are serine/threonines, putative phospho-acceptor sites.

Studies to understand the role of the HBc CTD in HBV replication have led to the proposal that electrostatic interactions between the highly basic CTD in assembled capsids and the viral RNA/DNA regulate reverse transcription of HBV DNA 10. Charge- driven destabilization of capsids, either by relaxed circular DNA (rcDNA) maturation and/or changes in post-transcriptional modifications of CTD, for example by phosphorylation, could explain capsid disassembly at the nuclear pores 11–14. On the

11 other hand, studies by Lewellyn et al favor the interpretation that the HBc CTD serves as a nucleic acid chaperone, i.e., CTD mediates changes in nucleic acid structure that enable pregenomic RNA (pgRNA) reverse transcription and rcDNA biosynthesis 15 .

However, these two models are not mutually exclusive, since the chaperone function of

HBc may be dependent on the overall charge of CTD.

A recent study employing cryo-EM scanning has determined a high-resolution 3D structure of assembled HBV capsids 16. It was proposed that the HBc CTD shuttles between the interior and exterior of capsids, evidenced by the presence of CTDs on the exterior of some capsids, and RNA-bound CTD to the interior of other capsids 16. This model is attractive because it provides a mechanism of how HBc can be accessible to interact with proteins involved in intracellular trafficking, capsid maturation, and envelopment, thus addressing the many roles of HBc. In this model, the positively charged CTD tail interacts transiently with the negatively charged Asp2 and Glu43 that line the local three-fold channel that presumably allows movement of CTD tails in and out of capsids 16. Studies supporting the presence of CTD in the capsid exterior include the cryo-EM study 16 as well as biochemical binding assays that demonstrate a nucleic acid sensitive and transient exposure of the CTD tails 17,18. Specifically, RNA filled capsids failed to bind serine arginine protein kinase (SRPK), a host chaperone that binds full

17 length HBc capsids but not HBc 1-149 capsids , suggesting dynamic exposure of CTDs are mediated by CTD-nucleic acid interactions. These results also agree with biochemical studies showing that the CTD tails of assembled capsids were sensitive to proteases, and accessible to host proteins 17,19,20. The exact mechanism of this dynamic CTD shuttling

12 remains unknown, although a role of phosphorylation has been suggested by site directed mutagenesis of HBc 18. However, a more detailed analysis of the effect of CTD phosphorylations and dephosphorylations in CTD shuttling is needed.

2.3 HBc, a nucleic acid chaperone

Nucleic acid chaperones are proteins that catalyze structural rearrangement of genetic material into a proper conformation 21. Binding of nucleic acid chaperones to their targets is characteristically weak, transient, lacks sequence specificity, and requires no ATP hydrolysis 21,22. A wide array of viral proteins display nucleic acid chaperone activity, for example, nucleo-capsid proteins of viruses including HIV 23, Dengue virus 24, and Hepatitis Delta virus 25. The CTD of HBc contains an arginine-rich domain that resembles histone tails with more than 50 % of the residues being arginines 15. The CTD binds to nucleic acids in vivo due to its positive charge 9,26. In addition to the characteristic positive charge, the CTD is also structurally disordered 16,27, another key feature of nucleic acid chaperones 28. Notably, the CTD has also been mapped to the interior of assembled capsids by cryo-EM 29. The localization of CTD to the interior of capsids and its ability to bind nucleic acids in vivo support the model that CTD works as a nucleic acid chaperone. The nucleic acid chaperone activity of HBc CTD was recently confirmed in vitro using recombinant full length HBc as well as CTD synthetic peptides 27.

HBc CTD facilitated annealing and unwinding of DNA and enhanced hammerhead ribozyme cleavage in vitro, rendering HBc CTD a bona fide nucleic acid chaperone 27.

However, the high degree of functional complexity associated with the CTD, makes it

13 difficult to demonstrate the nucleic acid chaperone activity of HBc in vivo, without disturbing the other HBc functions.

2.4 HBc localization

Studies investigating the subcellular localization of HBc showed that its subcellular localization is cell-cycle dependent 30,31. The amount of core protein in the nucleus increases in G1 phase, diminishes in S phase and increases again to detectable levels in non-proliferating cells 30. HBc shuttles between cytoplasm and nucleus rapidly and continuously 32. Although HBc is 21kDa, suggesting passive transport to and from the nucleus, it is unknown whether in infected cells HBc that is not assembled into capsids exists as a monomer. Thus, the signals that modulate HBc import and export from the nucleus are probably distinct from those that guide transport of assembled

HBc into viral capsids.

Accumulating evidence suggests the CTD tail is responsible for directing HBc subcellular localization 32–34. Several studies have attempted to map the nuclear localization sequence (NLS) to the arginine repeats of CTD, however the results are conflicting probably due to the redundancy of arginine repeats in the CTD 34–36.

Recently, an exhaustive mutagenesis analysis of the CTD localized the NLS sequence to the 1st and 3rd arginine repeats, while the 2nd and 4th repeats contained the cytoplasmic retention signals (CRS)- highlighted in Fig. 1 32. The presence of more than one NLS in a protein is known to maximize the import efficiency 37,38. However, one must consider the accessibility of these sequences in view of the dynamic structural changes of

14 assembled HBc in viral capsids. Other viral proteins such as the ICP27 of herpes simplex virus type 1 (HSV-1) 38 and the NS1 of Influenza 39 also contain multiple NLS sites.

The dynamic positioning of HBc CTD in assembled capsids makes it quite challenging to identify the different signals regulating HBc transport. Nuclear export of

HBc resembles ICP27 of HSV-1 and EBNA1 of Epstein Barr virus (EBV) in that they all bind to the protein and RNA export factor TAP, and all contain the characteristic arginine rich domains (Fig. 1) 40. Since the subcellular localization of ICP27 and EBNA1 are regulated by arginine methylation 41,42, it is intriguing to propose that HBc localization is driven by a similar mechanism. However, it is not yet known how the arginine repeats of CTD contribute to its subcellular localization, a hypothesis worthy of future investigation.

The contribution of the HBc CTD to capsid localization is still not well understood. The ambiguity is based on the long-standing notion that the CTD localizes to the interior of capsids based on cryo-EM studies 29. Furthermore, CTD binds nucleic acids and has a well-documented role in pgRNA packaging 9. However, more recent studies point towards a dynamic model where the HBc CTD shuttles between the interior and exterior of the capsid 17,18,32. According to this model, exposure of the CTD to the capsid exterior would then allow interaction with cellular factors to direct the subcellular localization of capsids. Transport of cargo across the nuclear envelope is often facilitated by regulatory phosphorylation/ dephosphorylation of specific residues

43. Earlier studies reported that phosphorylated HBc was only observed in the cytoplasm, based on subcellular fractionation of in vivo 32P-radiolabed cell lysates 30.

However, the molecular mechanism that regulates subcellular localization of monomeric

15 vs. assembled HBc in capsids remains to be understood. The contribution of HBc phosphorylation to the nuclear import of capsids likely recapitulates known import mechanisms for other viral proteins, including EBNA-1 41,44 and SV40 T-antigen 45.

Specifically, phosphorylation of EBNA-1 increases its affinity for the import adaptor importin alpha 5 which mediates the nuclear transport via interaction with importin beta 44. SV-40 large T antigen has the best characterized NLS, and phosphorylation upstream of the classic NLS enhances nuclear import 45. Thus, the phosphorylation status of HBc CTD could facilitate interaction with nuclear pore complex components.

Indeed, in pulldown experiments using HBc CTD-GST fusion proteins, serine to alanine substitution at the three serine/proline (S/P) sites (Fig. 2) showed increased binding to importin alpha 46. While the in vivo relevance of such observations remains to be determined, other groups have reported that HBc phosphorylated in vitro by PKC in assembled capsids preferentially bound importin beta 12,47. However, the phosphorylation sites of HBc required for interaction with importin beta had not been mapped47. Furthermore, it is unresolved whether PKC is the in vivo kinase that phosphorylates HBc CTD 48,49. Thus, more detailed studies are needed to address this issue, employing well-characterized and functionally documented mutants of HBc.

Alternatively, as shown by Schmitz et al, capsid maturation could be the driving force of HBV nuclear import 50. At the nuclear pore, HBc capsids dissociate into dimers followed by entry into the nucleus 11. Mature capsids directly interact with nucleoporin

153 (Nup153), an essential component of the nuclear basket that facilitates nuclear import via importin beta. Interaction of mature capsids with Nup153 resulted in capsid

16 disintegration followed by release of viral genomes into the nucleus 50. Interestingly, capsids with immature genomes (i.e., RNA- filled capsids) were halted at the nuclear pore. Recently, it was reported that serine to glutamate substitutions at the 3 S/P sites of the HBc CTD (Fig. 2) reduced CTD exposure to the exterior of capsids suggesting a role for these phosphorylations in directing CTD exposure inside or outside of the capsid 18.

However, the mechanism responsible for distinguishing mature capsids vs. RNA-filled capsids is not fully understood. Phosphorylation of HBc, shuttling of the HBc CTD to the interior vs. exterior of capsids, and maturation of the genome may regulate exposure of different localization signals to host transport machinery. Such a multilayer and dynamic regulation network can best explain the difficulty in deciphering the mechanism of HBc subcellular localization during the HBV life cycle. To understand these dynamic regulatory networks, it is essential to gain better understanding of HBc structure, HBc interacting partners, and HBc post-translational modifications.

2.5 HBc phosphorylation

Since the HBV genome does not encode a protein kinase, endogenous kinases could mediate post-translational HBc modifications 51–53. Protein kinase C (PKC)54,55, serine arginine protein kinase 1 (SRPK1) 48 and cyclin-dependent kinase 2 (CDK2)56 have been suggested as endogenous kinases that associate or packaged within viral capsids.

The robust phosphorylation of HBc has been demonstrated in vivo by phosphoproteomic approaches 17 and in vivo labeling with 32P-orthophosphate 57. The

HBc CTD contains multiple serine/threonine phosphoacceptor sites which are remarkably well-conserved among related viruses 57. Phosphoproteomic analyses of HBc

17 have identified at least 7 conserved serine and threonine sites that are phosphorylated in vivo 17. Notably, proline-directed serine (S/P) sites at positions 155, 162 and 170 are highly conserved and several S/P kinases have been reported to phosphorylate these sites (Fig. 2) 48,56. Putative kinases include SRPKs 48, Glyceraldehyde-3-phosphate

Dehydrogenase protein kinase (GAPD- protein kinase)58, PKC 54, and an unknown kinase of 46 kDa 59 . Yet none of those studies have mapped the phosphorylation to specific sites, with the exception of SRPK1 and 2 at the 3 S/P sites 48. However, the exact contribution of the SRPK-mediated phosphorylation to viral replication is not understood, because although overexpression of SRPK1 or SRPK2 impaired HBV replication, this effect was independent of kinase activity 60. These results support that the SRPK-mediated phosphorylation of HBc observed in vitro is due to promiscuous substrate specificity of these kinases 56, and that SRPKs exert other functions affecting viral replication. For example, SRPKs could act as non-canonical chaperones in capsid assembly17. Regarding PKC-mediated HBc phosphorylation, inactivation of PKC led to defective capsid envelopment but had no effect on genome maturation 55. There is however lack of consensus on PKC being the candidate host kinase since specific inhibition of PKC had no effect on CTD phosphorylation 48,49. Recently, CDK2 was shown to phosphorylate the core protein of HBV both in vitro and in vivo 56. Intriguingly CDK2 inhibitors had no observable effect on viral replication 56. The absence of such effect could be due to lack of specificity of the chemical inhibitors or more likely due to redundancy among host kinases in phosphorylating HBc. Knockout cell lines for CDK2 or

18 other kinases via expression of shRNAs could potentially elucidate their contribution to

HBc phosphorylation and viral replication.

Although the kinases that phosphorylate HBc are still to be determined, phosphorylations of HBc do regulate its function at many levels 47,57,61–63. Specifically, phosphorylation and de-phosphorylation of the HBc protein is required for capsid maturation as demonstrated by mass spectrometric analysis and site-directed mutagenesis studies of HBc 13,14. Phosphorylation of different serine residues of HBc CTD regulates reverse transcription, pgRNA encapsidation, DNA synthesis, subcellular localization and virion secretion 13,14,61,64. HBc phosphorylation is also required for the chaperone activity of the CTD 27. It was also reported that HBc phosphorylation status can influence capsid stability (17), since capsids with glutamate phospho-mimetic substitutions at the 3 S/P sites (S/P to E/P) were more stable than wildtype capsids. It is likely that during various stages in the life cycle of HBV or as a function of the metabolic and proliferative status of the infected hepatocyte distinct kinases target HBc phosphorylation, perhaps in an overlapping manner. The identification of those kinases and the mechanisms by which they regulate HBV replication is of great significance and will provide valuable targets for mechanism-based therapeutics.

Our studies have identified the mitotic Polo-like-kinase 1 (Plk1) as another cellular kinase that phosphorylates HBc in vitro (Diab et al 2016). Plk1 substrates are usually primed by phosphorylation at S/P directed sites mediated by CDKs, including

CDK1 or 2 65. Recombinant HBc as well as immuno-purified HBc from transfected mammalian cells serves as robust Plk1 substrate in vitro (Diab et al 2016). I have

19 mapped the phospho-acceptor sites to the S168, S176 and S178 (Fig. 2) (Diab et al

2016), all of which have been identified as in vivo phosphor-acceptor sites by an unknown kinase 57. Remarkably, inhibition of Plk1 by specific chemical inhibitors or siRNA-mediated knockdown of Plk1 suppressed viral replication, suggesting that HBc phosphorylation by Plk1 is functionally important in virus biosynthesis (Diab et al 2016).

Significantly, I made the interesting observation, in accordance with the known substrate preference of Plk1 that alanine substitutions at the 3 S/P sites (S/P to A/P) lead to loss of Plk1 phosphorylation in vitro. By contrast, Plk1 phosphorylation was robustly recovered in phospho-mimetic mutants, containing serine to aspartic acid substitutions at all three S/P sites (Diab et al 2016). This suggests that CDK2 functions as the priming kinase for HBc, mediating the phosphorylation of the S/P sites which in turn enables the subsequent phosphorylation by Plk1 65. Based on our results that Plk1 is a positive effector of HBV replication, a mechanistic understanding of the role of Plk1 phosphorylation of HBc in HBV replication promises a great therapeutic potential. So far,

Plk1 is the sole host kinase whose chemical inhibition results in impaired HBV replication in vivo at concentrations comparable to direct acting agents, including interferon- and entecavir (Diab et al, 2016).

2.6 HBc and interaction with host factors

Almost every stage of the HBV life cycle is modulated by HBc. With such intricate function, HBc has evolved to interact and modulate several host proteins. For example,

HBc dimers interact with HSP90, and HSP90 facilitates formation of HBV capsids both in

20 vitro and in vivo 66. On the other hand, the host HSP40 protein also binds but de- stabilizes HBc by accelerating its degradation 67. The binding of HBc to other cellular factors such as NIRF, an E3 ubiquitin ligase, is implicated in HBc degradation 68. Thus, dynamic interactions of HBc with host chaperones regulate both HBc stability and capsid assembly 66–68.

Viruses hijack host RNA processing machinery 69,70 and HBV is no exception. HBc utilizes the nuclear export factor 1 (NXF1/p15) and transcription export (TREX) machinery for nuclear export as ribonucleoprotein complex along with the viral pgRNA

40; however, TREX mediated export of HBc and pgRNA are independent of each other 40.

Other virus that hijack the NXF1/p15 pathway for mRNA processing and export include

EBV 71 and HSV- 1 72. However, depletion of different TREX components has no apparent effect on accumulation of viral DNA, suggesting redundancy among host factors utilized by the virus.

Another class of host proteins commonly targeted by viruses is serine, arginine rich-proteins 73 which, along with the kinases (SRPKs) that phosphorylate them, play central roles in alternative RNA splicing 74. Intriguingly, HBc CTD resembles serine, arginine-rich proteins and it is not surprising that SRPKs associate HBc 17,48,60.

Interestingly, mass spectrometry analyses of HBc interacting proteins identified SRPK1 as well as several known SRPK1 interacting partners, including SRSF1, SRSF9, DHX9,

CDKN2A, DDX21 and DDX50 (Diab et al unpublished results). The functional significance of these HBc interactions for the viral life cycle is unknown and worthy of investigation.

It could be speculated that HBV usurps the host RNA-processing machinery for

21 processing and production of its own viral RNA. It is also likely that such interactions might allow HBc to interfere with host gene expression. This has been evident in the case of the E1^E4 protein of the human papilloma type 1 virus which not only binds to

SRPK1 but also inhibits phosphorylation of its host serine arginine substrates 75. A yeast two-hybrid screen of a human liver cDNA library identified the human protein GIPC1 as another HBc interaction partner 76, but the significance of such interaction remains to be determined. GIPC1 contains a PDZ domain, a protein-protein interaction domain reviewed in 77. PDZ-containing host proteins are commonly targeted by viral proteins; examples include the Tax protein of the T-cell leukemia virus type 1 78 and the E6 protein of the human papilloma virus type 18 79.

It is reasonable to speculate that HBc exerts its various roles by interacting with specific host factors at different stages of the viral life cycle in a phosphorylation dependent manner. Indeed, phosphorylation of HBc has been reported to dictate some of HBc-host interactions. For example, binding of HBc to the nuclear pore complex is dependent on HBc phosphorylation 47,50,63. Phosphorylation of HBc is required for the interaction with importin beta47, whereas only the alanine substitution mutant at the

S/P sites of HBc CTD showed specific binding to importin alpha 46. Moreover, un- phosphorylated HBc was found to preferentially bind host proteins B23 and I2PP2A, while phosphorylation diminished these interactions 63. A better understanding of the host factors that interact with HBc will facilitate development of new antiviral strategies.

2.7 HBc as a regulator of transcription

In chronically infected patients, HBc is detected both in cytoplasm and nucleus of hepatocytes 80,81. In vitro models of HBV infection also show nuclear as well as cytoplasmic localization of HBc. Nuclear localization of HBc has been linked to high viremia and deficient immune response in biopsies from HBV infected patients 81–83.

These results prompted investigations on the nuclear function of HBc, both in terms of virus biosynthesis and effects on host gene expression.

In the nucleus of infected cells, the viral cccDNA assumes chromatin-like structure in association with histone and non-histone proteins, along with viral HBx and

HBc proteins 84,85. Association of HBc with the cccDNA mini-chromosome was reported to alter nucleosome spacing 84. Furthermore, it was reported that HBc preferentially associated with CpG island 2 of the HBV genome that overlaps enhancers (Enh) I and II involved in viral transcription; HBc binding to CpG island 2 positively correlated with the rcDNA/cccDNA ratio and serum levels of HBV DNA in infected patients 86. HBc also acts as transcriptional activator of the HBV pre-Core promoter by enhancing binding of NF-kB upstream of viral Enh II 87. However, how HBc modulates the functions of the cccDNA mini-chromosome is not yet understood. Since HBc is also essential for viral DNA replication, a definitive genetic approach involving mutagenesis or depletion of HBc is not feasible to delineate its role in cccDNA mini-chromosome functions. Therefore, novel approaches are needed to specifically target nuclear HBc, influencing its capacity to associate with the cccDNA mini-chromosome, without affecting HBc cytoplasmic functions.

In terms of effects of HBc on host gene expression, HBc enhanced cAMP-

Response-Element (CRE)-mediated transcription via the CRE/CREB/CBP pathway 88 in a manner similar but weaker to previously reported HBx-mediated CRE activation 89,90.

HBc may also function as a transcriptional repressor of host genes. For example, binding of HBc to E2F1 reduced the DNA-binding ability of E2F1 at the p53 promoter, thereby inhibiting p53 transcription 91. In hepatoma cell lines expressing the core protein, HBc blocked the human death receptor 5 (DR5) by repressing its promoter, and inhibition of

DR5 desensitized hepatocytes to TRAIL-induced apoptosis 92. These results suggest a role for HBc in the development of chronic infection by preventing hepatocyte death via blocking DR5 expression. The gene regulatory function of HBc may have evolved as an attempt by the virus to evade host immunity. HBc has been shown to downregulate IFN- induced host antiviral responses in hepatoma cell lines, by interacting with the promoter of MxA, an INF-inducible gene widely implicated in antiviral response 93,94.

However, these conclusions must be further confirmed by determining in vivo association of HBc with MxA promoter, in physiological cellular contexts of HBV infection. Interestingly, MxA has been recently shown to inhibit HBV replication by direct interaction with HBc, preventing capsid formation 95, yet another example of antagonism between viral and host proteins.

In continuing effort to understand the effect of HBc on host gene expression, recent studies have generated the genome-wide profile of HBc in HBV-infected hepatocytes employing chromatin immunoprecipitation microarray studies (ChIP-on-chip). These studies observed that the promoters of nearly 3,100 host genes exhibited increased

24 binding to HBc 96. However, further studies are necessary to understand the functional significance of HBc binding to the host genome in effecting HBV-mediated disease pathogenesis. Toward this goal, Lucifora et al recently demonstrated interaction of nuclear deaminases APOBEC3A and APOBEC3B with HBc-bound cccDNA in host nucleus, leading to cccDNA deamination and degradation 97. Whether HBc recruits these nuclear deaminases to host genes is unknown and of great importance, given the role of

APOBEC proteins as cancer driver genes in humans 98–100.

2.8 Unanswered questions and future directions

The core antigen (HBc) is involved in various aspects of HBV biosynthesis: formation of the viral capsid, pregenomic RNA (pgRNA) encapsidation followed by its reverse transcription, and as a component of the viral mini-chromosome contributing to its chromatin structure. The unique structure of HBc CTD and its modifications, mainly phosphorylation, are essential for viral biosynthesis. Therefore, HBc seems to be an excellent target for mechanism-based therapeutics. Yet many questions remain unanswered when it comes to HBc biology. A definitive answer on the identity of the kinase/kinases responsible of HBc phosphorylation in vivo is yet to be determined.

Additionally, systematic investigation of HBc-host interactions both in the context of physiologic infection as well as a single molecule will expand our knowledge not only of how HBc is regulated by post-translational modifications, but also how HBc regulates host genes. Moreover, given the unique structure of the CTD, the seemingly promiscuous affinity of HBc for RNA, and the observed interaction of HBc and ribonucleoproteins (Diab et al, unpublished results) collectively suggest potential

25 interactions between HBc and host RNAs. Thus I propose that HBc, as an RNA- interacting protein associates and regulates host RNAs and in particular long non-coding

RNAs (lncRNA). Accumulating evidence supports the regulatory role of lncRNAs in cellular gene expression 101; whether HBc/ lncRNA interactions also exert a role in hepatocyte physiology and virus biosynthesis is presently unknown 102 and the topic merits further investigation.

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2.10 Figures

Figure 2: Arginine rich motifs of different viral proteins are characterized by the abundance of arginine residues within short 10-20 residues. The arginine rich domain of the HBc protein has distinct nuclear localization and cytoplasmic retention signals.

Figure 3: HBc C-terminal domain (CTD) is a phosphorylation substrate for CDK2 and Plk1 at the indicated sites.

CHAPTER 3. MATERIALS AND METHODS

3.1 Cellular models

3.1.1 HepaRG cell lines

3.1.1.1 HepaRG cell culture:

- Proliferating HepaRG cells were maintained in complete William’s medium 1

containing:

 William’s E medium GIBCO (500ml bottle, stored @4°C)  50ml of HighClone FetalClone II, U.S. Origin, (10% final), stored and aliquoted @- 20°C,  Add 5ml of penicillin/streptomycin GIBCO (100 units/ml penicillin, 100 µg/ml streptomycin, aliquoted and stored @-20°C)  5ml of GlutaMax 100X GIBCO (stored @4°C)  250µl of human insulin solution (5 µg/ml final, I-9278, 5ml SIGMA) stored @4°C  500µl of hydrocortisone (50 µM final conc.)

Note: Hydrocortisone hemisuccinate (Sigma H2270-100MG or Stem Cell Technologies

07904) hydrocortisone hemisuccinate is prepared in sterile PBS (resuspend in 4ml of

PBS), 500µl aliquoted and stored @-20°C.

- To differentiate HepaRG cells, cells were not passaged for 2 weeks and media

was changed twice a week. After 2 weeks, cells were supplied with proliferation

medium supplemented with 1.8% DMSO (Hybri-Max Sigma D2650) for two more

weeks.

- A healthy monolayer of differentiated cells was obtained following this

procedure and was evaluated using phase-contrast microscope.

3.1.1.2 HepaRG-TR-PLK1 cell lines:

HepaRG cell lines stably expressing the tetracycline repressor (TR) in an inducible manner were obtained as described 2. HepaRG-TR cell lines over expressing wild type

PLK1, HepaRG-TR-PLK1WT, and constitutively active (CA) PLK1, HepaRG-TR-PLK1CA, were constructed by transducing HepaRG-TR cells with a lentiviral vector (pLenti4/TO) conferring zeocin resistance and containing either version of PLK1. PLK1WT or PLK1CA transgenes were cloned into pLenti4/TO vector under the control of the tetracycline- regulated (tet-on) promoter. Protein expression was confirmed by western blotting using PLK1 antibody and following standard protocol.

Note: For modified HepaRG, add corresponding antibiotic for selection (Zeocin

INVITROGEN 200µg/ml or Blasticidin INVITROGEN 20µg/ml).

3.1.1.3 Infection in HepaRG cells and Primary Human Hepatocytes

This protocol was used to carry out HBV infection in differentiated HepaRG cells as well as primary human hepatocytes.

- Calculations are for a single well of 12-well plate with an MOI of 100 and could

be scaled up based on the surface area to fit other platforms.

- For each well, 500 L of complete Williams medium containing 5% FBS and 2%

DMSO and 4% PEG was used to dilute the desired amount of virus.

- For mock infection, 500 L of medium without virus were used.

- Calculations of the required amount of virus were made according to Table 1

- For inoculation, diluted virus was added to the cells and incubated overnight

(16h max) at 37°C incubator.

- The following morning (Day1) the inoculum was removed and the cells were

washed 3x with wash buffer and fresh complete William’s medium

supplemented with 5% FBS and 2% DMSO was added.

- Day 4 after infection, the medium was changed.

Day 7 infection reaches plateau and viral parameters could be analyzed using standard protocols.

3.1.2 HepG2-NTCP cell line and infection

- HepG2hNTCP cell line (A3 clone) was obtained from Prof. Dr. Urban 3. HepG2hNTCP is

derived from HepG2 cell line and has been stably transduced by lentivirus

encoding human sodium taurocholate co-transporting polypeptide (NTCP) under

puromycin selection rendering them susceptible to HBV infection 3. Cells were

cultured in culture medium (DMEM 4.5g/L glucose +Glutamine, 10% FBS and 100

units/ml penicillin, 100 µg/ml streptomycin) and supplemented with 5µg/mL of

puromycin. Cells were passaged once a week in a ratio of 1:6 to avoid complete

confluence.

- After infection, cells were maintained in replication medium (culture medium

supplemented with 2.5 % DMSO.

- For infection, cells were inoculated with desired MOI of concentrated HBV (MOI

100 to 500 to achieve 20% to 100% infection efficiency) in presence of 4% PEG in

culture medium (alternatively replication medium).

- Inoculum was removed after 16 hours incubation and cells were washed 3x in

wash buffer and finally were kept in replication medium.

- Fresh replication medium was added every 2 days.

- Viral replication was measured by ELISA for HBe after day 7 of infection following

standard protocol.

3.2 Production of HBV inoculum in HepG2.2.15 cells:

3.2.1 Materials

- HepG2.2.15 cells 4 - Collagen I (Gibco, A10483-01) - DMEM 4,5 g/L glucose (Gibco, 11965126) - FBS 10% final - Pen/strep (Gibco) - Na pyruvate (Gibco) - DMSO for cell culture (Sigma) - G418 - PEG8000 (Sigma) at 40% in PBS.

3.2.2 Protocol

- HepG2.2.15 cells, a hepatoblastoma cell line with 4 integrated copies of HBV

genome (PMCID: PMC304350) under G418 selection, were grown in growth medium

(DMEM medium complemented with 10 % FBS, 1mM Na Pyruvate, 100 units/ml

penicillin, 100 µg/ml streptomycin, 250 g/mL G418), on pre-collagenated cell

culture flasks. Cells were passaged with 1/3 dilutions for propagation and

maintenance.

- Multiples of 175 cm2 cell culture flasks were grown to 90% confluence in growth

medium before switching to production medium.

- When cells reached confluence, cells were washed and replenished with

production medium (DMEM, 2% FBS, 2%DMSO, 1mM Na pyruvate, 100 units/ml

penicillin, 100 µg/ml streptomycin, without G418 as selection is no longer

necessary at this step).

- Cells were replenished with production medium every 3-4 days and old medium

was collected for concentration of virus and kept at 4°C until sufficient amount

was secured (1 L).

- Cells were maintained in production medium for 2-3 weeks or until cells started

to detach from the monolayer.

- Collected media was filtered (0.45 micron) to get rid of cell debris and 1x sodium

bicarbonate (Gibco) was added to stabilize pH.

- Virus titer before concentration is approximately 10^8 vge/mL.

- 40 % PEG8000 (sigma) stock was prepared and filtered (0.2 micron), collected

media with final conc. 8% PEG was incubated overnight at 4°C while shaking

gently to facilitate the concentration of the virus. (Each 50 mL falcon tube had

40mL media + 10 mL 40% PEG).

- A pellet of virus/PEG is obtained by centrifugation at 5000 g for 45 minutes at

4°C

- Pellets were re-suspended in 500 L of OptiMEM media, and were left to

dissociate overnight at 4°C.

- The following morning, the re-suspended pellets were passed through 200 L

filter tips several times until completely re-suspended.

- All aliquots were aggregated and re-aliquoted into 500 µL and stored at – 80 °C.

- An aliquot of 25 L was used for titration of virus. Viral DNA was extracted from

the sample using High Pure Viral Nucleic Acid Kit (Roche, 11858874001)

following manufacturer protocol.

- 5.0 and 0.5 L of eluted DNA were used in qPCR reaction using primers for HBV

genomic DNA to estimate viral genome equivalents. Ct values were compared to

another virus DNA sample with a pre-determined concentration. Alternatively, a

standard curve was generated by serially diluting HBV DNA vector starting with 1

ng, 0.1 ng, 0.01 ng etc…. (1 ng of genomic HBV DNA contains approximately 10^8

copies).

- Number of HBV molecules in 5L (vge) X dilution (5x)

= concentration (in vge) in the 25 L of purified inoculum

X 40

= concentration in vge per mL

- The following formula was used to calculate MOI for infection

vge [ ]volume ( ) x # cells /well cell = volume in mL x 1000 if you want it in µL [ ] in vge/ml of inoculum

- Only virus titer of 2x 10^9 or higher were used for infection assays.

- A functional assay was performed to evaluate the titer of the generated virus by

infecting HepG2-NTCP cell with 2, 5, 25, 50, 100 L of the newly concentrated

virus. Infection efficiency was evaluated by ELISA (for HBe) and qPCR (for

intracellular DNA) using standard protocols 5.

3.3 Tables

Table 1: Calculating MOI 100 for a single well in a 12 well plate Mix for 12 wells Estimated number of cells= 1x106 cell/well MOI 100= 1.108 vge/well Final concentration Cf = 2.108 vge/mL Vf = 0.5 ml (per well) Vp = … mL Medium (2% DMSO, 4% PEG) = …mL

3.4 References

1. Gripon P, Rumin S, Urban S, et al. Infection of a human hepatoma cell line by

hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A. 2002;99:15655–15660.

2. Lucifora J, Arzberger S, Durantel D, et al. Hepatitis B virus X protein is essential to

initiate and maintain virus replication after infection. J. Hepatol. 2011;55:996–

1003.

3. Ni Y, Lempp FA, Mehrle S, et al. Hepatitis B and D viruses exploit sodium

taurocholate co-transporting polypeptide for species-specific entry into

hepatocytes. Gastroenterology 2014;146:1070–1083.

4. Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells

transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. U. S. A.

1987;84:1005–1009.

5. Isorce N, Testoni B, Locatelli M, et al. Antiviral activity of various interferons and

pro-inflammatory cytokines in non-transformed cultured hepatocytes infected

with hepatitis B virus. Antiviral Res. 2016;130:36–45.

CHAPTER 4. POLO-LIKE-KINASE 1 IS A PROVIRAL HOST-FACTOR FOR HEPATITIS B VIRUS REPLICATION: THERAPUTIC IMPLICATIONS

4.1 Abstract

Background and aims: Chronic Hepatitis B Virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis, with nearly 250 million people chronically infected with HBV. Current treatments for HBV infection and HCC are ineffective.

Herein, I identified cellular Serine/Threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study is to demonstrate the proviral role of

PLK1 in HBV biosynthesis and explore PLK1 as an antiviral target.

Methods: To determine the role of PLK1 in HBV biosynthesis, I employed physiologically revenant HBV infection models of Primary Human Hepatocytes (PHH) and differentiated

HepaRG cells, in conjunction with pharmacologic PLK1 inhibitors, siRNA-mediated knockdown of PLK1, and overexpression of constitutively active PLK1(PLK1CA). In addition, humanized liver FRG mouse model was used to determine antiviral effect of

PLK1 inhibitor BI 2536 on HBV infection in vivo. Lastly, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate HBV core antigen (HBc) is

PLK1 substrate.

Conclusions. I demonstrate HBV infection activated cellular PLK1 in physiologically relevant HBV infection models of PHH and dHepaRG cells. PLK1 inhibition by BI 2536 (5-

50nM) or siRNA-mediated knockdown of PLK1 suppressed, whereas overexpression of

PLK1CA increased HBV DNA biosynthesis, supporting PLK1 effects on viral biosynthesis are specific and PLK1 is a proviral cellular factor. Significantly, BI 2536 administration to

HBV-infected humanized liver FRG mice suppressed HBV infection, identifying PLK1 as a novel antiviral target. Although how PLK1 regulates virus biosynthesis remains to be further delineated, our studies identified HBc as a PLK1 substrate in vitro, and mapped

PLK1 phosphorylation sites.

4.2 Introduction

Chronic hepatitis B virus (HBV) infection is an independent risk factor for development of hepatocellular carcinoma (HCC)1,2. Liver cancer is the 5th and 7th most common cancer worldwide according to gender, and the 3rd in terms of death. With approximately 250 million people chronically infected with HBV, according to the latest

World Health Organization (WHO) estimation, many patients with chronic hepatitis B

(CHB) are expected to progress to HCC, despite existing therapies1. Although a prophylactic vaccine is available, new infections still happen because vaccination campaigns are not always well-implemented, and the vaccine it is not always 100%2.

Furthermore, children born of infected mothers still become chronically infected in endemic areas2. Current treatments include use of Peg-IFN and/or antiviral nucleoside analogs; the latter require life-long administration, which can result in viral resistance when drugs with low genetic barrier are used3. New and effective therapies are urgently needed3. In particular, drugs that target both viral replication and prevent neoplastic transformation could represent a valuable addition to the therapeutic arsenal.

Replication of HBV within hepatocytes involves both nuclear and cytoplasmic events4. Following viral entry, nucleocapsids are transported to the nucleus, releasing the viral DNA and core/capsid protein (HBc) into the nucleoplasm. There, the viral genome, initially a relaxed-circular partially-double-stranded DNA (rcDNA), is converted into covalently-closed-circular dsDNA (cccDNA), responsible for viral persistence. This nuclear form of the genome serves as template for viral RNA transcription, including of pre-genomic RNA (pgRNA)4. In the cytoplasm, pgRNA interacts with HBV polymerase, is packaged into capsids where it is reverse-transcribed to generate newly synthesized rcDNA4. HBc plays an essential and dynamic role in HBV biosynthesis, including selective binding to pgRNA in association with viral polymerase, encapsidation and reverse- transcription of pgRNA, transport of mature capsids to the site of viral envelopment for virion generation, and transport to the nucleus for recycling5. Recent studies demonstrated this dynamic interplay between HBc structure and viral pgRNA reverse- transcription 6,7. Although, the mechanism by which HBc and capsid assembly regulate viral DNA synthesis is not fully understood, it is well established that phosphorylation of

HBc has an important role in various aspects of pgRNA reverse-transcription8. Kinases that mediate HBc phosphorylations include CDK2 (Cyclin-Dependent Kinase 2), which phosphorylates serine/proline (SP) sites located at the C-terminus of HBc, including

S155, S162 and S170 5,9. Another study has identified additional S/T sites as having a role in regulating all aspects of HBV replication, and SRPK1 (Serine/Arginine Protein Kinase 1) as another kinase that phosphorylates HBc in vitro10.

Our earlier studies, aiming at understanding the mechanism by which the HBV X protein (HBx) mediates oncogenic transformation of hepatocytes 11–13, led us to the study of Polo-like-kinase 1 (PLK1), a S/T kinase involved in cell cycle regulation14,15. In physiologic conditions, PLK1 is required for checkpoint recovery, mitotic entry and progression, whereas it is overexpressed in many human cancers, including HBV- mediated liver cancer14,15. Andrisani lab has shown that HBx activates PLK1 prematurely, allowing propagation of DNA damage to daughter cells by attenuating both DNA repair and p53 apoptosis 12,13. In a cellular model of HBx-mediated transformation and in liver tumors of animals modeling HBx- and HBV-mediated hepatocarcinogenesis, expression of PLK1 is elevated12,13,16,17. However, whether PLK1 is activated during physiologic HBV infection of normal hepatocytes, and whether it has a role in establishment and maintenance of infection is unknown. To address this issue, in this study, I used HBV infection models of non-transformed primary human hepatocytes

(PHH) and dHepaRG, a bipotential human de-differentiated hepatocyte cell line that upon differentiation supports HBV infection. In this study I demonstrate the cellular

PLK1 enzyme acts as a proviral host-factor for HBV replication in vitro, by favoring nucleocapsid formation and subsequent reverse transcription. Furthermore, I have identified the viral core protein HBc, as a PLK1 substrate in vitro, and mapped putative sites of PLK1 phosphorylation. Moreover, I provide evidence that PLK1 inhibitors serve as a novel class of anti-HBV molecules in vitro, and validated the antiviral effect of PLK1 inhibitor BI 2536 in HBV infected liver humanized mice, in vivo.

4.3 Results

4.3.1 PLK1 is activated by HBV infection in non-dividing/differentiated

hepatocytes

Our earlier studies demonstrated that i) HBx activates the mitotic S/T kinase

PLK1, in a conditional HBx-expressing cell line12, ii) PLK1 activation initiates proteasomal degradation of chromatin modifying nuclear proteins SUZ12 and ZNF15811, and iii)

SUZ12 downregulation in HBV replicating hepatocytes results in expression of hepatic cancer stem cell markers and pluripotency genes18. Andrisani lab has also shown activation of PLK1 in HBV replicating HepAD38 cells11, further suggesting a link between

HBV infection and PLK1 activation. However, it remained to be determined whether

PLK1 activation occurs in the context of physiologic infection of non-dividing, differentiated, and non-transformed hepatocytes. To this end, primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) were infected with HBV, and expression and activation of PLK1 was quantified. Upon infection of dHepaRG cells, PLK1 mRNA increased by15-fold 24hr post-infection (p.i.), followed by a constant level of expression of 3-to 5- fold from 48h to 168h p.i. (Figure 1A). This resulted in a transient increase in PLK1 protein levels (Fig. 1B). More interestingly, an increase in PLK1 phosphorylation on S137 and T210, indicative of PLK1 activation, was detected as a function of HBV infection by immunoblots (Fig. 1B), and immunofluorescence microscopy (Fig. 1C) employing phospho-specific PLK1 antibodies. Remarkably, this activation of PLK1 by HBV infection was also detected by immunoblots of lysates from various preparations of PHH (representative blots are shown; Fig. 1D).

4.3.2 PLK1 inhibitors, including BI 2536, suppress HBV DNA accumulation in

persistently HBV-infected hepatocytes

To test whether PLK1 activation has a proviral effect, dHepaRG cells were infected with HBV virions and on day-7 post-infection (7 d.p.i.), when infection had reached a “plateau” in terms of viral replication, cells were treated with various PLK1 inhibitors. Specifically, increasing concentration of ATP competitive (i.e. BI 2536 and BI

6727) or non-ATP competitive (i.e. MLN 0905 and ON 01910) PLK1 inhibitors were added for 3 days; accumulation of intracellular HBV DNA was measured by Southern blot and qPCR (Supplementary Figure 1A and 1B). Amongst all drugs tested, BI 2536 was the most active and selected for further studies, after monitoring batch-to-batch stability (Sup. Fig. 2). HBV infected dHepaRG treated with BI 2536 on day-7 p.i., exhibited no effect on levels of secreted HBe and HBs antigens, or intracellular viral RNA

(Fig. 2A). Interestingly, BI 2536 significantly inhibited synthesis of total HBV DNA, with an effective concentration at 50% (EC50) of approximately 4 nM (Fig. 2A), in the absence of any toxicity (cytotoxic concentration at 50%, CC50) of ~10 M; Fig. 2B). Interestingly BI

2536 reduced the level of capsid (i.e. assembled HBc/core protein) detected by immunofluorescence microscopy with an antibody recognizing only capsid assembled

HBc, and not monomeric or dimeric forms of HBc (Fig. 2C), and capsid migration assay

(Fig. 2D). These results suggest that inhibition of PLK1 by BI 2536 affects the assembly of

HBV capsids, thereby inhibiting rcDNA synthesis. Similar results were also obtained in

HBV infected PHH (Fig. 3), with an EC50 at ~ 50 nM. Interestingly, in this model, a

54 significant increase in intracellular viral RNA was observed, thus inversely correlating with total DNA decrease. This corroborates the hypothesis that rcDNA synthesis is likely inhibited by an impaired assembly of capsids around pgRNA.

4.3.3 Loss-of-function of PLK1 suppresses HBV DNA accumulation in infected

hepatocytes

To conclusively establish that PLK1 is a proviral factor, I investigated effect of loss of function of PLK1 on HBV replication by siRNA-mediated knockdown of PLK1 (siPLK1).

As controls, I employed siRNA targeting HBV transcripts (siHBV; positive control), and siRNA against HCV (siHCV) serving as negative control. The effectiveness of PLK1 knockdown by siRNA transfection was determined by quantification of PLK1 mRNA levels as a function of time after transfection (data not shown). Transfection of siPLK1 reduced by nearly 90% endogenous level of PLK1 mRNA at 72hr after transfection, while all non-targeting siRNAs had no effect on PLK1 mRNA levels (Fig. 4A). Transfection of increasing amounts of siPLK1, ranging from 5 to 25 nM in HBV-infected dHepaRG cells, resulted in a progressive reduction of 50 to nearly 70% in the accumulation of intracellular HBV DNA (Fig. 4A). By contrast transfection of 25nM siHCV had a minimal effect, while transfection of 25nM siHBV reduced HBV DNA by nearly 80% (Fig. 4A).

With siPLK1, I again observed a decreased accumulation of assembled HBc within infected cells by IF, using an antibody recognizing assembled capsids (Fig. 4B), thus confirming the potential relationship between proviral action of PLK1, formation of HBV capsids and resulting decrease in neo-synthetized rcDNA. Similar results were also obtained in HBV-infected PHH cultures, transfected for 72 h with siRNAs targeting PLK1,

HBV or an irrelevant target (Fig. 4C). Specifically, Southern blot analyses demonstrated a

50% reduction in HBV DNA by transfection of siPLK1, a 10% reduction with siHCV and

65% reduction with siHBV (Fig. 4D).

4.3.4 A modest gain-of-function of PLK1 is associated with an increased HBV

replication

To investigate the effect of gain-of-function of activated PLK1 on HBV replication,

I generated a HepaRG cell line expressing a constitutively active form of PLK1 (PLK1CA) in a tetracycline inducible manner (i.e. HepaRG-TR-PLK1CA) (Sup Fig. 4A). HBV infected dHepaRG-TR-PLK1CA cells were induced to express PLK1CA on day-7 p.i. by addition of increasing amount of tetracycline (0, 16, 80, and 400 ng/ml) for 3 days. Quantification of viral DNA and secreted antigens was carried out day-10 p.i. Under these conditions of established infection, a modest expression of PLK1CA enhanced HBV replication by 1.5- to 2- fold, whereas no effect on HBeAg secretion was observed (Sup Fig. 4B). This result suggests that active PLK1 enhances formation of new viral genomes by reverse- transcription of pregenomic RNA (pgRNA), i.e., it exerts a positive effect on capsid- assisted reverse transcription of pgRNA.

4.3.5 Reduction of HBV viremia in liver-humanized infected FRG mice treated

with BI 2536

To demonstrate the effect of BI 2536 on inhibition of HBV replication in vivo, I employed liver-humanized FRG mice36 infected with HBV (5x108 vge/mouse). Once serum viremia reached 7 log10 vge/mL, antigenemia 5 log10 IU/mL for HBsAg and 2,500

NCU/mL for HBeAg at week-4 post infection, treatment was administered by intra-

56 peritoneal injection of 10 mg/kg of BI 2536 (or same concentration of DMSO diluted in saline) bi-weekly for 4 weeks. Serum viremia and antigenemia were monitored at the end of weeks 1, 2, 4, 6, 7 and 8, while weeks 6, 7, and 8 were those under treatment.

Treated mice exhibited at week-8 a mean reduction of 1.406 log10  0.26 in viremia (Fig.

5C), whereas minimal effect was observed on antigenemia (Fig. 5A and B). It is worth noting that similar duration treatment with IFN- (25 g/kg/tw; 5 mice treated) or entecavir (ETV; 2.5g/mouse/day in drinking water; 3 mice treated) in liver-humanized

FRG mice led to a viremia reduction of 0.668 log10  0.18 or 1.919 log10  0.23 respectively (data not shown). These results indicate that a mean reduction of 1.406 log10  0.26 in mono-therapy obtained with BI 2536 was relevant and in between performance of IFN- and ETV in this model.

4.3.6 Effect of the combination of BI 2536 with approved or investigational

anti-HBV molecules

Our in vitro and in vivo data demonstrated that PLK1 inhibitors could be used as efficient and safe anti-HBV drugs. PLK1 inhibitor, including BI 2536 seems to target the capsid-associated reverse-transcription of pgRNA into rcDNA. Other drugs also target this step of the viral life cycle, including nucleoside/nucleotide analogues (NA) and core inhibitors (also called core protein allosteric modulators; CpAM). But all these drugs do have different molecular targets, and therefore could be theoretically used in combination to enhance the inhibitory phenotype. In order to establish whether BI 2536 could be used in combination with NA or CpAM without leading to antagonistic effect, I combined these molecules to treat infected dHepaRG cells. A combination of BI 2536

57 and IFN- was also included. As expected only BI 2536 + IFN- treatment led to a reduction in HBeAg secretion (Fig. 6A), whereas all other combinations led to a sharp reduction of intracellular HBV DNA (Fig. 6B). No obvious antagonism was evidenced for all combinations tested, thus suggesting that these drugs could be further considered for combination studies.

4.3.7 HBc is a substrate for PLK1 in vitro

During established infection, inhibition of PLK1 by treatment with BI 2536 suppressed only viral DNA synthesis (Fig. 2A, 3A, and 3B), suggesting that PLK1 may have a role in capsid-associated reverse-transcription of pgRNA into rcDNA. Conversely, inhibition of PLK1 could prevent these processes, based on capsid assembly. Since HBc has an essential and dynamic role in this process of capsid assembly, pgRNA encapsidation, and rcDNA biosynthesis, I investigated whether HBc is a PLK1 phosphorylation substrate.

To determine whether HBc is a direct target of PLK1, I performed in vitro kinase assays with purified recombinant HBc and PLK1 (Fig. 7). I observed in vitro PLK1 phosphorylation of HBc using either recombinant HBc or HBc isolated from HepaRG-TR-

HBc cells (Fig. 7A); furthermore, addition of BI 2536 at 500 nM inhibited this phosphorylation (Fig. 7A, left panel). The C-terminal domain of HBc (CTD), spanning amino acids 143 to 183 (Fig. 7B) contains eight S/T residues. I performed in vitro PLK1 kinase assays with GST-CTD fusion peptide isolated from transfected cells and featuring either wild type or mutated CTD sequences at all S/T sites. This approach was validated, by showing that fusion protein with 7 (not including T147) or all 8 S/T positions mutated

58 exhibited absence of PLK1 phosphorylation (Fig. 7C). PLK1 requires a priming phosphorylation of its substrate, usually by CDK1 or CDK2 kinases at S/P sites.

Interestingly, HBc is known to be phosphorylated by CDK2 at three SP phosphorylation sites (Fig. 7B). Accordingly, I tested whether HBc proteins containing S/A and S/D substitutions at all three SP positions (labeled as 3AP or 3DP) could serve as PLK1 substrates. Interestingly, only the phosphomimetic substitutions, i.e., S/D at positions

155, 162, and 170 (referred to as 3DP), exhibited phosphorylation by PLK1 (Fig. 7C).

Next, I observed that S168A exhibited significantly reduced phosphorylation in the context of the phosphomimetic substitutions at SP sites, whereas no difference in the phosphorylation by PLK1 was detected by T146A and T147A substitutions (Fig. 7D).

Single point mutations at residues 168, 176 and 178 all resulted in significant reduction of HBc phosphorylation by PLK1 (Fig. 7E). Therefore, I conclude that S168, S176 and

S178 are PLK1 phosphorylation sites in vitro.

4.3.8 S/A substitution of in vitro PLK1 phosphorylation sites diminish HBV

replication

To assess the in vivo significance for the identified in vitro Plk1 phosphorylation sites, I employed a trans-complementation assay for HBV replication. HuH7 cells were co-transfected with a plasmid that provided the HBV genome that had a point mutation in the HBc gene, inactivating the latter. Another plasmid provided WT or mutant variants of HBc. Co-transfection of HBc WT and the HBV genome lacking HBc resulted in successful HBV replication reflected in the production of cccDNA (Fig. 7F). Co- transfection of HBc variants with alanine (3AP) or aspartic acid (3DP) substitutions at the

3SP sites did not result in observable change in cccDNA production (Fig. 7F).

Interestingly, alanine substitutions at the 3 identified in vitro PLK1 phosphorylation sites

(168, 176 and 178) in the 3DP background resulted in almost 50% decrease in cccDNA production (Fig. 7F).

4.4 Discussion

The hepatitis B virus (HBV) is one of the most important human oncogenic virus

1,2,19. Approximately 250 million individuals worldwide are chronically infected by HBV, having increased risk for developing hepatocellular carcinoma (HCC)19. If clinicians dispose of several anti-HBV drugs, they have limited chemotherapeutic options for HCC.

It is thus crucial to identify novel molecules that target both virus biosynthesis and neoplastic transformation. In this study I provide evidence that the polo-like-kinase 1

(PLK1), a kinase already demonstrated to be involved in HCC, could represent a potential target to tackle both HBV replication and liver oncogenesis.

Earlier studies demonstrated that HBx activates PLK1 in a hepatocyte model cell line12, and that PLK1 was activated in the HepAD38 cellular model of HBV11. However, this cellular model of viral replication does not model physiologic infection. In this study, using physiologically relevant HBV infection models comprised of primary human hepatocytes and the HepaRG cell line, I show that indeed, HBV infection leads to early activation of PLK1. Here, I show that PLK1 phosphorylation occurs upon HBV infection in non-dividing and differentiated cells; the mechanism of PLK1 activation during HBV infection is yet to be determined. HBx protein, which is expressed very early after HBV infection, remains the main candidate for this activation, likely mediating PLK1

60 activation via the p38MAKP12,20. Here I provide evidence of the functional advantage to the viral life cycle of this early activation of PLK1 by HBV infection.

Employing PLK1 pharmacological inhibitors and an RNA interference approach, I have shown that PLK1 enzymatic activity is crucial for maintenance of HBV replication in non-dividing hepatocytes. Indeed, BI 2536, an ATP-dependent PLK1 inhibitor, drastically inhibited HBV replication in vitro in both HepaRG cells and PHH, as well as in vivo, in liver humanized FRG mice, without toxicity. Interestingly, the antiviral activity of BI 2536 was very potent with an EC50 ranging from 5 and 50 nM, depending on cell type used; such an antiviral efficacy is rarely obtained with host targeting agents (HTA) and corresponds to the efficacy of direct acting agents (DAA; such as nucleoside analogues or assembly inhibitors). Accordingly, an in vivo antiviral activity was also obtained with BI 2536 in

HBV-infected liver humanized FRG mice, and this antiviral activity was comparable to that obtained with entecavir or IFN- in the same model. The RNAi approach confirmed the PLK1 specificity, as kinase inhibitors are often non-specific. The antiviral phenotype obtained by RNAi targeting PLK1 was also very strong in vitro. The use of such an approach in vivo using siRNA that could be specifically targeted to infected hepatocytes could represent an interesting alternative to pharmacological inhibitors, which may be quite toxic when administrated systemically. Recently, it has been shown that RNAi nanoparticles in complex with PLK1 siRNA, administered systemically, efficiently delivers

PLK1 siRNA to the liver with no observable toxicity21.

The molecular mechanism underlying the proviral role of PLK1 has not been completely uncovered in this study. Nevertheless, I have shown that a target of PLK1 is

61 the capsid/core protein (HBc), responsible for capsid assembly around the pregenomic

RNA, and indirectly pgRNA reverse-transcription, as the latter occurs only within capsids. PLK1-mediated phosphorylation requires prior phosphorylated “primed” substrates22. Here I presented evidence that HBc is a direct target of PLK1 in vitro, and importantly, efficient phosphorylation of HBc required prior phosphorylation at positions S155/S162/S170, sites shown to be phosphorylated by CDK25,9. Our studies identified S168, 176 and 178 as potential sites for PLK1 phosphorylation in vitro, dependent on prior phosphorylation of S/P sites at positions S155, S162 and S170. Thus,

PLK1-mediated phosphorylation of HBc in a sequential phosphorylation process could directly mediate the dynamic events associated with capsid formation, pgRNA encapsidation and reverse transcription. Indeed, substitution of serines at these sites with alanines resulted in major reduction in cccDNA production. Further mechanistic studies and molecular tools (phospho-specific antibodies) are required to fully delineate this mechanism of PLK1 involvement.

Many other viral proteins are directly phosphorylated by PLK1, including the nonstructural 5A protein from HCV23, the mump virus nucleoprotein24, and the parainfluenza virus 5 (PIV5) P protein25. PLK1-mediated phosphorylation of the target can either lead to its degradation or change in function; therefore, PLK1 can either act as a proviral or antiviral factor. For HCV, PLK1 seems to be a proviral factor, as it contributes to the hyper-phosphoryaltion of NS5A23, which is a crucial event to switch from a replicative cycle (i.e. amplification of viral RNA) to a productive one (i.e. generation and secretion of novel virions). Nevertheless, the use of anti-PLK1 drugs has

62 only limited value to inhibit HCV replication, because the virus replicates better in vitro in dividing cells, thus precluding non-toxic PLK1 inhibition. But in the case of HBV, this framework for a potential use of PLK1 targeting drugs does exist. Indeed, HBV only replicates in non-dividing and highly differentiated cells. Although I have shown here that an anti-HBV phenotype could be obtained in vivo, more experiments are warranted to further demonstrate the safety of such an HTA-based approach. Many PLK1 pharmacological inhibitors as well as strategies based on PLK1 silencing are in current development for treating several cancers26,27; a repositioning of such drugs for the treatment of CHB patients could be foreseen. The fact that PLK1 inhibitors could reinforce the inhibition of the neo-synthesis of encapsidated rcDNA, as nucleoside analogues already do, represent an excellent framework for the potential development of combination therapies.

4.5 Materials and Methods

4.5.1 Chemicals, antibodies, and others reagents

All chemicals were purchased from Sigma Aldrich unless otherwise specified. PLK1 inhibitors (BI 2536, BI 6727, MLN 0905 and ON 01910) were from Selleckchem.

Polyclonal HBc28, and commercially available HBc antibodies were used for immunofluorescent staining and immunoblots. Rabbit polyclonal anti-HBc was from

Dako, and monoclonal anti-HBc from Abcam (Clone C1). PLK1 antibodies used: rabbit polyclonal (Abcam 21738-100) for detection of phospho-PLK1-S137 in WB and IF; rabbit monoclonal (Ab115095) for detection of phospho-PLK1-T210 in WB and IF, and rabbit polyclonal (Ab109777) for detection of PLK1. Small interfering RNA (SmartPool or

63 sequence designed) were purchased from Dharmacon, transfected into dHepaRG or

PHH using Darmafect-1 reagent, following manufacturer’s instructions. Tenofovir and

HBV capsid assembly inhibitors Bay41-410929 were kindly provided by Gilead Sciences and Novira Pharmaceutics, respectively. IFN- (i.e. Roferon) was from Hoffmann-La-

Roche.

4.5.2 HepaRG cells and primary human hepatocytes & infection by HBV

Human liver progenitor HepaRG cells30 were cultured for 2 weeks in complete William’s medium (Gibco) supplemented with 10% FCS Fetal Clone II serum (Thermo scientific), 50

U/mL penicillin/streptomycin (Gibco), 2 mM glutaMax (Gibco), 5 μg/mL human insulin

(Sigma), and 5 × 10−5 M hydrocortisone hemisuccinate (UpJohn, SERB) at 37°C in humidified CO2 (5%) incubators and differentiated into hepatocyte-like cells by a treatment with 1.8% DMSO (Hybrid-max, Sigma) for two weeks as described31.

Engineered HepaRG cell lines were also used. HepaRG-TR-PLK1CA cell line enabling tetracycline-inducible expression of a constitutively active form of PLK1 (i.e. T210D mutant 32) was constructed as described for HepaRG-TR-HBc cell line 33. Primary human hepatocytes (PHHs) were from surgical liver resections, after informed consent of patients (kindly provided by Pr. Rivoire (CLB, Lyon); agreement numbers DC-2008-99 and DC-2008-101). PHH were prepared as previously described34 and cultured in complete William’s medium supplemented with 1.8 % of DMSO.

4.5.3 HBV infection and analysis of viral parameters during replication

Differentiated HepaRG cells (dHepaRG) and PHH were infected as previously described

35, with HBV inoculum prepared either from HepG2.2.15 or HepaAD38 cells. Intracellular

64 accumulation of viral RNA and DNA, secretion of HBe and HBs antigens were monitored by qPCR, RTqPCR, southern blotting, and ELISA, as described35. Briefly, HBeAg and

HBsAg were quantified in culture medium by ELISA, using a chemiluminescence immunoassay kit (Autobio, China) following manufacturer’s instructions. Total DNA was purified from infected cells using MasterPure™ Complete DNA Purification Kit

(Epicentre). Southern blot analysis of HBV DNA was performed using equal amounts of total DNA, electrophoresed on 1.1% agarose gels, hybridized to HBV DNA radiolabelled with [α-32P]CTP 31 and analyzed by autoradiography. Total RNA was extracted from infected cells using NucleoSpin® RNA (Macherey-Nagel) and transcribed into cDNA using

SuperScript III reverse transcriptase (Invitrogen, Carlsbad, USA). Real-time PCR for total

HBV DNA and cccDNA was performed using LightCycler 96 (Roche) as described35.

4.5.4 Transfections, plasmids and siRNAs

Transient transfections in HEK293T cells were carried out employing Lipofectamine 2000

(Invitrogen) as described by manufacturer. HBc-WT (EL43), HBc-3A (EL113) and HBc-3D

(EL114) were kindly provided by Dr. D. Loeb, derived from EL43 plasmid 7. The CTD-GST fusion protein plasmids: GST-HCTD141 (CTD WT) and, the GST-HCTD141-AAAAAAA (CTD

7A) 5 were a kind gift from Dr. Hu. SiRNAs (Smart pool grade; Dharmacon) for PLK1, HBV and HCV (5-25nM) were transfected in HBV-infected HepaRG or PHH using Dharmafect I

(Dharmacon) following manufacturer’s protocol.

4.5.5 Humanized FRG mouse model and HBV infection

All animal studies were reviewed and approved by a local ethical committee. The highly immunosuppressed FRG mice (Fah-/-/Rag2-/-/Il2rg-/-), deficient for T-, B- and NK-cells

65 were used 36 and maintained in pathogen-free facility. High quality, cryopreserved human hepatocytes were purchased (BD, Biosciences) and injected via an intra-splenic route into 2 to 3 month old mice as described previously 36. Liver humanized Fah-/-/Rag2-

/-/Il2rg-/- mice featuring serum production of human albumin, at least 5 mg/mL, were infected with 200 μl of HBV inoculums (1.108 veg to 1.109 veg in PBS) via intra-peritoneal route 37.

Mice were treated by intraperitoneal injection of BI 2536 (10mg/kg/twice a week) for a month. Serum was harvested every week by retro-orbital bleeding and stored at -80°C in aliquots for further antigenemia and viremia analysis. Mice were sacrificed at week 8 post-infection and hepatic tissues were frozen and processed for virologic parameter analyses or fixed in formalin and embedded in paraffin for immune-staining.

4.5.6 In vitro PLK1 kinase assays

Assays were performed as previously described 11using recombinant PLK1 (BPS

Bioscience, Protein One). Core protein was immuno-purified from HepaRG-TR-HBc cell line or purchased from Meridian Life Science, Inc. Site-directed mutagenesis of putative

PLK1 phosphorylation sites in HBc-WT and HBc-3D was performed employing the Quick- change Lightning site-directed mutagenesis Kit (Agilent). Point mutations in the GST-

CTD-WT and GST-CTD-7A plasmids were introduced following the same procedure.

Mutations were confirmed by DNA sequencing. For protein staining, PageBlue™ Protein

Staining Solution (ThermoFischer) was used following manufacturer’s protocol.

4.5.7 Trans-complementation assay

HuH7 cells were grown in DMEM media, 10% FBS and 50 U/mL penicillin/streptomycin

(Gibco). Transient transfections in HEK293T cells were carried out employing

Lipofectamine 2000 (Invitrogen) as described by manufacturer. DsRed-HBV1.1-∆HBc plasmid provided 1.1 copy of HBV genome lacking HBc expression due to a point mutation introducing a stop codon. (DsRed-HBV1.1 plasmids were kindly provided by Dr.

Tsai-Yu Lin. cccDNA was measured by qPCR, HBeAg and HBsAg by ELISA 2-3 days after transfection.

4.5.8 Statistical analysis

Statistical analysis was performed using two-way Anova, t tests, or nonparametric

Mann-Whitney tests using the GraphPad Prism software. For all tests, p-value ≤0.05 (*),

≤0.01 (**), and ≤0.001 (***) were considered as significant.

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4.7 Acknowledgments

I thank Lydie Lefrançois, Judith Fresquet and Maud Michelet for isolation of primary human hepatocytes, as well as the staff from Pr Michel Rivoire’s surgery room for providing us with liver resection.

Thanks to Adrien Foca and David Durantel for help with experiments for figures 1, 2B,

2C, 4, 3S and 6. Experiments for figures 5, 5S and 6 were performed by Floriane Fusil and analyzed by Adrien Foca.

This work was supported by grants from ANRS (French national agency for research on

AIDS and viral hepatitis; several grants from CSS4) and by INSERM core grants. This work was also supported by the DEVweCAN LABEX (ANR-10-LABX-0061) of the “Université de

Lyon”, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), and NIH grant DK044533-19 to OA. AD received a Chateaubriand Fellowship from the French Embassy in Washington DC.

4.8 Figure legends

Figure 1: HBV infection activates PLK1. dHepaRG cells (A, B and C) or PHH (D) were infected with low dose (100 vge/cell) or high dose (1000 vge/cell) HBV. A) Cells were harvested at indicated time points, RNA extracted and subjected to RT-qPCR. Fold induction of mRNA expression level of PLK1 and HBV were normalized to housekeeping genes, compared to mock infection. B) Immunoblot of PLK1 and phosphorylated PLK1

(S137 and T210) using whole cell extracts (WCE) of mock- or HBV-infected dHepaRG cells isolated at indicated time points post-infection (p.i.). Quantification by

73 chemiluminescence was done with a ChemiDoc XRS+ system (Biorad). C)

Immunofluorescence microscopy of indicated proteins +/- HBV infection in dHepaRG cells at different time p.i. Cells were fixed by 2% PFA and stained with indicated antibodies. D) Immunoblots of PLK1 and phosphorylated PLK1 using WCE from mock- or

HBV-infected PHH cells.

Figure 2: Effect of BI 2536 on HBV replication in non-dividing and non-transformed dHepaRG. Differentiated HepaRG cell (A, B, C, and D) were infected with 100 vge/cell of

HBV for 7 days followed by treatment with increasing concentration of PLK1 inhibitor BI

2536, as indicated, for 3 days. A) Secreted antigens HBsAg and HBeAg quantified by ELISA from supernatants of HBV infected dHepaRG cells, day-10 p.i. Total RNA and DNA extracted from infected cells, day-10 p.i., were analyzed by HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes and compared to mock-infected cells. Results are presented as fold change in expression or secretion, compared to untreated controls and are mean ± SEM of at least 3 independent experiments. B) Cell viability of HepaRG cells measured using Cell Titer Glo One Solution Assay, with increasing concentration of BI

2536, as indicated. DMSO and puromycin were used as controls. C) Immunofluorescence microscopy of indicated proteins, +/- BI 2536 in infected dHepaRG cells. D).

Figure 3: Effect of BI 2536 on HBV replication in non-dividing and non-transformed PHH.

PHH (A, B, and C) were infected with 100 vge/cell of HBV for 4 days followed by treatment with increasing concentration of PLK1 inhibitor BI 2536, as indicated, for 3 days. A)

Quantification of secreted HBsAg and HBeAg by ELISA using supernatants of HBV infected

PHH on day 7 p.i. Total RNA and DNA extracted from HBV infected cells, day-7 p.i., were analyzed by HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes, and compared to mock-infected cells. Fold change in expression or secretion quantified relative to untreated controls represent mean ± SEM of at least 3 independent experiments. B) A representative Southern blot is shown (n=3). Total DNA extracted from cells on day-7 p.i., was analyzed by agarose gel electrophoresis, transferred onto nylon membrane, and hybridized to radioactive HBV probe as previously described31.

Figure 4: Effect of PLK1 knockdown on HBV replication in dHepaRG and PHH. PHH or dHepaRG cells infected with 100 vge/cell of HBV for 7 days were transfected with siRNA

(5 or 25 nM) targeting PLK1(siPLK1) or HBV (siHBV) or HCV (siHCV), as indicated. A and C)

Total RNA and DNA were extracted and subjected to RT-qPCR or qPCR with PLK1 or HBV primers. Quantification of PLK1 mRNA and HBV DNA is relative to absence of siRNA transfection (no siRNA). Results represent the mean ± SEM of at least 3 independent experiments. B) Immunofluorescence microscopy of indicated proteins, +/- siPLK1 transfection in HBV infected dHepaRG cells. Cells were fixed by 2% PFA at day 10 p.i. and stained with indicated antibodies. D) A representative Southern blot is shown. DNA was extracted from infected PHH transfected by indicated siRNA.

Figure 5: Effect of BI 2536 on HBV replication in liver humanized HBV-infected FRG mice.

Eight mice, engrafted with human PHH for two months were infected with HBV (5.10e8

75 vge/mouse). On week-4 p.i., treatment was initiated as indicated. BI 2536 (10mg/kg) was injected twice per week intraperitoneally (IP) for 4 weeks. Blood was collected at the end of week-1, 2, 4, 6, 7, and 8 and antigenemia (A and B) and viremia (C) monitored by ELISA and qPCR. D) E)

Figure 6: Combination treatment of BI 2536 and tenofovir, IFN-a, or Bay41-4109.

Differentiated HepaRG were infected with 100 vge/cell of HBV for 7 days, followed by treatment with indicated concentration of drugs for 3 days. A) HBeAg secretion in supernatant was monitored by ELISA. B) Intracellular HBV DNA was quantified by qPCR.

Figure 7: HBc is a phosphorylation substrate of PLK1 in vitro. In vitro PLK1 kinase assays of HBc and site-directed mutants. A), recombinant HBc or immune-affinity purified HBc isolated from HepaRG-TR-HBc cells, and PLK1 were used in in vitro PLK1 kinase assays.

Reactions were performed with (+) or without (-) PLK1 inhibitor BI 2536 (500nM), as indicated, in the presence of γ32P-ATP and analyzed by SDS-PAGE and autoradiography.

B) Amino acid sequence of C-terminal domain (CTD) of HBc. S/T residues are numbered

1-8; CDK2 SP sites, and putative PLK1 phosphorylation sites are indicated. C, D and E) In vitro kinase assays of WT HBc and site-directed mutants, as indicated. Commassie blue staining shows the same gel used for autoradiography. Relative intensity quantified by

ImageJ software is ratio of signal from in vitro kinase reaction versus corresponding

Commassie blue-stained band, expressed relative to WT (n = 3).

4.9 Supplementary figure legends

Supplementary Figure 1: Dose response effect of competitive and non-competitive PLK1 inhibitors on HBV replication in non-dividing hepatocytes. PHH were infected with 100 vge/cell of HBV for 7 days followed by treatment with different concentration (0, 5, 50,

500 and 5000 nM) of indicated PLK1 inhibitors for 3 days. A) Total intracellular HBV DNA was extracted and analyzed by Southern blot. B) Infected dHepaRG cells on day-7 p.i. were treated with 500 nM of different PLK1 inhibitors for 3 days; total HBV DNA was extracted and quantified by qPCR. Results are expressed as fold change normalized to housekeeping genes and are from 3 independent experiments.

Supplementary Figure 2: Comparison of batches of BI 2536. Differentiated HepaRG cells infected with HBV (100 vge/cell) were treated with increasing concentration of BI 2536

(0, 0.5, 5, 50, 500 and 5000 nM) on day-7 p.i., for 3 days. Total DNA was extracted and

HBV DNA was quantified by qPCR relative to housekeeping genes. Results are shown as fold change in HBV DNA, compared to mock- treated. Two different batches of BI 2536, from different providers were used as indicated. Results are represented as the mean ±

SEM from at least 3 independent experiments.

Supplementary Figure 3: Effect of BI 2536 on establishment of HBV infection in dHepaRG. A) Diagram of infection and treatment protocol. B) Differentiated HepaRG cell were treated with increasing concentration of BI 2536 for 3 days, then infected with 100 vge/cell of HBV for 7 days. Secreted antigens HBsAg and HBeAg were quantified by

ELISA from supernatants of HBV infected dHepaRG cells at day-10 p.i. Total RNA and

DNA extracted from infected cells on day-10 p.i. were analyzed by HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes, and compared to mock-treated cells. Fold change in expression or secretion was quantified relative to untreated controls, and are mean ± SEM of at least 3 independent experiments. C) Immunofluorescence microscopy of indicated proteins, +/- transfection of siPLK1 in HepaRG cells. siRNA was transfected for 3 days prior to infection and cells were fixed on days-3 or 7 p.i.

Supplementary Figure 4: Limited over-expression of PLK1 leads to an increase of HBV replication in dHepaRG. A) Differentiated HepaRG-TR-PLK1CAcells were induced by the addition of tetracycline (0, 16, 80 400, 1000 or 2000 ng/mL) for 72 hours and PLK1 levels analyzed by immunoblotting. B) dHepaRG-TR-PLK1CA cells were infected with 100 vge/cell of HBV for 7 days followed by induction of PLK1 by the addition of indicated amount of tetracyline for 3 days. HBeAg secretion was analyzed by ELISA. Total DNA was extracted and HBV DNA was measured by qPCR. Results are shown as fold change in

HBV DNA as compared to control cell line. Results are represented as the mean ± SEM of

2 independent experiments.

Supplementary Figure 5: Effect of IFN-α and entecavir on HBV replication in liver humanized HBV-infected FRG mice. Eleven mice, engrafted with human PHH for two months, were infected with HBV (5.10e8 vge/mouse). At week-4 post infection treatment was started in 8 mice and 3 other mice were mock treated. IFN- (25 g/kg) was injected twice a week by IP injections, for 4 weeks. ETV (2.5 g/day) was

78 administrated in drinking water. Blood was taken at the end of weeks 4 and 8 and viremia monitored by qPCR.

4.10 Figures

CHAPTER 5. HOST CELL INTERACTOME OF HBV CORE PROTEIN REVEALS A NOVEL LINK TO PARASPECKLE COMPONENTS

5.1 Introduction

Studies of virus-host interactions continue to provide critical insights into host pathobiology upon infection. Viruses manipulate specific host pathways to allow efficient viral amplification and/or evasion of host immune defenses. Due to genomes size constrains, the HBV genome is only 3.2KB, HBV proteins such as the X protein (HBx) and the core antigen (HBc) have evolved various functional roles beyond their contribution to viral replication 1,2. For example, previous studies have demonstrated how HBx activates host mitogenic pathways eventually leading to liver cell transformation3,4. The viral HBc protein, which forms the viral capsids, plays many roles in viral replication beyond its structural function. Notably, several biochemical studies have demonstrated the interaction between HBc and host cell components that help facilitate efficient viral replication (reviewed in chapter 2). HBc has a unique C-terminal domain, which is characteristically rich in arginine and is capable of binding viral and host RNAs 5 with similar levels of cooperativity 6. Arginine rich domains with nucleic acid binding ability are commonly shared features among several viral proteins 7. Such motifs enable viral proteins like the human immunodeficiency virus-1 (HIV-1) Rev to interact with host ribonucleoprotein complexes 8 therefore enabling HIV-1 to hijack host RNA

91 splicing machinery. Given the structural similarity, HBc may interact with host proteins in a similar fashion and accordingly, I employed proteomics-based approaches as well as biochemical assays to investigate this hypothesis.

Affinity purification of viral proteins combined with mass spectrometry-based proteomics is a powerful tool to study virus-host interaction, as it helps identify putative host partners and likely cellular pathways deregulated by the virus. Aided by affinity purification/mass spectrometry (AP/MS) researchers were able to study systematically protein complexes hijacked by several viruses including human immunodeficiency virus

1 (HIV-1)8, hepatitis C virus (HCV)9 and Kaposi’s sarcoma-associated herpesvirus

(KSHV)10. As for HBV, HBx remains the best-characterized protein in terms of global host-interaction partners. Proteomic- based approaches identified several novel host factors hijacked by HBx in HBV replication and infection models, including the interferon-inducible phospholipid scramblase 1 (PLSCR1), apolipoprotein A-I (apoA-I) and the apoptosis induced factor (AIF) 11–14. The role of HBx as a week oncogene is well- established, and the cellular pathways manipulated by HBx during transformation are well-studied15. When it comes to HBc, the global host interactome map remains an unexplored territory. Aside from text-mining studies to catalogue HBc-host interactions

16 and traditional yeast-two-hybrid screens of human liver cDNA library 17, no comprehensive analysis of global HBc-host interactions has been reported to date. In this study, I employed affinity purification of epitope-tagged HBc protein from the human bipotential progenitor liver cell line (HepaRG) 18. To provide a comprehensive analysis of HBc interactome, I combined affinity purification with mass spectrometry-

92 based proteomics to identify novel physical in vivo interaction partners of HBc. This analysis identified previously reported as well as novel putative HBc interacting partners.

Interestingly, HBc interacting proteins include components of the nuclear speckle and paraspeckle bodies. Nuclear speckles and paraspeckles are dynamic nuclear subdomains, enriched for serine- and arginine-rich (SR) proteins that comprise the mRNA-splicing machinery 19. The distinct paraspeckles are located in close proximity to the nuclear speckles, hence the name 20. Both nuclear speckle and paraspeckle bodies are known to associate with long noncoding (lnc)RNAs21,22. In a screen for nuclear transcripts associated with the SR-rich splicing machinery, two lncRNAs, nuclear enriched abundant transcripts (NEAT), NEAT2 an NEAT1 were identified 21. The same study demonstrated, using RNA fluorescence in situ hybridization (FISH), that NEAT2 localizes to the nuclear speckles while NEAT2 localizes to their periphery21, now a distinct domain called paraspeckles. It is well-accepted now that paraspeckle formation proceeds in conjugation with the biogenesis of lncRNA NEAT123,24. Genetic Loss of

NEAT2 has been recently associated with slower tumor growth and reduction in metastasis in mammary tumors25. Both NEAT1 and NEAT2 expression levels were reported to increase in response to viral infection26,27 implying a role in viral infection.

Recently, lncRNA NEAT2 and NEAT1 were shown to physically interact with proteins that are resident components of the nuclear speckles and paraspeckles, respectively, thereby confirming their structural role in the organization of these nuclear bodies 10. Our mass- spec analysis identified some of these proteins as putative HBc interacting partners.

Given the capacity of HBc to bind RNA6, I hypothesized that HBc associates with lncRNAs that are part of these nuclear complexes. Ribonucleoprotein immunoprecipitation (RIP) of HBc confirmed that HBc associated with NEAT1 and NEAT2 in cellular models of HBc replication and infection.

5.2 Results

5.2.1 Identification of HBc interacting proteins by AP-MS

Assembly competent strep-tagged HBc protein was expressed from an HBc expressing HepaRG cell line (HepaRG-TR-HBc). Affinity purified and processed HBc was subjected to mass spec analysis. I compared the HBc interactome from this study with

HBc interactomes curated from the literature in a previous study16 (Fig. 1). Among the previously reported interacting partners, Serine/Arginine-rich protein-specific kinase 1

(SRPK1)28, PDZ domain-containing protein GIPC117, nucleophosmin29, Phosphatase 2A inhibitor (I2PP2A)/protein SET30 and Cyclin-dependent kinase inhibitor 1 (CDKN1A)31 were also identified in my study. My study also identified additional putative HBc interacting proteins (Table 1). In addition to identifying SRPK1, a well-documented HBc binding partner 28,32,33, I also identified several proteins that are known to interact with

SRPK1 namely SRSF134, SRSF9, DHX9, CDKN2A, DDX21 and DDX50 (BioGRID3.4 database).

A complex of these proteins generally known as serine arginine rich protein (SR-rich) complexes with SRPK1 play key roles in alternative RNA splicing and contribute pleiotropically to regulation of gene expression 35. Interestingly, SR-rich proteins are also known to serve as adaptor proteins for mRNA- and ribonucleoprotein- export factor nuclear RNA export factor 1 (NFX-1 or TAP)36. Given that the core protein and viral pre-

94 genomic RNA (pgRNA) may exploit NFX-1/TAP machinery for nuclear exit 37, my studies have likely identified additional factors involved in this process and they deserve biochemical validation.

5.2.2 An overlap between interactomes of HBV core protein and HIV-1 Rev

protein

Both HBV and HIV have common routes of transmission (blood borne) and persons at high risk for infection of HIV are also likely to be at high risk for HBV infection. Remarkably, there is evidence of prior HBV infection in approximately 90% of

HIV-infected persons38, 5-15 % among whom are chronic carriers of HBV. This observation suggests that some of the cellular pathways hijacked by both viruses may be common. I noticed a notable degree of overlap in high confidence interactors between HIV-1 Rev8 and HBc with a common group of 32 proteins (Fig. 2). These common interacting proteins are likely involved in cellular processes broadly exploited by viruses for purposes of their replication or evasion from host immunity regardless of how distinct these viruses are in their pathobiology. Further study of these proteins and associated pathways promises to expand our understanding of such pathways.

5.2.3 STRING network analysis reveals paraspeckle proteins as HBc interacting

partners

To gain a global understanding of the human HBc interactome and to identify novel complexes and pathways hijacked by HBc, I performed a STRING network analysis, which provided a global map of HBc interactome (Fig. 3) as well as gene ontology (GO) analysis. I observed that HBc interacted with several members of the nuclear speckle

95 and paraspeckle complexes. In particular, I identified heterogeneous nuclear ribonucleoprotein M HNRNPM, splicing factor proline/glutamine-rich (SFPQ) and non-

POU domain containing protein (NONO) as HBc binding proteins; all are resident components of paraspeckle nuclear bodies 20,23. Of the proteins that localize to the nuclear specks, I found peptidyl prolyl cis-trans isomerase 1 (PIN1), RNA binding motif protein 39 (RBM39) and DEAD box polypeptide 3, X-linked RNA helicase (DDX3X) of particular interest, as they have also been identified independently by colleagues in

France (Durantel, personal communication) as HBc interacting partners. Other newly identified HBc binding proteins, although not biochemically validated, such as

HNRNPA1, HNRNPF, HNRNPH, HNRNPU, FUS and RBM14 that localize both to nuclear speckle and paraspeckle bodies were also identified. Interestingly, these proteins were recently identified as interacting partners for lncRNAs NEAT1 and NEAT2 in a pull-down study of these lncRNAs using specific oligonucleotides followed by MS analysis (Table 2)

22. Whether HBc physically associated with all these proteins and/or lncRNAs is yet to be determined.

5.2.4 HBc associates with lncRNAs NEAT1 and NEAT2

MS analysis implicated a novel association between HBc and proteins that are structural components of the nuclear speckle and paraspeckle bodies as well as proteins that localize to both nuclear subdomains. Therefore, it was intriguing to test whether

HBc also associated with NEAT1 and NEAT2 in vivo. I performed ribonucleoprotein immunopericipitation (RIP) assays to pull down HBc from lysates of HBc expressing liver cells, and I isolated and quantified the associated RNAs. Remarkably, both lncRNAs

NEAT1 and NEAT2 associated with the core protein (Fig. 4). This indirectly supports a model where HBc associates or localizes at nuclear speckles and paraspeckles. However, this point is yet to be demonstrated by direct co-immunoprecipitation (co-IP) or co- immunofluorescence experiments.

5.2.5 HBc binds lncRNAs NEAT1 and NEAT2 during HBV replication and

infection

Since the HBc interactome / MS analysis (Table 1) and HBc RIP experiments (Fig.

4) were performed in the absence of viral replication, to gain more insights into HBc function in the context of the viral machinery, I performed HBc-RIP experiments using

HBV replicating hepatoblastoma cells (HepAD38)39 as well as HBV-infected HepG2hNTCP cells. In both models of HBV replication and infection, HBc associated with NEAT1 and

NEAT2 implying a role for NEAT1 and NEAT2 in HBV pathogenesis (Fig. 5). Interestingly, no change in NEAT1 or NEAT2 expression level was observed upon HBV infection in

HepG2hNTCP cells (Fig. 6).

5.2.6 Phosphorylation status of HBc regulates interaction with lncRNAs

Phosphorylation of HBc is a key regulator of HBc-mediated RNA encapsidation40 and is particularly essential for facilitating HBc nucleic acid chaperone activity41. I wondered if HBc phosphorylation influences HBc binding to NEAT1 and NEAT2. To investigate this hypothesis, I performed RIP experiments with HBc variants carrying the following substitution mutations: HBc-3AP with 3 Ser/Ala substitutions at the 3 SP sites,

HBc-3DP with 3 Ser/Asp substitutions at the 3 SP sites, HBc-3DP/3A with 3 Ser/Asp substitutions at the 3 SP sites, as well as 3 Ser/Ala substitutions at Plk1 phosphorylation

97 sites 176, 178 and 181 and finally HBc 4A with 4 Ser/Ala substitutions at sites 168, 176,

178 and 181 in addition to HBc WT. While no significant difference was observed among the different HBc variants with regards to their ability to bind NEAT1 and NEAT2, a consistent decrease with the HBc-3DP/3A binding to NEAT1 was observed when compared to WT or 3DP (Fig. 7). I interpret this to mean that loss of Plk1 phosphorylation may regulate HBc binding to NEAT1, although additional studies are required in support of this preliminary finding.

5.3 Discussion

This study provides the first global analysis of HBc human interactome. Using affinity-purification and mass spectrometry, I identified HBc interacting partners that have been previously described as well as novel putative one. In particular, I observed several SR-rich proteins as potential HBc interacting partners. Previous studies have identified SR-rich proteins such as SRSF1 as regulators of viral biogenesis42–45. While

SRPK1 is an established interacting partner of HBc 28,33, the significance of this interaction seems ambiguous and probably secondary for the viral life cycle- since the kinase activity has no impact on viral replication32. To our interest, SRSF1, which is a canonical SRPK1 phosphorylation substrate, could shed more light on the significance of

HBc-SRPK1 interaction. While SPRK1-HBc interaction may seem redundant, HBc could simply function as a competitive inhibitor of SRPK1 substrates. Other viral proteins such as Human papillomavirus type 1 E1^E4 protein is a documented example of such case46.

Interaction between HBc and SRSF1, a canonical substrate of SRPK1, could also be interpreted as an inhibitory interaction. Indeed, phosphorylation status of SR-rich

98 proteins not only modulates their splicing activity and subcellular localization but also regulates their interaction with NFX-1/TAP complex 47. Remarkably, NEAT2 is another key regulator of SR-rich protein phosphorylation 48; whether NEAT2-HBc interaction is an additional regulatory layer for SR-rich protein functions remains to be investigated. A more comprehensive analysis of all such players and how HBc affects/regulates their function will certainly further expand our understanding of the pathobiology of HBV infection.

Only a subset of previously reported HBc interacting proteins was identified by my

MS analysis. In my study, I only used immuno-purified HBc protein in absence of other viral components. The presence of other HBV replication intermediates may be required for HBc interaction with certain host molecules. Future MS studies using HBV infection and replication cellular models will facilitate the identification and future characterization of such molecules. It is also important to note that some of these protein-protein interactions are transient in nature and may therefore fall outside the range of MS based detection methods. To enrich for week and transient interactions, it may be useful to specifically enrich for certain subcellular domains and/or use synchronized cell cultures.

Study of virus-host interaction during infection using host cells with replication competent-virus is optimal for the identification of critical host factors. Yet, such approach is not always practical. Alternatively, the use of ectopically expressed epitope- tagged individual viral proteins has proven valuable particularly when trying to decipher the function of specific viral proteins independent of other viral factors49–52. However, it

99 is noteworthy to remember that this approach does not recapitulate the viral pathogenesis in its entirety. In order to assess the in vivo relevance of such interactions in the context of HBV infection, future analysis using loss of function genetic approach will be useful. A knockdown or knockout of the different protein or RNA components will help characterize their significance for the viral life cycle. While no change in NEAT1 and NEAT2 expression was observed with HBV infection, HBV infection may result in other changes with regards to localization of HBc and/or HBV mini-chromosome inside the nucleus. The use of RNA FISH and other intracellular imaging techniques may help elucidate any changes in HBc and NEAT1/NEAT2 localization during infection.

Flavivirus infection- either by Japanese encephalitis virus or West Nile virus- reportedly caused elevated NEAT2 expression in mammalian cells26. Similarly, NEAT1 levels were elevated upon viral infection including influenza virus and herpes simplex virus as well as poly I:C 27. Notably, induction of NEAT1 was associated with enlarged paraspeckles and involved binding of NEAT1 to SFPQ, displacing the latter from the IL-8 promoter region. This resulted in the activation of IL-8 as well as IL-8 dependent innate- immunity genes 27.My studies show that HBc associates with NEAT1 and potentially with

SFPQ so it is reasonable to envisage a similar mechanism taking place during HBV infection.

5.4 Materials and Methods

5.4.1 Chemicals, antibodies, and others reagents

All chemicals were purchased from Sigma Aldrich unless otherwise specified.

Rabbit polyclonal anti-HBc was from Dako (B058601-1). Strep-coated MagStrep XT

100 magnetic beads were from (IBA, 2-4090-002). Protein G Dynabeads® were purchased from Thermo (10003D). 10X cell lysis buffer (Cell Signaling #9803) was prepared according to manufacturer protocol. Ambion Purelink DNase (12-185-010) kit was purchased from Thermo. Proteinase K (EO0492) was purchased from Thermo. Purelink

RNA Mini Kit (12183018A) from Thermo was used to isolate RNA. iScript cDNA Synthesis

Kit (170-8891; Bio-Rad) was used to synthesize cDNA.

5.4.2 Cell culture

Strep-tagged HBc protein was overexpressed in a HepaRG cell line (HepaRG-TR-

HBc) in a tetracycline-regulated fashion. Culture and maintenance of HepaRG cells was performed as previously described 1. Cells were collected in CST lysis buffer supplemented with protease inhibitor cocktail (sigma) following manufacturer protocol.

After brief sonication, lysates were cleared by centrifugation at 14000 rpm at 4°C.

Expression of strep-HBc was confirmed by western blotting of whole cell extracts obtained 2-3 days after tetracycline treatment. HepAD38 cells, HepG2hNTCP cells and

HEK293T cells were all maintained as described previously 53,54.

5.4.3 Affinity purification and Protein Digestion

Cleared lysates were incubated overnight at 4°C with 50 µL of MagStrep XT beads. Beads were washed 3x with cold lysis buffer then once with PBS. Proteins were denatured and reduced in 50 mM trimethyl ammounium bicarbonate containing 8M

Urea and 5 mM dithiothreitol for 30 min at 37 °C. The proteins were further alkylated in

15 mM iodoacetamide for 1 h in the dark at room temperature and digested with proteomics grade trypsin at 1:50 ratio overnight at 37 °C. 10% trifluoroacetic acid (TFA)

101 was added to the peptide mix to lower the pH below 3 and loaded onto a Stage-Tip 55 to efficiently remove buffer and small molecules. The peptides were eluted with 0.1% TFA in 80% acetonitrile and dried completely using a SpeedVac.

5.4.4 LC-MS/MS Analysis

Peptides were dissolved in 4 µL of 0.3% formic acid (FA) with 3% acetonitrile (ACN) and injected into an Easy-nLC 1000 (Thermo Fisher Scientific). Peptides were separated on a 45 cm in-house packed column (360 µm OD × 75 µm ID) containing C18 resin (2.2

µm, 100Å, Michrom Bioresources) with a 30 cm column heater (Analytical Sales and

Services) and the temperature was set at 50 °C. The mobile phase buffer consisted of 0.1%

FA in ultra-pure water (buffer A) with an eluting buffer of 0.1% FA in 80% ACN (buffer B) run over either with a linear 45 min gradient of 6%-30% buffer B at flow rate of 250 nL/min.

The Easy-nLC 1000 was coupled online with a Velos LTQ-Orbitrap mass spectrometer

(Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode in which a full-scan MS (from m/z 350-1500 with the resolution of 30,000 at m/z

400). The 10 most intense ions were subjected to collision-induced dissociation (CID) fragmentation (normalized collision energy (NCE) 30%, AGC 3e4, max injection time 100 ms).

5.4.5 Data analysis

Data analysis was performed with SEQUEST algorithms using Proteome

Discoverer software V2.0 (ThermoFisher Scientific, San Jose, CA, USA). RAW files were searched against Homo sapiens database with no redundant entries and manual added

HBV proteins sequence. Peptide mass tolerance was set at 10 ppm, and MS/MS

102 tolerance was set at 0.8 Da (0.05 Da for HCD data). Search criteria included a static modification of cysteine residues of +57.0214 Da, variable modifications of +15.9949 Da to include potential oxidation of methionines. Search was performed with Trypsin/P digestion and allowed a maximum of two missed cleavages on the peptides analyzed from the sequence database. The false discovery rates of proteins, peptides and phosphosites were set at 0.01. The minimum peptide length was six amino acids, and a minimum Andromeda score was set at 40 for modified peptides.

5.4.6 Bioinformatics

Common contaminants and non-specific binders (including keratins, actin, myosin, IgG etc…) were removed manually and the list was further filtered using the

Crapome (Contaminant Repository for Affinity Purification and Mass Spectrometry) online platform 56. I obtained protein-protein interaction information using the STRING database v.10.0 57 which includes direct (physical) as well as indirect (functional) associations. I used STRING in protein mode, the Homo sapiens interactome and only high confidence interactions (>0.7) were taken into consideration. Data was visualized using the STRING platform and high-resolution confidence views were downloaded and saved.

5.4.7 Ribonucleoprotein Immunoprecipitation (RNP-IP)

Cells were grown in 100mm cell culture plates. Cells were washed once with cold

PBS and 1 mL of cold PBS was added to each plate. Cells were then cross-linked by UV- irradiation, scrapped off the plates and centrifuged at 1000 rpm for 5 minutes. Pellets

103 were then resuspended in 600 µL of CST lysis buffer, briefly sonicated before centrifugation at 14000 rpm for 15 minutes at 4°C.

Cleared lysates were incubated with protein G beads conjugated with HBc antibody or rabbit IgG. 5 µL of antibody were used for every 20 µL of bead slurry. 1 % of whole cell lysates were kept as input for RNA extraction. After overnight incubation with the beads, RIP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5%

NP40) was used to wash beads 3X. The RNP complexes (on beads) were incubated with

30 units of RNase free DNase (Thermo) for 15 minutes at room temperature and further incubated with 0.1% SDS and 0.5 mg/mL proteinase K (Thermo) for 15 minutes at 55°C to degrade DNA and proteins respectively.

RNA was isolated from the immunoprecipitated material using Purelink RNA Mini

Kit as per manufacturer’s protocol. Primers for lncRNAs NEAT1, NEAT2 and HOTAIR were used to assess RNA levels by RT-qPCR. Analysis was performed using input enrichment method relative to IgG, normalized to 1.0% input.

104

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5.6 Figure Legends

Figure 1: Interactome analysis identifies previously known and novel HBc interacting partners. V- diagrams showing overlap between newly identified potential HBc interacting partners and previously reported HBV interacting proteins.

Figure 2: Overlap between HIV-1 Rev and HBc interactomes. A list of 32 different proteins that showed up as both HBc and HIV-1 REV interacting proteins.

Figure 3: Global map of HBc interactome. Map was created using STRING online platform using 226 top scoring HBV interacting partners.

Figure 4: HBc binds lncRNAs NEAT1 and NEAT2 in HepaRG-TR-HBc cells. HBc-RIP analysis shows binding of HBc to lncRNAs NEAT1 and NEAT2, HOTAIR is a control lncRNA. % input method was used relative to IgG.

Figure 5: HBc binds lncRNAs NEAT1 and NEAT2 in HBV replication and infection. HBc-RIP analysis in HepAD38 cells (A, B and C) after 5 days of HBV replication (n=2) and

HepG2hNTCP cells (E, F and G) as shown in D.

Figure 6: HBV infection does not induce NEAT1 and NEAT2. RT-qPCR analysis of NEAT1 and NEAT2 expression in HepG2hNTCP cells with and without infection (n=7).

Figure 7: HBc mutants bind lncRNAs NEAT1 and NEAT2. HBc-RIP analysis in HEK293T cells over-expressing different HBc mutants or WT. 3AP has 3 Ala substitutions at SP sites, 3DP has 3 Asp substitutions at SP sites, 3DP 3A has additional 3 Ala substitutions at

Plk1 sites 168, 176 and 178 and finally WT 4A has 4 Ala substitutions at sites 168, 176,

178 and 181.

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5.7 Figures

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116

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119

5.1 Tables

Table 2: NEAT1 and NEAT2 interacting proteins that are part of HBc interactome

120

Table 2: HBc interactome raw table Protein FDR Accession Description Exp. q- Confidence value High P35579 Myosin-9 OS=Homo sapiens GN=MYH9 PE=1 0 SV=4 High Q15149 Plectin OS=Homo sapiens GN=PLEC PE=1 SV=3 0 High Q13813 Spectrin alpha chain, non-erythrocytic 1 0 OS=Homo sapiens GN=SPTAN1 PE=1 SV=3 High Q01082 Spectrin beta chain, non-erythrocytic 1 0 OS=Homo sapiens GN=SPTBN1 PE=1 SV=2 High P35580 Myosin-10 OS=Homo sapiens GN=MYH10 PE=1 0 SV=3 High Q13085 Acetyl-CoA carboxylase 1 OS=Homo sapiens 0 GN=ACACA PE=1 SV=2 High Q96RQ3 Methylcrotonoyl-CoA carboxylase subunit alpha, 0 mitochondrial OS=Homo sapiens GN=MCCC1 PE=1 SV=3 High O43707 Alpha-actinin-4 OS=Homo sapiens GN=ACTN4 0 PE=1 SV=2 High P11498 Pyruvate carboxylase, mitochondrial OS=Homo 0 sapiens GN=PC PE=1 SV=2 High Q9HCC0 Methylcrotonoyl-CoA carboxylase beta chain, 0 mitochondrial OS=Homo sapiens GN=MCCC2 PE=1 SV=1 High P60709 Actin, cytoplasmic 1 OS=Homo sapiens GN=ACTB 0 PE=1 SV=1 High P05165 Propionyl-CoA carboxylase alpha chain, 0 mitochondrial OS=Homo sapiens GN=PCCA PE=1 SV=4 High A0A087WVQ6 Clathrin heavy chain OS=Homo sapiens GN=CLTC 0 PE=3 SV=1 High P12814 Alpha-actinin-1 OS=Homo sapiens GN=ACTN1 0 PE=1 SV=2 High O00763 Acetyl-CoA carboxylase 2 OS=Homo sapiens 0 GN=ACACB PE=1 SV=3 High E9PMS6 LIM domain only protein 7 OS=Homo sapiens 0 GN=LMO7 PE=1 SV=1 High P04264 Keratin, type II cytoskeletal 1 OS=Homo sapiens 0 GN=KRT1 PE=1 SV=6 High F8WD26 LIM domain only protein 7 OS=Homo sapiens 0 GN=LMO7 PE=1 SV=2 High E9PDR0 Propionyl-CoA carboxylase beta chain, 0 mitochondrial OS=Homo sapiens GN=PCCB PE=1 SV=1

121

High E9PMT2 LIM domain only protein 7 OS=Homo sapiens 0 GN=LMO7 PE=1 SV=1 High P05787 Keratin, type II cytoskeletal 8 OS=Homo sapiens 0 GN=KRT8 PE=1 SV=7 High Q6WCQ1 Myosin phosphatase Rho-interacting protein 0 OS=Homo sapiens GN=MPRIP PE=1 SV=3 High O00159 Unconventional myosin-Ic OS=Homo sapiens 0 GN=MYO1C PE=1 SV=4 High O75369 Filamin-B OS=Homo sapiens GN=FLNB PE=1 0 SV=2 High P35527 Keratin, type I cytoskeletal 9 OS=Homo sapiens 0 GN=KRT9 PE=1 SV=3 High Q92614 Unconventional myosin-XVIIIa OS=Homo sapiens 0 GN=MYO18A PE=1 SV=3 High Q15746 Myosin light chain kinase, smooth muscle 0 OS=Homo sapiens GN=MYLK PE=1 SV=4 High P35908 Keratin, type II cytoskeletal 2 epidermal 0 OS=Homo sapiens GN=KRT2 PE=1 SV=2 High O94929 Actin-binding LIM protein 3 OS=Homo sapiens 0 GN=ABLIM3 PE=1 SV=3 High P05783 Keratin, type I cytoskeletal 18 OS=Homo sapiens 0 GN=KRT18 PE=1 SV=2 High P08727 Keratin, type I cytoskeletal 19 OS=Homo sapiens 0 GN=KRT19 PE=1 SV=4 High P68032 Actin, alpha cardiac muscle 1 OS=Homo sapiens 0 GN=ACTC1 PE=1 SV=1 High P13645 Keratin, type I cytoskeletal 10 OS=Homo sapiens 0 GN=KRT10 PE=1 SV=6 High A0A0A0MRL6 Actin-binding LIM protein 1 OS=Homo sapiens 0 GN=ABLIM1 PE=4 SV=1 High O14639 Actin-binding LIM protein 1 OS=Homo sapiens 0 GN=ABLIM1 PE=1 SV=3 High P21333 Filamin-A OS=Homo sapiens GN=FLNA PE=1 0 SV=4 High A0A0A0MRM8 Unconventional myosin-VI OS=Homo sapiens 0 GN=MYO6 PE=4 SV=1 High Q7Z406 Myosin-14 OS=Homo sapiens GN=MYH14 PE=1 0 SV=2 High H0Y2S9 Myosin phosphatase Rho-interacting protein 0 (Fragment) OS=Homo sapiens GN=MPRIP PE=1 SV=3 High P07437 Tubulin beta chain OS=Homo sapiens GN=TUBB 0 PE=1 SV=2 High P68371 Tubulin beta-4B chain OS=Homo sapiens 0 GN=TUBB4B PE=1 SV=1

122

High O43795 Unconventional myosin-Ib OS=Homo sapiens 0 GN=MYO1B PE=1 SV=3 High O00515 Ladinin-1 OS=Homo sapiens GN=LAD1 PE=1 0 SV=2 High P25705 ATP synthase subunit alpha, mitochondrial 0 OS=Homo sapiens GN=ATP5A1 PE=1 SV=1 High O15042 U2 snRNP-associated SURP motif-containing 0 protein OS=Homo sapiens GN=U2SURP PE=1 SV=2 High A0A087WWU8 Tropomyosin alpha-3 chain OS=Homo sapiens 0 GN=TPM3 PE=1 SV=1 High P02538 Keratin, type II cytoskeletal 6A OS=Homo 0 sapiens GN=KRT6A PE=1 SV=3 High P09493 Tropomyosin alpha-1 chain OS=Homo sapiens 0 GN=TPM1 PE=1 SV=2 High P02533 Keratin, type I cytoskeletal 14 OS=Homo sapiens 0 GN=KRT14 PE=1 SV=4 High P67936 Tropomyosin alpha-4 chain OS=Homo sapiens 0 GN=TPM4 PE=1 SV=3 High Q9UPQ0 LIM and calponin homology domains-containing 0 protein 1 OS=Homo sapiens GN=LIMCH1 PE=1 SV=4 High Q9ULV4 Coronin-1C OS=Homo sapiens GN=CORO1C PE=1 0 SV=1 High P04259 Keratin, type II cytoskeletal 6B OS=Homo 0 sapiens GN=KRT6B PE=1 SV=5 High O95425 Supervillin OS=Homo sapiens GN=SVIL PE=1 0 SV=2 High Q6ZN40 Tropomyosin 1 (Alpha), isoform CRA_f 0 OS=Homo sapiens GN=TPM1 PE=2 SV=1 High J3QSX6 Actin-binding LIM protein 1 OS=Homo sapiens 0 GN=ABLIM1 PE=1 SV=2 High Q16643 Drebrin OS=Homo sapiens GN=DBN1 PE=1 SV=4 0 High H7BYY1 Tropomyosin 1 (Alpha), isoform CRA_m 0 OS=Homo sapiens GN=TPM1 PE=3 SV=1 High Q96I24 Far upstream element-binding protein 3 0 OS=Homo sapiens GN=FUBP3 PE=1 SV=2 High J3KN67 Tropomyosin alpha-3 chain OS=Homo sapiens 0 GN=TPM3 PE=1 SV=1 High O15020 Spectrin beta chain, non-erythrocytic 2 0 OS=Homo sapiens GN=SPTBN2 PE=1 SV=3 High P13647 Keratin, type II cytoskeletal 5 OS=Homo sapiens 0 GN=KRT5 PE=1 SV=3 High I3L204 Unconventional myosin-Ic (Fragment) OS=Homo 0 sapiens GN=MYO1C PE=1 SV=2

123

High P11021 78 kDa glucose-regulated protein OS=Homo 0 sapiens GN=HSPA5 PE=1 SV=2 High K7EJH8 Alpha-actinin-4 (Fragment) OS=Homo sapiens 0 GN=ACTN4 PE=1 SV=1 High Q9Y2D5 A-kinase anchor protein 2 OS=Homo sapiens 0 GN=AKAP2 PE=1 SV=3 High Q13045 Protein flightless-1 homolog OS=Homo sapiens 0 GN=FLII PE=1 SV=2 High J3QRS3 Myosin regulatory light chain 12A OS=Homo 0 sapiens GN=MYL12A PE=4 SV=1 High H0YK48 Tropomyosin alpha-1 chain OS=Homo sapiens 0 GN=TPM1 PE=1 SV=1 High O94832 Unconventional myosin-Id OS=Homo sapiens 0 GN=MYO1D PE=1 SV=2 High J3KND3 Myosin light polypeptide 6 OS=Homo sapiens 0 GN=MYL6 PE=1 SV=1 High Q9ULV0 Unconventional myosin-Vb OS=Homo sapiens 0 GN=MYO5B PE=1 SV=3 High P08779 Keratin, type I cytoskeletal 16 OS=Homo sapiens 0 GN=KRT16 PE=1 SV=4 High G8JLA2 Myosin light polypeptide 6 OS=Homo sapiens 0 GN=MYL6 PE=1 SV=1 High J3KN01 Afadin OS=Homo sapiens GN=MLLT4 PE=1 SV=1 0 High B1AK88 Capping protein (Actin filament) muscle Z-line, 0 beta, isoform CRA_d OS=Homo sapiens GN=CAPZB PE=1 SV=1 High Q8N3V7 Synaptopodin OS=Homo sapiens GN=SYNPO 0 PE=1 SV=2 High P38646 Stress-70 protein, mitochondrial OS=Homo 0 sapiens GN=HSPA9 PE=1 SV=2 High P15144 Aminopeptidase N OS=Homo sapiens 0 GN=ANPEP PE=1 SV=4 High Q9NS25 Sperm protein associated with the nucleus on 0 the X chromosome B/F OS=Homo sapiens GN=SPANXB1 PE=2 SV=1 High J3QK89 Calcium homeostasis endoplasmic reticulum 0 protein OS=Homo sapiens GN=CHERP PE=1 SV=1 High P49411 Elongation factor Tu, mitochondrial OS=Homo 0 sapiens GN=TUFM PE=1 SV=2 High P68363 Tubulin alpha-1B chain OS=Homo sapiens 0 GN=TUBA1B PE=1 SV=1 High P11142 Heat shock cognate 71 kDa protein OS=Homo 0 sapiens GN=HSPA8 PE=1 SV=1 High P02768 Serum albumin OS=Homo sapiens GN=ALB PE=1 0 SV=2

124

High O00571 ATP-dependent RNA helicase DDX3X OS=Homo 0 sapiens GN=DDX3X PE=1 SV=3 High Q9Y2W1 Thyroid hormone receptor-associated protein 3 0 OS=Homo sapiens GN=THRAP3 PE=1 SV=2 High Q69YQ0 Cytospin-A OS=Homo sapiens GN=SPECC1L PE=1 0 SV=2 High E9PGZ1 Caldesmon OS=Homo sapiens GN=CALD1 PE=1 0 SV=1 High O95782 AP-2 complex subunit alpha-1 OS=Homo sapiens 0 GN=AP2A1 PE=1 SV=3 High P19474 E3 ubiquitin-protein ligase TRIM21 OS=Homo 0 sapiens GN=TRIM21 PE=1 SV=1 High Q96PK6 RNA-binding protein 14 OS=Homo sapiens 0 GN=RBM14 PE=1 SV=2 High Q562R1 Beta-actin-like protein 2 OS=Homo sapiens 0 GN=ACTBL2 PE=1 SV=2 High Q05682 Caldesmon OS=Homo sapiens GN=CALD1 PE=1 0 SV=3 High P05141 ADP/ATP translocase 2 OS=Homo sapiens 0 GN=SLC25A5 PE=1 SV=7 High P47755 F-actin-capping protein subunit alpha-2 0 OS=Homo sapiens GN=CAPZA2 PE=1 SV=3 High P06576 ATP synthase subunit beta, mitochondrial 0 OS=Homo sapiens GN=ATP5B PE=1 SV=3 High Q9UHB6 LIM domain and actin-binding protein 1 0 OS=Homo sapiens GN=LIMA1 PE=1 SV=1 High P68366 Tubulin alpha-4A chain OS=Homo sapiens 0 GN=TUBA4A PE=1 SV=1 High P52907 F-actin-capping protein subunit alpha-1 0 OS=Homo sapiens GN=CAPZA1 PE=1 SV=3 High O94875 Sorbin and SH3 domain-containing protein 2 0 OS=Homo sapiens GN=SORBS2 PE=1 SV=3 High Q86YZ3 Hornerin OS=Homo sapiens GN=HRNR PE=1 0 SV=2 High P07355 Annexin A2 OS=Homo sapiens GN=ANXA2 PE=1 0 SV=2 High Q9NYL9 Tropomodulin-3 OS=Homo sapiens GN=TMOD3 0 PE=1 SV=1 High Q04695 Keratin, type I cytoskeletal 17 OS=Homo sapiens 0 GN=KRT17 PE=1 SV=2 High P62269 40S ribosomal protein S18 OS=Homo sapiens 0 GN=RPS18 PE=1 SV=3 High A0A0A0MR36 Unconventional myosin-Vb (Fragment) 0 OS=Homo sapiens GN=MYO5B PE=4 SV=1

125

High G3V1L9 Tight junction protein 1 (Zona occludens 1), 0 isoform CRA_a OS=Homo sapiens GN=TJP1 PE=4 SV=1 High H7BXR3 Sorbin and SH3 domain-containing protein 2 0 (Fragment) OS=Homo sapiens GN=SORBS2 PE=1 SV=1 High Q5D862 Filaggrin-2 OS=Homo sapiens GN=FLG2 PE=1 0 SV=1 High P68104 Elongation factor 1-alpha 1 OS=Homo sapiens 0 GN=EEF1A1 PE=1 SV=1 High P78386 Keratin, type II cuticular Hb5 OS=Homo sapiens 0 GN=KRT85 PE=1 SV=1 High P36578 60S ribosomal protein L4 OS=Homo sapiens 0 GN=RPL4 PE=1 SV=5 High P23246 Splicing factor, proline- and glutamine-rich 0 OS=Homo sapiens GN=SFPQ PE=1 SV=2 High P28799 Granulins OS=Homo sapiens GN=GRN PE=1 SV=2 0 High F8WE88 Unconventional myosin-Va OS=Homo sapiens 0 GN=MYO5A PE=1 SV=2 High Q96C19 EF-hand domain-containing protein D2 0 OS=Homo sapiens GN=EFHD2 PE=1 SV=1 High Q9BX66 Sorbin and SH3 domain-containing protein 1 0 OS=Homo sapiens GN=SORBS1 PE=1 SV=3 High P46783 40S ribosomal protein S10 OS=Homo sapiens 0 GN=RPS10 PE=1 SV=1 High Q9Y4K1 Absent in melanoma 1 protein OS=Homo 0 sapiens GN=AIM1 PE=1 SV=3 High Q13425 Beta-2-syntrophin OS=Homo sapiens GN=SNTB2 0 PE=1 SV=1 High P02545 Prelamin-A/C OS=Homo sapiens GN=LMNA PE=1 0 SV=1 High P11137 Microtubule-associated protein 2 OS=Homo 0 sapiens GN=MAP2 PE=1 SV=4 High D3DTX6 Neurabin-2 OS=Homo sapiens GN=PPP1R9B 0 PE=4 SV=1 High P62136 Serine/threonine-protein phosphatase PP1- 0 alpha catalytic subunit OS=Homo sapiens GN=PPP1CA PE=1 SV=1 High O43790 Keratin, type II cuticular Hb6 OS=Homo sapiens 0 GN=KRT86 PE=1 SV=1 High Q06830 Peroxiredoxin-1 OS=Homo sapiens GN=PRDX1 0 PE=1 SV=1 High Q7Z5L9 Interferon regulatory factor 2-binding protein 2 0 OS=Homo sapiens GN=IRF2BP2 PE=1 SV=2 High O95678 Keratin, type II cytoskeletal 75 OS=Homo 0 sapiens GN=KRT75 PE=1 SV=2

126

High P04406 Glyceraldehyde-3-phosphate dehydrogenase 0 OS=Homo sapiens GN=GAPDH PE=1 SV=3 High P63010 AP-2 complex subunit beta OS=Homo sapiens 0 GN=AP2B1 PE=1 SV=1 High P06748 Nucleophosmin OS=Homo sapiens GN=NPM1 0 PE=1 SV=2 High P14373 Zinc finger protein RFP OS=Homo sapiens 0 GN=TRIM27 PE=1 SV=1 High P62979 Ubiquitin-40S ribosomal protein S27a OS=Homo 0 sapiens GN=RPS27A PE=1 SV=2 High P14618 Pyruvate kinase PKM OS=Homo sapiens 0 GN=PKM PE=1 SV=4 High P23396 40S ribosomal protein S3 OS=Homo sapiens 0 GN=RPS3 PE=1 SV=2 High Q9BY89 Uncharacterized protein KIAA1671 OS=Homo 0 sapiens GN=KIAA1671 PE=1 SV=2 High P33121 Long-chain-fatty-acid--CoA ligase 1 OS=Homo 0 sapiens GN=ACSL1 PE=1 SV=1 High A0A087X130 Ig kappa chain C region OS=Homo sapiens 0 GN=IGKC PE=4 SV=1 High P62701 40S ribosomal protein S4, X isoform OS=Homo 0 sapiens GN=RPS4X PE=1 SV=2 High Q14677 Clathrin interactor 1 OS=Homo sapiens 0 GN=CLINT1 PE=1 SV=1 High P29590 Protein PML OS=Homo sapiens GN=PML PE=1 0 SV=3 High P61247 40S ribosomal protein S3a OS=Homo sapiens 0 GN=RPS3A PE=1 SV=2 High P35222 Catenin beta-1 OS=Homo sapiens GN=CTNNB1 0 PE=1 SV=1 High P62987 Ubiquitin-60S ribosomal protein L40 OS=Homo 0 sapiens GN=UBA52 PE=1 SV=2 High E7EMB3 Calmodulin OS=Homo sapiens GN=CALM2 PE=1 0 SV=1 High Q02878 60S ribosomal protein L6 OS=Homo sapiens 0 GN=RPL6 PE=1 SV=3 High P36542 ATP synthase subunit gamma, mitochondrial 0 OS=Homo sapiens GN=ATP5C1 PE=1 SV=1 High O00151 PDZ and LIM domain protein 1 OS=Homo 0 sapiens GN=PDLIM1 PE=1 SV=4 High P12235 ADP/ATP translocase 1 OS=Homo sapiens 0 GN=SLC25A4 PE=1 SV=4 High Q15233 Non-POU domain-containing octamer-binding 0 protein OS=Homo sapiens GN=NONO PE=1 SV=4 High O94973 AP-2 complex subunit alpha-2 OS=Homo sapiens 0 GN=AP2A2 PE=1 SV=2

127

High Q9Y608 Leucine-rich repeat flightless-interacting protein 0 2 OS=Homo sapiens GN=LRRFIP2 PE=1 SV=1 High P48047 ATP synthase subunit O, mitochondrial 0 OS=Homo sapiens GN=ATP5O PE=1 SV=1 High P35221 Catenin alpha-1 OS=Homo sapiens GN=CTNNA1 0 PE=1 SV=1 High Q00839 Heterogeneous nuclear ribonucleoprotein U 0 OS=Homo sapiens GN=HNRNPU PE=1 SV=6 High P18124 60S ribosomal protein L7 OS=Homo sapiens 0 GN=RPL7 PE=1 SV=1 High P16402 Histone H1.3 OS=Homo sapiens GN=HIST1H1D 0 PE=1 SV=2 High P62249 40S ribosomal protein S16 OS=Homo sapiens 0 GN=RPS16 PE=1 SV=2 High P62851 40S ribosomal protein S25 OS=Homo sapiens 0 GN=RPS25 PE=1 SV=1 High P38159 RNA-binding motif protein, X chromosome 0 OS=Homo sapiens GN=RBMX PE=1 SV=3 High P62424 60S ribosomal protein L7a OS=Homo sapiens 0 GN=RPL7A PE=1 SV=2 High F5GYR0 Actin-binding LIM protein 2 OS=Homo sapiens 0 GN=ABLIM2 PE=4 SV=2 High Q13501 Sequestosome-1 OS=Homo sapiens GN=SQSTM1 0 PE=1 SV=1 High P06733 Alpha-enolase OS=Homo sapiens GN=ENO1 0 PE=1 SV=2 High P50395 Rab GDP dissociation inhibitor beta OS=Homo 0 sapiens GN=GDI2 PE=1 SV=2 High P62140 Serine/threonine-protein phosphatase PP1-beta 0 catalytic subunit OS=Homo sapiens GN=PPP1CB PE=1 SV=3 High Q12792 Twinfilin-1 OS=Homo sapiens GN=TWF1 PE=1 0 SV=3 High E9PFW3 AP-2 complex subunit mu OS=Homo sapiens 0 GN=AP2M1 PE=1 SV=1 High P22626 Heterogeneous nuclear ribonucleoproteins 0 A2/B1 OS=Homo sapiens GN=HNRNPA2B1 PE=1 SV=2 High H3BLZ8 Probable ATP-dependent RNA helicase DDX17 0 OS=Homo sapiens GN=DDX17 PE=1 SV=1 High Q08380 Galectin-3-binding protein OS=Homo sapiens 0 GN=LGALS3BP PE=1 SV=1 High Q86X51 Uncharacterized protein CXorf67 OS=Homo 0 sapiens GN=CXorf67 PE=2 SV=1 High P62241 40S ribosomal protein S8 OS=Homo sapiens 0 GN=RPS8 PE=1 SV=2

128

High J3KS54 Protein flightless-1 homolog OS=Homo sapiens 0 GN=FLII PE=1 SV=1 High G8JLB6 Heterogeneous nuclear ribonucleoprotein H 0 OS=Homo sapiens GN=HNRNPH1 PE=1 SV=1 High Q02543 60S ribosomal protein L18a OS=Homo sapiens 0 GN=RPL18A PE=1 SV=2 High P26373 60S ribosomal protein L13 OS=Homo sapiens 0 GN=RPL13 PE=1 SV=4 High P62753 40S ribosomal protein S6 OS=Homo sapiens 0 GN=RPS6 PE=1 SV=1 High P35268 60S ribosomal protein L22 OS=Homo sapiens 0 GN=RPL22 PE=1 SV=2 High Q6ZRV2 Protein FAM83H OS=Homo sapiens 0 GN=FAM83H PE=1 SV=3 High P52272 Heterogeneous nuclear ribonucleoprotein M 0 OS=Homo sapiens GN=HNRNPM PE=1 SV=3 High J3KTA4 Probable ATP-dependent RNA helicase DDX5 0 OS=Homo sapiens GN=DDX5 PE=1 SV=1 High E9PKC0 Pleckstrin homology domain-containing family A 0 member 7 OS=Homo sapiens GN=PLEKHA7 PE=1 SV=1 High Q9NS26 Sperm protein associated with the nucleus on 0 the X chromosome A OS=Homo sapiens GN=SPANXA1 PE=2 SV=1 High O14974 Protein phosphatase 1 regulatory subunit 12A 0 OS=Homo sapiens GN=PPP1R12A PE=1 SV=1 High P98082 Disabled homolog 2 OS=Homo sapiens 0 GN=DAB2 PE=1 SV=3 High P06396 Gelsolin OS=Homo sapiens GN=GSN PE=1 SV=1 0 High Q08431 Lactadherin OS=Homo sapiens GN=MFGE8 PE=1 0 SV=2 High P61313 60S ribosomal protein L15 OS=Homo sapiens 0 GN=RPL15 PE=1 SV=2 High U3KQK0 Histone H2B OS=Homo sapiens GN=HIST1H2BN 0 PE=3 SV=1 High A0A0A0MRV0 Ribosome-binding protein 1 OS=Homo sapiens 0 GN=RRBP1 PE=4 SV=1 High O60941 Dystrobrevin beta OS=Homo sapiens GN=DTNB 0 PE=1 SV=1 High P62937 Peptidyl-prolyl cis-trans isomerase A OS=Homo 0 sapiens GN=PPIA PE=1 SV=2 High P62913 60S ribosomal protein L11 OS=Homo sapiens 0 GN=RPL11 PE=1 SV=2 High P62829 60S ribosomal protein L23 OS=Homo sapiens 0 GN=RPL23 PE=1 SV=1

129

High O43166 Signal-induced proliferation-associated 1-like 0 protein 1 OS=Homo sapiens GN=SIPA1L1 PE=1 SV=4 High P09496 Clathrin light chain A OS=Homo sapiens 0 GN=CLTA PE=1 SV=1 High Q5VWP3 Muscular LMNA-interacting protein OS=Homo 0 sapiens GN=MLIP PE=1 SV=3 High P04908 Histone H2A type 1-B/E OS=Homo sapiens 0 GN=HIST1H2AB PE=1 SV=2 High Q9P266 Junctional protein associated with coronary 0 artery disease OS=Homo sapiens GN=KIAA1462 PE=1 SV=3 High Q96E39 RNA binding motif protein, X-linked-like-1 0 OS=Homo sapiens GN=RBMXL1 PE=1 SV=1 High Q99569 Plakophilin-4 OS=Homo sapiens GN=PKP4 PE=1 0 SV=2 High P21397 Amine oxidase [flavin-containing] A OS=Homo 0 sapiens GN=MAOA PE=1 SV=1 High P62805 Histone H4 OS=Homo sapiens GN=HIST1H4A 0 PE=1 SV=2 High Q6FI13 Histone H2A type 2-A OS=Homo sapiens 0 GN=HIST2H2AA3 PE=1 SV=3 High A0A087X2I6 Keratin, type I cuticular Ha3-II OS=Homo sapiens 0 GN=KRT33B PE=3 SV=1 High P60866 40S ribosomal protein S20 OS=Homo sapiens 0 GN=RPS20 PE=1 SV=1 High Q15323 Keratin, type I cuticular Ha1 OS=Homo sapiens 0 GN=KRT31 PE=2 SV=3 High P62280 40S ribosomal protein S11 OS=Homo sapiens 0 GN=RPS11 PE=1 SV=3 High Q4KMQ1 Taperin OS=Homo sapiens GN=TPRN PE=1 SV=2 0 High J3KQN4 60S ribosomal protein L36a OS=Homo sapiens 0 GN=RPL36A PE=3 SV=1 High P62917 60S ribosomal protein L8 OS=Homo sapiens 0 GN=RPL8 PE=1 SV=2 High P39019 40S ribosomal protein S19 OS=Homo sapiens 0 GN=RPS19 PE=1 SV=2 High P19013 Keratin, type II cytoskeletal 4 OS=Homo sapiens 0 GN=KRT4 PE=1 SV=4 High Q969Q0 60S ribosomal protein L36a-like OS=Homo 0 sapiens GN=RPL36AL PE=1 SV=3 High Q71DI3 Histone H3.2 OS=Homo sapiens GN=HIST2H3A 0 PE=1 SV=3 High P27348 14-3-3 protein theta OS=Homo sapiens 0 GN=YWHAQ PE=1 SV=1

130

High A0A0A0PLW7 Core protein (Fragment) OS=Hepatitis B virus 0 GN=C PE=4 SV=1 High Q01105 Protein SET OS=Homo sapiens GN=SET PE=1 0 SV=3 High P84090 Enhancer of rudimentary homolog OS=Homo 0 sapiens GN=ERH PE=1 SV=1 High J3QQ67 60S ribosomal protein L18 (Fragment) OS=Homo 0 sapiens GN=RPL18 PE=1 SV=1 High P27105 Erythrocyte band 7 integral membrane protein 0 OS=Homo sapiens GN=STOM PE=1 SV=3 High P55795 Heterogeneous nuclear ribonucleoprotein H2 0 OS=Homo sapiens GN=HNRNPH2 PE=1 SV=1 High A0A0A6YYJ8 Putative RNA-binding protein Luc7-like 2 0 OS=Homo sapiens GN=LUC7L2 PE=4 SV=1 High P63173 60S ribosomal protein L38 OS=Homo sapiens 0 GN=RPL38 PE=1 SV=2 High P31151 Protein S100-A7 OS=Homo sapiens GN=S100A7 0 PE=1 SV=4 High M0R2L9 40S ribosomal protein S19 (Fragment) OS=Homo 0 sapiens GN=RPS19 PE=1 SV=1 High O60701 UDP-glucose 6-dehydrogenase OS=Homo 0 sapiens GN=UGDH PE=1 SV=1 High Q8TA86 Retinitis pigmentosa 9 protein OS=Homo 0 sapiens GN=RP9 PE=1 SV=2 High Q6KC79 Nipped-B-like protein OS=Homo sapiens 0 GN=NIPBL PE=1 SV=2 High P05109 Protein S100-A8 OS=Homo sapiens GN=S100A8 0 PE=1 SV=1 High P06899 Histone H2B type 1-J OS=Homo sapiens 0 GN=HIST1H2BJ PE=1 SV=3 High P62263 40S ribosomal protein S14 OS=Homo sapiens 0 GN=RPS14 PE=1 SV=3 High P39023 60S ribosomal protein L3 OS=Homo sapiens 0 GN=RPL3 PE=1 SV=2 High P84243 Histone H3.3 OS=Homo sapiens GN=H3F3A PE=1 0 SV=2 High O75494 Serine/arginine-rich splicing factor 10 OS=Homo 0 sapiens GN=SRSF10 PE=1 SV=1 High P0CW22 40S ribosomal protein S17-like OS=Homo 0 sapiens GN=RPS17L PE=1 SV=1 High P08238 Heat shock protein HSP 90-beta OS=Homo 0 sapiens GN=HSP90AB1 PE=1 SV=4 High Q6NZI2 Polymerase I and transcript release factor 0 OS=Homo sapiens GN=PTRF PE=1 SV=1 High P09651 Heterogeneous nuclear ribonucleoprotein A1 0 OS=Homo sapiens GN=HNRNPA1 PE=1 SV=5

131

High H0Y2W2 ATPase family AAA domain-containing protein 0 3A (Fragment) OS=Homo sapiens GN=ATAD3A PE=1 SV=1 High Q9NQX4 Unconventional myosin-Vc OS=Homo sapiens 0 GN=MYO5C PE=1 SV=2 High P30050 60S ribosomal protein L12 OS=Homo sapiens 0 GN=RPL12 PE=1 SV=1 High P09497 Clathrin light chain B OS=Homo sapiens 0 GN=CLTB PE=1 SV=1 High Q9NSY1 BMP-2-inducible protein kinase OS=Homo 0 sapiens GN=BMP2K PE=1 SV=2 High P45880 Voltage-dependent anion-selective channel 0 protein 2 OS=Homo sapiens GN=VDAC2 PE=1 SV=2 High Q9NWB6 Arginine and glutamate-rich protein 1 OS=Homo 0 sapiens GN=ARGLU1 PE=1 SV=1 High Q6NYC8 Phostensin OS=Homo sapiens GN=PPP1R18 0 PE=1 SV=1 High P62266 40S ribosomal protein S23 OS=Homo sapiens 0 GN=RPS23 PE=1 SV=3 High P61158 Actin-related protein 3 OS=Homo sapiens 0 GN=ACTR3 PE=1 SV=3 High P10599 Thioredoxin OS=Homo sapiens GN=TXN PE=1 0 SV=3 High P10809 60 kDa heat shock protein, mitochondrial 0 OS=Homo sapiens GN=HSPD1 PE=1 SV=2 High P62879 Guanine nucleotide-binding protein 0 G(I)/G(S)/G(T) subunit beta-2 OS=Homo sapiens GN=GNB2 PE=1 SV=3 High H0Y8C2 60S ribosomal protein L22-like 1 (Fragment) 0 OS=Homo sapiens GN=RPL22L1 PE=4 SV=1 High P46776 60S ribosomal protein L27a OS=Homo sapiens 0 GN=RPL27A PE=1 SV=2 High O43734 Adapter protein CIKS OS=Homo sapiens 0 GN=TRAF3IP2 PE=1 SV=3 High P62854 40S ribosomal protein S26 OS=Homo sapiens 0 GN=RPS26 PE=1 SV=3 High F8W1S1 Keratin, type II cytoskeletal 74 OS=Homo 0 sapiens GN=KRT74 PE=1 SV=1 High Q15637 Splicing factor 1 OS=Homo sapiens GN=SF1 PE=1 0 SV=4 High P04899 Guanine nucleotide-binding protein G(i) subunit 0 alpha-2 OS=Homo sapiens GN=GNAI2 PE=1 SV=3 High G1E7Q7 External core antigen OS=Hepatitis B virus PE=3 0 SV=1 High Q9P2M7 Cingulin OS=Homo sapiens GN=CGN PE=1 SV=2 0

132

High P61254 60S ribosomal protein L26 OS=Homo sapiens 0 GN=RPL26 PE=1 SV=1 High P80297 Metallothionein-1X OS=Homo sapiens GN=MT1X 0 PE=1 SV=1 High A0A0A6YYL6 Protein RPL17-C18orf32 OS=Homo sapiens 0 GN=RPL17-C18orf32 PE=4 SV=1 High P00338 L-lactate dehydrogenase A chain OS=Homo 0 sapiens GN=LDHA PE=1 SV=2 High P05388 60S acidic ribosomal protein P0 OS=Homo 0 sapiens GN=RPLP0 PE=1 SV=1 High Q92804 TATA-binding protein-associated factor 2N 0 OS=Homo sapiens GN=TAF15 PE=1 SV=1 High P04792 Heat shock protein beta-1 OS=Homo sapiens 0 GN=HSPB1 PE=1 SV=2 High H0Y449 Nuclease-sensitive element-binding protein 1 0 (Fragment) OS=Homo sapiens GN=YBX1 PE=1 SV=1 High M0R0R2 40S ribosomal protein S5 OS=Homo sapiens 0 GN=RPS5 PE=1 SV=1 High Q92764 Keratin, type I cuticular Ha5 OS=Homo sapiens 0 GN=KRT35 PE=2 SV=5 High P15880 40S ribosomal protein S2 OS=Homo sapiens 0 GN=RPS2 PE=1 SV=2 High Q9H477 Ribokinase OS=Homo sapiens GN=RBKS PE=1 0 SV=1 High P51991 Heterogeneous nuclear ribonucleoprotein A3 0 OS=Homo sapiens GN=HNRNPA3 PE=1 SV=2 High P62277 40S ribosomal protein S13 OS=Homo sapiens 0 GN=RPS13 PE=1 SV=2 High Q9NR12 PDZ and LIM domain protein 7 OS=Homo 0 sapiens GN=PDLIM7 PE=1 SV=1 High P83731 60S ribosomal protein L24 OS=Homo sapiens 0 GN=RPL24 PE=1 SV=1 High L0H7I3 Core protein OS=Hepatitis B virus GN=C PE=4 0 SV=1 High P23284 Peptidyl-prolyl cis-trans isomerase B OS=Homo 0 sapiens GN=PPIB PE=1 SV=2 High Q6UWP8 Suprabasin OS=Homo sapiens GN=SBSN PE=2 0 SV=2 High Q9BR76 Coronin-1B OS=Homo sapiens GN=CORO1B 0 PE=1 SV=1 High P02795 Metallothionein-2 OS=Homo sapiens GN=MT2A 0 PE=1 SV=1 High P46939 Utrophin OS=Homo sapiens GN=UTRN PE=1 0 SV=2

133

High P01023 Alpha-2-macroglobulin OS=Homo sapiens 0 GN=A2M PE=1 SV=3 High B1ALY0 Protein PALM2-AKAP2 (Fragment) OS=Homo 0 sapiens GN=PALM2-AKAP2 PE=4 SV=1 High P46781 40S ribosomal protein S9 OS=Homo sapiens 0 GN=RPS9 PE=1 SV=3 High E9PR17 CD59 glycoprotein OS=Homo sapiens GN=CD59 0 PE=1 SV=1 High Q9UDY2 Tight junction protein ZO-2 OS=Homo sapiens 0 GN=TJP2 PE=1 SV=2 High O76031 ATP-dependent Clp protease ATP-binding 0 subunit clpX-like, mitochondrial OS=Homo sapiens GN=CLPX PE=1 SV=2 High Q9UGI8 Testin OS=Homo sapiens GN=TES PE=1 SV=1 0 High P01891 HLA class I histocompatibility antigen, A-68 0 alpha chain OS=Homo sapiens GN=HLA-A PE=1 SV=4 High Q9Y6N5 Sulfide:quinone oxidoreductase, mitochondrial 0 OS=Homo sapiens GN=SQRDL PE=1 SV=1 High H7BY58 Protein-L-isoaspartate O-methyltransferase 0 OS=Homo sapiens GN=PCMT1 PE=1 SV=1 High A0A0A0MTH9 TATA-binding protein-associated factor 172 0 OS=Homo sapiens GN=BTAF1 PE=4 SV=1 High Q9Y5B9 FACT complex subunit SPT16 OS=Homo sapiens 0 GN=SUPT16H PE=1 SV=1 High F8VVM2 Phosphate carrier protein, mitochondrial 0 OS=Homo sapiens GN=SLC25A3 PE=1 SV=1 High A0A871 External core antigen OS=Hepatitis B virus 0 GN=PreC/C PE=3 SV=1 High M0QYS1 60S ribosomal protein L13a (Fragment) 0 OS=Homo sapiens GN=RPL13A PE=1 SV=2 High E9PB61 THO complex subunit 4 OS=Homo sapiens 0 GN=ALYREF PE=1 SV=1 High Q9P0K7 Ankycorbin OS=Homo sapiens GN=RAI14 PE=1 0 SV=2 High Q08211 ATP-dependent RNA helicase A OS=Homo 0 sapiens GN=DHX9 PE=1 SV=4 High E7EVA0 Microtubule-associated protein OS=Homo 0 sapiens GN=MAP4 PE=1 SV=1 High Q8NC51 Plasminogen activator inhibitor 1 RNA-binding 0 protein OS=Homo sapiens GN=SERBP1 PE=1 SV=2 High H0Y948 Afadin (Fragment) OS=Homo sapiens GN=MLLT4 0 PE=1 SV=1 High P46940 Ras GTPase-activating-like protein IQGAP1 0 OS=Homo sapiens GN=IQGAP1 PE=1 SV=1

134

High P84098 60S ribosomal protein L19 OS=Homo sapiens 0 GN=RPL19 PE=1 SV=1 High H3BN98 Uncharacterized protein (Fragment) OS=Homo 0 sapiens PE=4 SV=2 High Q9UNX3 60S ribosomal protein L26-like 1 OS=Homo 0 sapiens GN=RPL26L1 PE=1 SV=1 High H3BPE7 RNA-binding protein FUS OS=Homo sapiens 0 GN=FUS PE=1 SV=1 High P31946 14-3-3 protein beta/alpha OS=Homo sapiens 0 GN=YWHAB PE=1 SV=3 High P05387 60S acidic ribosomal protein P2 OS=Homo 0 sapiens GN=RPLP2 PE=1 SV=1 High P02751 Fibronectin OS=Homo sapiens GN=FN1 PE=1 0 SV=4 High Q16527 Cysteine and glycine-rich protein 2 OS=Homo 0 sapiens GN=CSRP2 PE=1 SV=3 High O76021 Ribosomal L1 domain-containing protein 1 0 OS=Homo sapiens GN=RSL1D1 PE=1 SV=3 High P14649 Myosin light chain 6B OS=Homo sapiens 0 GN=MYL6B PE=1 SV=1 High O75367 Core histone macro-H2A.1 OS=Homo sapiens 0 GN=H2AFY PE=1 SV=4 High Q9NYF8 Bcl-2-associated transcription factor 1 OS=Homo 0 sapiens GN=BCLAF1 PE=1 SV=2 High P61978 Heterogeneous nuclear ribonucleoprotein K 0 OS=Homo sapiens GN=HNRNPK PE=1 SV=1 High P48735 Isocitrate dehydrogenase [NADP], mitochondrial 0 OS=Homo sapiens GN=IDH2 PE=1 SV=2 High P62906 60S ribosomal protein L10a OS=Homo sapiens 0 GN=RPL10A PE=1 SV=2 High Q16666 Gamma-interferon-inducible protein 16 0 OS=Homo sapiens GN=IFI16 PE=1 SV=3 High A0A087WXF8 Nucleolar protein of 40 kDa OS=Homo sapiens 0 GN=ZCCHC17 PE=1 SV=1 High P60842 Eukaryotic initiation factor 4A-I OS=Homo 0 sapiens GN=EIF4A1 PE=1 SV=1 High I3XMW9 External core antigen OS=Hepatitis B virus GN=C 0 PE=3 SV=1 High O14908 PDZ domain-containing protein GIPC1 OS=Homo 0 sapiens GN=GIPC1 PE=1 SV=2 High Q9NSB4 Keratin, type II cuticular Hb2 OS=Homo sapiens 0 GN=KRT82 PE=1 SV=3 High A0A075B6K9 Ig lambda-2 chain C regions (Fragment) 0 OS=Homo sapiens GN=IGLC2 PE=4 SV=1 High P61981 14-3-3 protein gamma OS=Homo sapiens 0 GN=YWHAG PE=1 SV=2

135

High G3V4C1 Heterogeneous nuclear ribonucleoproteins 0 C1/C2 OS=Homo sapiens GN=HNRNPC PE=1 SV=1 High P62750 60S ribosomal protein L23a OS=Homo sapiens 0 GN=RPL23A PE=1 SV=1 High Q6YHK3 CD109 antigen OS=Homo sapiens GN=CD109 0 PE=1 SV=2 High Q95604 HLA class I histocompatibility antigen, Cw-17 0 alpha chain OS=Homo sapiens GN=HLA-C PE=1 SV=1 High P07900 Heat shock protein HSP 90-alpha OS=Homo 0 sapiens GN=HSP90AA1 PE=1 SV=5 High Q14247 Src substrate cortactin OS=Homo sapiens 0 GN=CTTN PE=1 SV=2 High O00567 Nucleolar protein 56 OS=Homo sapiens 0 GN=NOP56 PE=1 SV=4 High Q6J326 External core antigen OS=Hepatitis B virus PE=3 0 SV=1 High P23141 Liver carboxylesterase 1 OS=Homo sapiens 0 GN=CES1 PE=1 SV=2 High K7QJE2 External core antigen OS=Hepatitis B virus GN=C 0 PE=3 SV=1 High H6V5L5 HBcAg OS=Hepatitis B virus PE=4 SV=1 0 High P02787 Serotransferrin OS=Homo sapiens GN=TF PE=1 0 SV=3 High P31040 Succinate dehydrogenase [ubiquinone] 0 flavoprotein subunit, mitochondrial OS=Homo sapiens GN=SDHA PE=1 SV=2 High P09874 Poly [ADP-ribose] polymerase 1 OS=Homo 0 sapiens GN=PARP1 PE=1 SV=4 High Q8IUE6 Histone H2A type 2-B OS=Homo sapiens 0 GN=HIST2H2AB PE=1 SV=3 High L8B1Z8 External core antigen OS=Hepatitis B virus 0 GN=PC-C PE=3 SV=1 High P52597 Heterogeneous nuclear ribonucleoprotein F 0 OS=Homo sapiens GN=HNRNPF PE=1 SV=3 High P0C0L4 Complement C4-A OS=Homo sapiens GN=C4A 0 PE=1 SV=2 High B4Z2V2 Core protein (Fragment) OS=Hepatitis B virus 0 GN=C PE=4 SV=1 High P61513 60S ribosomal protein L37a OS=Homo sapiens 0 GN=RPL37A PE=1 SV=2 High P04080 Cystatin-B OS=Homo sapiens GN=CSTB PE=1 0 SV=2 High E9PK25 Cofilin-1 OS=Homo sapiens GN=CFL1 PE=1 SV=1 0

136

High P81605 Dermcidin OS=Homo sapiens GN=DCD PE=1 0 SV=2 High Q9Y3U8 60S ribosomal protein L36 OS=Homo sapiens 0 GN=RPL36 PE=1 SV=3 High Q9Y3Y2 Chromatin target of PRMT1 protein OS=Homo 0 sapiens GN=CHTOP PE=1 SV=2 High K7ELC2 40S ribosomal protein S15 OS=Homo sapiens 0 GN=RPS15 PE=1 SV=1 High P62888 60S ribosomal protein L30 OS=Homo sapiens 0 GN=RPL30 PE=1 SV=2 High H7BYT1 Casein kinase I isoform delta OS=Homo sapiens 0 GN=CSNK1D PE=1 SV=2 High Q5JSZ5 Protein PRRC2B OS=Homo sapiens GN=PRRC2B 0 PE=1 SV=2 High H0YMV8 40S ribosomal protein S27 OS=Homo sapiens 0 GN=RPS27L PE=1 SV=1 High P04732 Metallothionein-1E OS=Homo sapiens GN=MT1E 0 PE=1 SV=1 High Q8TF72 Protein Shroom3 OS=Homo sapiens 0 GN=SHROOM3 PE=1 SV=2 High P15924 Desmoplakin OS=Homo sapiens GN=DSP PE=1 0 SV=3 High E0ZS18 Core protein OS=Hepatitis B virus GN=C PE=4 0 SV=1 High H0YKD8 60S ribosomal protein L28 OS=Homo sapiens 0 GN=RPL28 PE=1 SV=1 High P09382 Galectin-1 OS=Homo sapiens GN=LGALS1 PE=1 0 SV=2 High Q9Y281 Cofilin-2 OS=Homo sapiens GN=CFL2 PE=1 SV=1 0 High Q0VF96 Cingulin-like protein 1 OS=Homo sapiens 0 GN=CGNL1 PE=1 SV=2 High P47914 60S ribosomal protein L29 OS=Homo sapiens 0 GN=RPL29 PE=1 SV=2 High Q96Q07 BTB/POZ domain-containing protein 9 OS=Homo 0 sapiens GN=BTBD9 PE=2 SV=2 High Q6KB66 Keratin, type II cytoskeletal 80 OS=Homo 0 sapiens GN=KRT80 PE=1 SV=2 High P38936 Cyclin-dependent kinase inhibitor 1 OS=Homo 0 sapiens GN=CDKN1A PE=1 SV=3 High P53007 Tricarboxylate transport protein, mitochondrial 0 OS=Homo sapiens GN=SLC25A1 PE=1 SV=2 High Q9Y2X3 Nucleolar protein 58 OS=Homo sapiens 0 GN=NOP58 PE=1 SV=1 High P46778 60S ribosomal protein L21 OS=Homo sapiens 0 GN=RPL21 PE=1 SV=2

137

High O14773 Tripeptidyl-peptidase 1 OS=Homo sapiens 0 GN=TPP1 PE=1 SV=2 High P04183 Thymidine kinase, cytosolic OS=Homo sapiens 0 GN=TK1 PE=1 SV=2 High Q14498 RNA-binding protein 39 OS=Homo sapiens 0 GN=RBM39 PE=1 SV=2 High H7C0V5 Protein ANKHD1-EIF4EBP3 (Fragment) OS=Homo 0 sapiens GN=ANKHD1-EIF4EBP3 PE=4 SV=1 High F8W727 60S ribosomal protein L32 OS=Homo sapiens 0 GN=RPL32 PE=1 SV=1 High P06702 Protein S100-A9 OS=Homo sapiens GN=S100A9 0 PE=1 SV=1 High Q66PJ3 ADP-ribosylation factor-like protein 6-interacting 0 protein 4 OS=Homo sapiens GN=ARL6IP4 PE=1 SV=2 High P22087 rRNA 2'-O-methyltransferase fibrillarin 0 OS=Homo sapiens GN=FBL PE=1 SV=2 High A0A096ZUQ1 Core protein (Fragment) OS=Hepatitis B virus 0 PE=4 SV=1 High Q03135 Caveolin-1 OS=Homo sapiens GN=CAV1 PE=1 0 SV=4 High P24539 ATP synthase F(0) complex subunit B1, 0 mitochondrial OS=Homo sapiens GN=ATP5F1 PE=1 SV=2 High P62857 40S ribosomal protein S28 OS=Homo sapiens 0 GN=RPS28 PE=1 SV=1 High P49207 60S ribosomal protein L34 OS=Homo sapiens 0 GN=RPL34 PE=1 SV=3 High Q9BWE0 Replication initiator 1 OS=Homo sapiens 0 GN=REPIN1 PE=1 SV=1 High Q9E0S7 Core protein (Fragment) OS=Hepatitis B virus 0 GN=prec/C PE=4 SV=1 High C7AYN4 Core protein OS=Hepatitis B virus GN=C PE=4 0 SV=1 High Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 0 OS=Homo sapiens GN=ITIH3 PE=1 SV=2 High Q13151 Heterogeneous nuclear ribonucleoprotein A0 0 OS=Homo sapiens GN=HNRNPA0 PE=1 SV=1 High P00966 Argininosuccinate synthase OS=Homo sapiens 0 GN=ASS1 PE=1 SV=2 High P50914 60S ribosomal protein L14 OS=Homo sapiens 0 GN=RPL14 PE=1 SV=4 High B1AHD1 NHP2-like protein 1 OS=Homo sapiens 0 GN=NHP2L1 PE=1 SV=1 High A0A087WYJ9 Ig mu chain C region OS=Homo sapiens 0 GN=IGHM PE=1 SV=1

138

High C9JWC3 Sorbin and SH3 domain-containing protein 2 0 (Fragment) OS=Homo sapiens GN=SORBS2 PE=1 SV=1 High Q9NZT1 Calmodulin-like protein 5 OS=Homo sapiens 0 GN=CALML5 PE=1 SV=2 High P63096 Guanine nucleotide-binding protein G(i) subunit 0 alpha-1 OS=Homo sapiens GN=GNAI1 PE=1 SV=2 High Q13451 Peptidyl-prolyl cis-trans isomerase FKBP5 0 OS=Homo sapiens GN=FKBP5 PE=1 SV=2 High P19823 Inter-alpha-trypsin inhibitor heavy chain H2 0 OS=Homo sapiens GN=ITIH2 PE=1 SV=2 High Q96C36 Pyrroline-5-carboxylate reductase 2 OS=Homo 0 sapiens GN=PYCR2 PE=1 SV=1 High S5N100 External core antigen OS=Hepatitis B virus GN=C 0 PE=3 SV=1 High Q14444 Caprin-1 OS=Homo sapiens GN=CAPRIN1 PE=1 0 SV=2 High P04179 Superoxide dismutase [Mn], mitochondrial 0 OS=Homo sapiens GN=SOD2 PE=1 SV=2 High Q13526 Peptidyl-prolyl cis-trans isomerase NIMA- 0 interacting 1 OS=Homo sapiens GN=PIN1 PE=1 SV=1 High P27635 60S ribosomal protein L10 OS=Homo sapiens 0 GN=RPL10 PE=1 SV=4 High E7EX29 14-3-3 protein zeta/delta (Fragment) OS=Homo 0 sapiens GN=YWHAZ PE=1 SV=1 High Q02978 Mitochondrial 2-oxoglutarate/malate carrier 0 protein OS=Homo sapiens GN=SLC25A11 PE=1 SV=3 High Q53HC5 Kelch-like protein 26 OS=Homo sapiens 0 GN=KLHL26 PE=1 SV=2 High O15511 Actin-related protein 2/3 complex subunit 5 0 OS=Homo sapiens GN=ARPC5 PE=1 SV=3 High Q14764 Major vault protein OS=Homo sapiens GN=MVP 0 PE=1 SV=4 High P30837 Aldehyde dehydrogenase X, mitochondrial 0 OS=Homo sapiens GN=ALDH1B1 PE=1 SV=3 High E9PAU2 Ribonucleoprotein PTB-binding 1 OS=Homo 0 sapiens GN=RAVER1 PE=4 SV=1 High Q02413 Desmoglein-1 OS=Homo sapiens GN=DSG1 PE=1 0 SV=2 High U5JI55 External core antigen (Fragment) OS=Hepatitis B 0 virus GN=C PE=3 SV=1 High Q5T4U5 Acyl-Coenzyme A dehydrogenase, C-4 to C-12 0 straight chain, isoform CRA_a OS=Homo sapiens GN=ACADM PE=3 SV=1

139

High P42766 60S ribosomal protein L35 OS=Homo sapiens 0 GN=RPL35 PE=1 SV=2 High P01024 Complement C3 OS=Homo sapiens GN=C3 PE=1 0 SV=2 High P22695 Cytochrome b-c1 complex subunit 2, 0 mitochondrial OS=Homo sapiens GN=UQCRC2 PE=1 SV=3 High Q9J1L7 Core protein OS=Hepatitis B virus PE=4 SV=1 0 High A0A0A6YYG9 Protein ARPC4-TTLL3 OS=Homo sapiens 0 GN=ARPC4-TTLL3 PE=4 SV=1 High P62314 Small nuclear ribonucleoprotein Sm D1 0 OS=Homo sapiens GN=SNRPD1 PE=1 SV=1 High Q5VTI5 Pleckstrin homology domain-containing family A 0 member 6 OS=Homo sapiens GN=PLEKHA6 PE=1 SV=1 High Q8N1N4 Keratin, type II cytoskeletal 78 OS=Homo 0 sapiens GN=KRT78 PE=2 SV=2 High P50402 Emerin OS=Homo sapiens GN=EMD PE=1 SV=1 0 High P01594 Ig kappa chain V-I region AU OS=Homo sapiens 0 PE=1 SV=1 High Q96QZ7 Membrane-associated guanylate kinase, WW 0 and PDZ domain-containing protein 1 OS=Homo sapiens GN=MAGI1 PE=1 SV=3 High Q92522 Histone H1x OS=Homo sapiens GN=H1FX PE=1 0 SV=1 High P35754 Glutaredoxin-1 OS=Homo sapiens GN=GLRX 0 PE=1 SV=2 High P61353 60S ribosomal protein L27 OS=Homo sapiens 0 GN=RPL27 PE=1 SV=2 High P11413 Glucose-6-phosphate 1-dehydrogenase 0 OS=Homo sapiens GN=G6PD PE=1 SV=4 High Q9BU76 Multiple myeloma tumor-associated protein 2 0 OS=Homo sapiens GN=MMTAG2 PE=1 SV=1 High P20930 Filaggrin OS=Homo sapiens GN=FLG PE=1 SV=3 0 High O75190 DnaJ homolog subfamily B member 6 OS=Homo 0 sapiens GN=DNAJB6 PE=1 SV=2 High Q5M775 Cytospin-B OS=Homo sapiens GN=SPECC1 PE=1 0 SV=1 High Q01844 RNA-binding protein EWS OS=Homo sapiens 0 GN=EWSR1 PE=1 SV=1 High B8PTF7 External core antigen OS=Hepatitis B virus GN=C 0 PE=3 SV=1 High Q04837 Single-stranded DNA-binding protein, 0 mitochondrial OS=Homo sapiens GN=SSBP1 PE=1 SV=1

140

High Q9BXS6 Nucleolar and spindle-associated protein 1 0 OS=Homo sapiens GN=NUSAP1 PE=1 SV=1 High F8VWL3 FYVE, RhoGEF and PH domain-containing 0 protein 4 OS=Homo sapiens GN=FGD4 PE=4 SV=1 High B4DPQ0 Complement C1r subcomponent OS=Homo 0 sapiens GN=C1R PE=2 SV=1 High P32969 60S ribosomal protein L9 OS=Homo sapiens 0 GN=RPL9 PE=1 SV=1 High P11387 DNA topoisomerase 1 OS=Homo sapiens 0 GN=TOP1 PE=1 SV=2 High Q07065 Cytoskeleton-associated protein 4 OS=Homo 0 sapiens GN=CKAP4 PE=1 SV=2 High P40261 Nicotinamide N-methyltransferase OS=Homo 0 sapiens GN=NNMT PE=1 SV=1 High O15235 28S ribosomal protein S12, mitochondrial 0 OS=Homo sapiens GN=MRPS12 PE=1 SV=1 High Q5R363 SRSF protein kinase 1 OS=Homo sapiens 0 GN=SRPK1 PE=1 SV=2 High Q6YN16 Hydroxysteroid dehydrogenase-like protein 2 0.000621 OS=Homo sapiens GN=HSDL2 PE=1 SV=1 High P36957 Dihydrolipoyllysine-residue succinyltransferase 0.000621 component of 2-oxoglutarate dehydrogenase complex, mitochondrial OS=Homo sapiens GN=DLST PE=1 SV=4 High B7Z4C8 60S ribosomal protein L31 OS=Homo sapiens 0.000621 GN=RPL31 PE=2 SV=1 High Q9C0C2 182 kDa tankyrase-1-binding protein OS=Homo 0.000621 sapiens GN=TNKS1BP1 PE=1 SV=4 High W0SK71 Core protein OS=Hepatitis B virus GN=C PE=4 0.000621 SV=1 High P62891 60S ribosomal protein L39 OS=Homo sapiens 0.000621 GN=RPL39 PE=1 SV=2 High O14976 Cyclin-G-associated kinase OS=Homo sapiens 0.000621 GN=GAK PE=1 SV=2 High P57721 Poly(rC)-binding protein 3 OS=Homo sapiens 0.000621 GN=PCBP3 PE=2 SV=2 High Q12959 Disks large homolog 1 OS=Homo sapiens 0.000621 GN=DLG1 PE=1 SV=2 High P01602 Ig kappa chain V-I region HK102 (Fragment) 0.000621 OS=Homo sapiens GN=IGKV1-5 PE=4 SV=1 High Q9NZR1 Tropomodulin-2 OS=Homo sapiens GN=TMOD2 0.000621 PE=1 SV=1 High P43490 Nicotinamide phosphoribosyltransferase 0.000621 OS=Homo sapiens GN=NAMPT PE=1 SV=1

141

High A0A0A0MT30 Aldo-keto reductase family 1 member C1 0.000621 OS=Homo sapiens GN=AKR1C1 PE=4 SV=1 High O75947 ATP synthase subunit d, mitochondrial 0.000621 OS=Homo sapiens GN=ATP5H PE=1 SV=3 High Q9NR30 Nucleolar RNA helicase 2 OS=Homo sapiens 0.000621 GN=DDX21 PE=1 SV=5 High P62081 40S ribosomal protein S7 OS=Homo sapiens 0.000621 GN=RPS7 PE=1 SV=1 High Q15293 Reticulocalbin-1 OS=Homo sapiens GN=RCN1 0.000621 PE=1 SV=1 High P04196 Histidine-rich glycoprotein OS=Homo sapiens 0.000621 GN=HRG PE=1 SV=1 High P20700 Lamin-B1 OS=Homo sapiens GN=LMNB1 PE=1 0.000621 SV=2 High D4I4S1 C protein OS=Hepatitis B virus GN=C PE=4 SV=1 0.000621 High P01613 Ig kappa chain V-I region Ni OS=Homo sapiens 0.000621 PE=1 SV=1 High P20073 Annexin A7 OS=Homo sapiens GN=ANXA7 PE=1 0.000621 SV=3 High P19022 Cadherin-2 OS=Homo sapiens GN=CDH2 PE=1 0.000621 SV=4 High P25398 40S ribosomal protein S12 OS=Homo sapiens 0.000621 GN=RPS12 PE=1 SV=3 High P13639 Elongation factor 2 OS=Homo sapiens GN=EEF2 0.000621 PE=1 SV=4 High P61160 Actin-related protein 2 OS=Homo sapiens 0.000621 GN=ACTR2 PE=1 SV=1 High P05452 Tetranectin OS=Homo sapiens GN=CLEC3B PE=1 0.000621 SV=3 High P13797 Plastin-3 OS=Homo sapiens GN=PLS3 PE=1 SV=4 0.000621 High Q16698 2,4-dienoyl-CoA reductase, mitochondrial 0.000621 OS=Homo sapiens GN=DECR1 PE=1 SV=1 High P62995 Transformer-2 protein homolog beta OS=Homo 0.000621 sapiens GN=TRA2B PE=1 SV=1 High B4DLN1 Uncharacterized protein OS=Homo sapiens PE=2 0.000621 SV=1 High O75964 ATP synthase subunit g, mitochondrial 0.000621 OS=Homo sapiens GN=ATP5L PE=1 SV=3 High P00450 Ceruloplasmin OS=Homo sapiens GN=CP PE=1 0.000621 SV=1 High O76013 Keratin, type I cuticular Ha6 OS=Homo sapiens 0.000621 GN=KRT36 PE=1 SV=1 High D0EZ03 Core protein OS=Hepatitis B virus GN=C PE=4 0.000621 SV=1 High S5TQ29 External core antigen (Fragment) OS=Hepatitis B 0.000621 virus GN=C PE=3 SV=1

142

High Q5T5U3 Rho GTPase-activating protein 21 OS=Homo 0.000621 sapiens GN=ARHGAP21 PE=1 SV=1 High P0C0S5 Histone H2A.Z OS=Homo sapiens GN=H2AFZ 0.000621 PE=1 SV=2 High P78527 DNA-dependent protein kinase catalytic subunit 0.000621 OS=Homo sapiens GN=PRKDC PE=1 SV=3 High P27169 Serum paraoxonase/arylesterase 1 OS=Homo 0.000621 sapiens GN=PON1 PE=1 SV=3 High P30481 HLA class I histocompatibility antigen, B-44 0.000621 alpha chain OS=Homo sapiens GN=HLA-B PE=1 SV=1 High D2U640 External core antigen OS=Hepatitis B virus PE=4 0.000621 SV=1 High G1E7X4 Core protein OS=Hepatitis B virus PE=4 SV=1 0.000621 High P53621 Coatomer subunit alpha OS=Homo sapiens 0.000621 GN=COPA PE=1 SV=2 High J3KQL8 Apolipoprotein L2 OS=Homo sapiens GN=APOL2 0.000621 PE=1 SV=2 High Q86SQ0 Pleckstrin homology-like domain family B 0.000621 member 2 OS=Homo sapiens GN=PHLDB2 PE=1 SV=2 High E9PRY8 Elongation factor 1-delta OS=Homo sapiens 0.000621 GN=EEF1D PE=1 SV=1 High P21980 Protein-glutamine gamma-glutamyltransferase 2 0.000621 OS=Homo sapiens GN=TGM2 PE=1 SV=2 High Q1MSW4 External core antigen (Fragment) OS=Hepatitis B 0.000621 virus GN=preC/C PE=3 SV=1 High P01604 Ig kappa chain V-I region Kue OS=Homo sapiens 0.000621 PE=1 SV=1 High Q86V48 Leucine zipper protein 1 OS=Homo sapiens 0.000621 GN=LUZP1 PE=1 SV=2 High H7C3D8 COX assembly mitochondrial protein homolog 0.000621 (Fragment) OS=Homo sapiens GN=CMC1 PE=1 SV=1 High Q16629 Serine/arginine-rich splicing factor 7 OS=Homo 0.000621 sapiens GN=SRSF7 PE=1 SV=1 High F5H6S0 Keratin-associated protein 2-2 OS=Homo 0.000621 sapiens GN=KRTAP2-2 PE=4 SV=1 High Q86X95 Corepressor interacting with RBPJ 1 OS=Homo 0.000621 sapiens GN=CIR1 PE=1 SV=1 High P31944 Caspase-14 OS=Homo sapiens GN=CASP14 PE=1 0.000621 SV=2 High Q92828 Coronin-2A OS=Homo sapiens GN=CORO2A 0.000621 PE=2 SV=2 High Q92499 ATP-dependent RNA helicase DDX1 OS=Homo 0.000621 sapiens GN=DDX1 PE=1 SV=2

143

High A6MGA7 Core protein OS=Hepatitis B virus GN=C PE=4 0.000621 SV=1 High Q7Z417 Nuclear fragile X mental retardation-interacting 0.000621 protein 2 OS=Homo sapiens GN=NUFIP2 PE=1 SV=1 High P29401 Transketolase OS=Homo sapiens GN=TKT PE=1 0.000621 SV=3 High Q8WWM7 Ataxin-2-like protein OS=Homo sapiens 0.000621 GN=ATXN2L PE=1 SV=2 High E7ETY2 Treacle protein OS=Homo sapiens GN=TCOF1 0.000621 PE=1 SV=1 High Q9NZN4 EH domain-containing protein 2 OS=Homo 0.000621 sapiens GN=EHD2 PE=1 SV=2 High A0A075B6I1 Protein IGLV4-60 (Fragment) OS=Homo sapiens 0.000621 GN=IGLV4-60 PE=4 SV=1 High J3QRU1 Non-specific protein-tyrosine kinase OS=Homo 0.000621 sapiens GN=YES1 PE=1 SV=1 High Q96HS1 Serine/threonine-protein phosphatase PGAM5, 0.000621 mitochondrial OS=Homo sapiens GN=PGAM5 PE=1 SV=2 High Q8IUC1 Keratin-associated protein 11-1 OS=Homo 0.000621 sapiens GN=KRTAP11-1 PE=1 SV=1 High B3KS98 Eukaryotic translation initiation factor 3 subunit 0.000621 H OS=Homo sapiens GN=EIF3S3 PE=2 SV=1 High Q13867 Bleomycin hydrolase OS=Homo sapiens 0.000621 GN=BLMH PE=1 SV=1 High Q9BZE4 Nucleolar GTP-binding protein 1 OS=Homo 0.000621 sapiens GN=GTPBP4 PE=1 SV=3 High Q96IX5 Up-regulated during skeletal muscle growth 0.000621 protein 5 OS=Homo sapiens GN=USMG5 PE=1 SV=1 High P61626 Lysozyme C OS=Homo sapiens GN=LYZ PE=1 0.000621 SV=1 High P60953 Cell division control protein 42 homolog 0.000621 OS=Homo sapiens GN=CDC42 PE=1 SV=2 High Q10589 Bone marrow stromal antigen 2 OS=Homo 0.000621 sapiens GN=BST2 PE=1 SV=1 High P14174 Macrophage migration inhibitory factor 0.000621 OS=Homo sapiens GN=MIF PE=1 SV=4 High O43852 Calumenin OS=Homo sapiens GN=CALU PE=1 0.000621 SV=2 High G3V2S9 SRA stem-loop-interacting RNA-binding protein, 0.000621 mitochondrial OS=Homo sapiens GN=SLIRP PE=1 SV=1 High E9PR30 40S ribosomal protein S30 OS=Homo sapiens 0.000621 GN=FAU PE=1 SV=1

144

High P49458 Signal recognition particle 9 kDa protein 0.000621 OS=Homo sapiens GN=SRP9 PE=1 SV=2 High Q14974 Importin subunit beta-1 OS=Homo sapiens 0.000621 GN=KPNB1 PE=1 SV=2 High P01605 Ig kappa chain V-I region Lay OS=Homo sapiens 0.000621 PE=1 SV=1 High Q14151 Scaffold attachment factor B2 OS=Homo sapiens 0.000621 GN=SAFB2 PE=1 SV=1 High P62316 Small nuclear ribonucleoprotein Sm D2 0.000621 OS=Homo sapiens GN=SNRPD2 PE=1 SV=1 High P29508 Serpin B3 OS=Homo sapiens GN=SERPINB3 PE=1 0.000621 SV=2 High O00479 High mobility group nucleosome-binding 0.000621 domain-containing protein 4 OS=Homo sapiens GN=HMGN4 PE=1 SV=3 High P02788 Lactotransferrin OS=Homo sapiens GN=LTF PE=1 0.000621 SV=6 High Q14160 Protein scribble homolog OS=Homo sapiens 0.000621 GN=SCRIB PE=1 SV=4 High P02042 Hemoglobin subunit delta OS=Homo sapiens 0.000621 GN=HBD PE=1 SV=2 High A0A087WUD1 Zinc finger protein 787 OS=Homo sapiens 0.000621 GN=ZNF787 PE=1 SV=1 High Q9UM22 Mammalian ependymin-related protein 1 0.000621 OS=Homo sapiens GN=EPDR1 PE=1 SV=2 High P62942 Peptidyl-prolyl cis-trans isomerase FKBP1A 0.000621 OS=Homo sapiens GN=FKBP1A PE=1 SV=2 High Q5T749 Keratinocyte proline-rich protein OS=Homo 0.000621 sapiens GN=KPRP PE=1 SV=1 High P52943 Cysteine-rich protein 2 OS=Homo sapiens 0.000621 GN=CRIP2 PE=1 SV=1 High Q68073 External core antigen OS=Hepatitis B virus 0.000621 GN=core PE=3 SV=1 High Q9Y2Q3 Glutathione S-transferase kappa 1 OS=Homo 0.000621 sapiens GN=GSTK1 PE=1 SV=3 High P13010 X-ray repair cross-complementing protein 5 0.000621 OS=Homo sapiens GN=XRCC5 PE=1 SV=3 High O75400 Pre-mRNA-processing factor 40 homolog A 0.000621 OS=Homo sapiens GN=PRPF40A PE=1 SV=2 High Q7Z4L5 Tetratricopeptide repeat protein 21B OS=Homo 0.000621 sapiens GN=TTC21B PE=1 SV=2 High P53597 Succinyl-CoA ligase [ADP/GDP-forming] subunit 0.000621 alpha, mitochondrial OS=Homo sapiens GN=SUCLG1 PE=1 SV=4 High Q9Y6C9 Mitochondrial carrier homolog 2 OS=Homo 0.000621 sapiens GN=MTCH2 PE=1 SV=1

145

High Q96I25 Splicing factor 45 OS=Homo sapiens GN=RBM17 0.000621 PE=1 SV=1 High P07305 Histone H1.0 OS=Homo sapiens GN=H1F0 PE=1 0.000621 SV=3 High P62258 14-3-3 protein epsilon OS=Homo sapiens 0.000621 GN=YWHAE PE=1 SV=1 High P49748 Very long-chain specific acyl-CoA 0.001848 dehydrogenase, mitochondrial OS=Homo sapiens GN=ACADVL PE=1 SV=1 High P18859 ATP synthase-coupling factor 6, mitochondrial 0.001848 OS=Homo sapiens GN=ATP5J PE=1 SV=1 High P07858 Cathepsin B OS=Homo sapiens GN=CTSB PE=1 0.001848 SV=3 High P56182 Ribosomal RNA processing protein 1 homolog A 0.001848 OS=Homo sapiens GN=RRP1 PE=1 SV=1 High O14949 Cytochrome b-c1 complex subunit 8 OS=Homo 0.001848 sapiens GN=UQCRQ PE=1 SV=4 High Q92576 PHD finger protein 3 OS=Homo sapiens 0.002443 GN=PHF3 PE=1 SV=3 High P62847 40S ribosomal protein S24 OS=Homo sapiens 0.002443 GN=RPS24 PE=1 SV=1 High P55084 Trifunctional enzyme subunit beta, 0.002642 mitochondrial OS=Homo sapiens GN=HADHB PE=1 SV=3 High Q12797 Aspartyl/asparaginyl beta-hydroxylase 0.002642 OS=Homo sapiens GN=ASPH PE=1 SV=3 High O00443 Phosphatidylinositol 4-phosphate 3-kinase C2 0.002642 domain-containing subunit alpha OS=Homo sapiens GN=PIK3C2A PE=1 SV=2 High Q9HAV7 GrpE protein homolog 1, mitochondrial 0.002642 OS=Homo sapiens GN=GRPEL1 PE=1 SV=2 High Q96EV2 RNA-binding protein 33 OS=Homo sapiens 0.002642 GN=RBM33 PE=1 SV=3 High H7C4P4 U2 snRNP-associated SURP motif-containing 0.002642 protein (Fragment) OS=Homo sapiens GN=U2SURP PE=1 SV=1 High C7DNM0 External core antigen OS=Hepatitis B virus GN=C 0.002642 PE=3 SV=1 High P63244 Guanine nucleotide-binding protein subunit 0.002642 beta-2-like 1 OS=Homo sapiens GN=GNB2L1 PE=1 SV=3 High Q14331 Protein FRG1 OS=Homo sapiens GN=FRG1 PE=1 0.002642 SV=1 High Q96G21 U3 small nucleolar ribonucleoprotein protein 0.002642 IMP4 OS=Homo sapiens GN=IMP4 PE=1 SV=1

146

High E0ZNI6 Core protein (Fragment) OS=Hepatitis B virus 0.002642 PE=4 SV=1 High P01603 Ig kappa chain V-I region Ka OS=Homo sapiens 0.002642 PE=1 SV=1 High Q15773 Myeloid leukemia factor 2 OS=Homo sapiens 0.002642 GN=MLF2 PE=1 SV=1 High P21796 Voltage-dependent anion-selective channel 0.002642 protein 1 OS=Homo sapiens GN=VDAC1 PE=1 SV=2 High H7C0N4 Splicing factor 1 (Fragment) OS=Homo sapiens 0.003225 GN=SF1 PE=1 SV=1 High Q4KMP7 TBC1 domain family member 10B OS=Homo 0.003225 sapiens GN=TBC1D10B PE=1 SV=3 High O60884 DnaJ homolog subfamily A member 2 OS=Homo 0.006108 sapiens GN=DNAJA2 PE=1 SV=1 High G3V325 Protein ATP5J2-PTCD1 OS=Homo sapiens 0.006108 GN=ATP5J2-PTCD1 PE=4 SV=1 High Q01081 Splicing factor U2AF 35 kDa subunit OS=Homo 0.006108 sapiens GN=U2AF1 PE=1 SV=3 High P08473 Neprilysin OS=Homo sapiens GN=MME PE=1 0.006108 SV=2 High P19338 Nucleolin OS=Homo sapiens GN=NCL PE=1 SV=3 0.006108 High P62273 40S ribosomal protein S29 OS=Homo sapiens 0.006108 GN=RPS29 PE=1 SV=2 High Q99959 Plakophilin-2 OS=Homo sapiens GN=PKP2 PE=1 0.006108 SV=2 High P27708 CAD protein OS=Homo sapiens GN=CAD PE=1 0.006108 SV=3 High P30048 Thioredoxin-dependent peroxide reductase, 0.006108 mitochondrial OS=Homo sapiens GN=PRDX3 PE=1 SV=3 High Q9Y5J1 U3 small nucleolar RNA-associated protein 18 0.006108 homolog OS=Homo sapiens GN=UTP18 PE=1 SV=3 High D3J5Z4 Precore/core protein (Fragment) OS=Hepatitis B 0.006108 virus GN=C PE=4 SV=1 High P27658 Collagen alpha-1(VIII) chain OS=Homo sapiens 0.006108 GN=COL8A1 PE=1 SV=2 High P28290 Sperm-specific antigen 2 OS=Homo sapiens 0.006108 GN=SSFA2 PE=1 SV=3 High P18206 Vinculin OS=Homo sapiens GN=VCL PE=1 SV=4 0.006108 High Q7Z7G8 Vacuolar protein sorting-associated protein 13B 0.006648 OS=Homo sapiens GN=VPS13B PE=1 SV=2 High Q9Y3I0 tRNA-splicing ligase RtcB homolog OS=Homo 0.006648 sapiens GN=RTCB PE=1 SV=1

147

High Q9P2D8 Protein unc-79 homolog OS=Homo sapiens 0.006648 GN=UNC79 PE=2 SV=4 High P51114 Fragile X mental retardation syndrome-related 0.007434 protein 1 OS=Homo sapiens GN=FXR1 PE=1 SV=3 High A0A0A0MRM9 Nucleolar and coiled-body phosphoprotein 1 0.007434 (Fragment) OS=Homo sapiens GN=NOLC1 PE=4 SV=1 High P49761 Dual specificity protein kinase CLK3 OS=Homo 0.007434 sapiens GN=CLK3 PE=1 SV=3 High Q8TDN6 Ribosome biogenesis protein BRX1 homolog 0.007434 OS=Homo sapiens GN=BRIX1 PE=1 SV=2 High A0A075B6S8 Ig kappa chain V-I region HK102 (Fragment) 0.007434 OS=Homo sapiens GN=IGKV1-5 PE=4 SV=1 High P00387 NADH-cytochrome b5 reductase 3 OS=Homo 0.008199 sapiens GN=CYB5R3 PE=1 SV=3 High Q6UN15 Pre-mRNA 3'-end-processing factor FIP1 0.008199 OS=Homo sapiens GN=FIP1L1 PE=1 SV=1 High H0YIV9 Uncharacterized protein (Fragment) OS=Homo 0.008199 sapiens PE=3 SV=1 High Q1MSQ5 External core antigen (Fragment) OS=Hepatitis B 0.008199 virus GN=preC/C PE=3 SV=1 High Q52LJ0 Protein FAM98B OS=Homo sapiens GN=FAM98B 0.008199 PE=1 SV=1 High Q6P1L8 39S ribosomal protein L14, mitochondrial 0.008199 OS=Homo sapiens GN=MRPL14 PE=1 SV=1 High P14854 Cytochrome c oxidase subunit 6B1 OS=Homo 0.008199 sapiens GN=COX6B1 PE=1 SV=2 High P37802 Transgelin-2 OS=Homo sapiens GN=TAGLN2 0.008199 PE=1 SV=3 High E7EPK1 Septin-7 OS=Homo sapiens GN=SEPT7 PE=1 0.008199 SV=2 High B9ZVT1 RNA-binding protein 12B OS=Homo sapiens 0.008199 GN=RBM12B PE=1 SV=2 High P13497 Bone morphogenetic protein 1 OS=Homo 0.008465 sapiens GN=BMP1 PE=1 SV=2 Medium H3BLT4 Baculoviral IAP repeat-containing protein 5 0.011147 OS=Homo sapiens GN=BIRC5 PE=1 SV=1 Medium Q93052 Lipoma-preferred partner OS=Homo sapiens 0.011147 GN=LPP PE=1 SV=1 Medium Q9Y4P3 Transducin beta-like protein 2 OS=Homo sapiens 0.013137 GN=TBL2 PE=1 SV=1 Medium Q96DI7 U5 small nuclear ribonucleoprotein 40 kDa 0.013137 protein OS=Homo sapiens GN=SNRNP40 PE=1 SV=1

148

Medium Q9Y277 Voltage-dependent anion-selective channel 0.013137 protein 3 OS=Homo sapiens GN=VDAC3 PE=1 SV=1 Medium Q9UQ35 Serine/arginine repetitive matrix protein 2 0.013137 OS=Homo sapiens GN=SRRM2 PE=1 SV=2 Medium P18077 60S ribosomal protein L35a OS=Homo sapiens 0.013137 GN=RPL35A PE=1 SV=2 Medium P00390 Glutathione reductase, mitochondrial OS=Homo 0.013137 sapiens GN=GSR PE=1 SV=2 Medium Q9H0D6 5'-3' exoribonuclease 2 OS=Homo sapiens 0.013137 GN=XRN2 PE=1 SV=1 Medium P03973 Antileukoproteinase OS=Homo sapiens GN=SLPI 0.013137 PE=1 SV=2 Medium O15143 Actin-related protein 2/3 complex subunit 1B 0.013137 OS=Homo sapiens GN=ARPC1B PE=1 SV=3 Medium H0Y300 Haptoglobin OS=Homo sapiens GN=HP PE=1 0.013137 SV=4 Medium P14923 Junction plakoglobin OS=Homo sapiens GN=JUP 0.013137 PE=1 SV=3 Medium X6RAL5 Histone deacetylase complex subunit SAP18 0.013137 OS=Homo sapiens GN=SAP18 PE=1 SV=1 Medium Q9H444 Charged multivesicular body protein 4b 0.013137 OS=Homo sapiens GN=CHMP4B PE=1 SV=1 Medium B3VAK2 Core protein OS=Hepatitis B virus PE=4 SV=1 0.013137 Medium M0QYZ2 AP-2 complex subunit sigma OS=Homo sapiens 0.013137 GN=AP2S1 PE=1 SV=1 Medium Q8ND56 Protein LSM14 homolog A OS=Homo sapiens 0.013351 GN=LSM14A PE=1 SV=3 Medium P0C263 Serine/threonine-protein kinase SBK2 OS=Homo 0.013351 sapiens GN=SBK2 PE=3 SV=2 Medium P35232 Prohibitin OS=Homo sapiens GN=PHB PE=1 SV=1 0.013351 Medium E9PI68 Signal peptidase complex subunit 2 OS=Homo 0.013351 sapiens GN=SPCS2 PE=1 SV=1 Medium Q9UJC5 SH3 domain-binding glutamic acid-rich-like 0.01356 protein 2 OS=Homo sapiens GN=SH3BGRL2 PE=1 SV=2 Medium Q96T37 Putative RNA-binding protein 15 OS=Homo 0.01356 sapiens GN=RBM15 PE=1 SV=2 Medium P01608 Ig kappa chain V-I region Roy OS=Homo sapiens 0.01356 PE=1 SV=1 Medium Q8TC07 TBC1 domain family member 15 OS=Homo 0.01356 sapiens GN=TBC1D15 PE=1 SV=2 Medium P22735 Protein-glutamine gamma-glutamyltransferase K 0.01356 OS=Homo sapiens GN=TGM1 PE=1 SV=4 Medium P69905 Hemoglobin subunit alpha OS=Homo sapiens 0.01356 GN=HBA1 PE=1 SV=2

149

Medium P01612 Ig kappa chain V-I region Mev OS=Homo sapiens 0.01356 PE=1 SV=1 Medium P05187 Alkaline phosphatase, placental type OS=Homo 0.01356 sapiens GN=ALPP PE=1 SV=2 Medium Q9NWH9 SAFB-like transcription modulator OS=Homo 0.014308 sapiens GN=SLTM PE=1 SV=2 Medium Q93074 Mediator of RNA polymerase II transcription 0.014308 subunit 12 OS=Homo sapiens GN=MED12 PE=1 SV=4 Medium P11940 Polyadenylate-binding protein 1 OS=Homo 0.014308 sapiens GN=PABPC1 PE=1 SV=2 Medium Q70EL1 Inactive ubiquitin carboxyl-terminal hydrolase 0.014737 54 OS=Homo sapiens GN=USP54 PE=1 SV=4 Medium U5JMW5 External core antigen (Fragment) OS=Hepatitis B 0.014737 virus GN=C PE=3 SV=1 Medium P12268 Inosine-5'-monophosphate dehydrogenase 2 0.014737 OS=Homo sapiens GN=IMPDH2 PE=1 SV=2 Medium Q92734 Protein TFG OS=Homo sapiens GN=TFG PE=1 0.015441 SV=2 Medium Q9BZL4 Protein phosphatase 1 regulatory subunit 12C 0.015441 OS=Homo sapiens GN=PPP1R12C PE=1 SV=1 Medium H0YL18 Beta-2-microglobulin OS=Homo sapiens 0.016441 GN=B2M PE=1 SV=1 Medium Q8WXE9 Stonin-2 OS=Homo sapiens GN=STON2 PE=1 0.016441 SV=1 Medium P01611 Ig kappa chain V-I region Wes OS=Homo sapiens 0.016441 PE=1 SV=1 Medium J3KNN5 Probable ATP-dependent RNA helicase DDX41 0.016499 (Fragment) OS=Homo sapiens GN=DDX41 PE=1 SV=1 Medium Q14774 H2.0-like homeobox protein OS=Homo sapiens 0.016499 GN=HLX PE=1 SV=3 Medium Q9H0E2 Toll-interacting protein OS=Homo sapiens 0.016499 GN=TOLLIP PE=1 SV=1 Medium Q4L180 Filamin A-interacting protein 1-like OS=Homo 0.016499 sapiens GN=FILIP1L PE=1 SV=2 Medium Q96L91 E1A-binding protein p400 OS=Homo sapiens 0.016499 GN=EP400 PE=1 SV=4 Medium Q12830 Nucleosome-remodeling factor subunit BPTF 0.016499 OS=Homo sapiens GN=BPTF PE=1 SV=3 Medium Q9H6R4 Nucleolar protein 6 OS=Homo sapiens GN=NOL6 0.016499 PE=1 SV=2 Medium Q8N9E0 Protein FAM133A OS=Homo sapiens 0.016499 GN=FAM133A PE=2 SV=1 Medium Q8N9Q2 Protein SREK1IP1 OS=Homo sapiens 0.016499 GN=SREK1IP1 PE=1 SV=1

150

Medium B4DKF8 PH and SEC7 domain-containing protein 3 0.016499 OS=Homo sapiens GN=PSD3 PE=2 SV=1 Medium Q8WUH2 Transforming growth factor-beta receptor- 0.016731 associated protein 1 OS=Homo sapiens GN=TGFBRAP1 PE=1 SV=1 Medium A0A060CND0 External core antigen OS=Hepatitis B virus GN=C 0.016871 PE=3 SV=1 Medium O75083 WD repeat-containing protein 1 OS=Homo 0.016871 sapiens GN=WDR1 PE=1 SV=4 Medium F5H423 Uncharacterized protein OS=Homo sapiens PE=3 0.016871 SV=1 Medium Q96T51 RUN and FYVE domain-containing protein 1 0.016871 OS=Homo sapiens GN=RUFY1 PE=1 SV=2 Medium Q96JI7 Spatacsin OS=Homo sapiens GN=SPG11 PE=1 0.016871 SV=3 Medium Q8N8E3 Centrosomal protein of 112 kDa OS=Homo 0.016871 sapiens GN=CEP112 PE=1 SV=2 Medium P57740 Nuclear pore complex protein Nup107 0.016871 OS=Homo sapiens GN=NUP107 PE=1 SV=1 Medium P00734 Prothrombin OS=Homo sapiens GN=F2 PE=1 0.016871 SV=2 Medium P13073 Cytochrome c oxidase subunit 4 isoform 1, 0.016871 mitochondrial OS=Homo sapiens GN=COX4I1 PE=1 SV=1 Medium Q5VZ46 Uncharacterized protein KIAA1614 OS=Homo 0.016871 sapiens GN=KIAA1614 PE=2 SV=3 Medium Q96CN9 GRIP and coiled-coil domain-containing protein 0.018135 1 OS=Homo sapiens GN=GCC1 PE=1 SV=1 Medium P0C7P4 Putative cytochrome b-c1 complex subunit 0.018135 Rieske-like protein 1 OS=Homo sapiens GN=UQCRFS1P1 PE=5 SV=1 Medium P00747 Plasminogen OS=Homo sapiens GN=PLG PE=1 0.018344 SV=2 Medium P01011 Alpha-1-antichymotrypsin OS=Homo sapiens 0.018344 GN=SERPINA3 PE=1 SV=2 Medium Q96QR8 Transcriptional activator protein Pur-beta 0.019309 OS=Homo sapiens GN=PURB PE=1 SV=3 Medium P30536 Translocator protein OS=Homo sapiens 0.019309 GN=TSPO PE=1 SV=3 Medium P13584 Cytochrome P450 4B1 OS=Homo sapiens 0.019309 GN=CYP4B1 PE=1 SV=2 Medium P51397 Death-associated protein 1 OS=Homo sapiens 0.019309 GN=DAP PE=1 SV=3 Medium Q3ZCV2 Uncharacterized protein C1orf177 OS=Homo 0.019309 sapiens GN=C1orf177 PE=2 SV=3

151

Medium Q9Y2F5 Little elongation complex subunit 1 OS=Homo 0.019309 sapiens GN=ICE1 PE=1 SV=5 Medium P50416 Carnitine O-palmitoyltransferase 1, liver isoform 0.019543 OS=Homo sapiens GN=CPT1A PE=1 SV=2 Medium Q8WXW3 Progesterone-induced-blocking factor 1 0.019543 OS=Homo sapiens GN=PIBF1 PE=1 SV=2 Medium Q9BRX2 Protein pelota homolog OS=Homo sapiens 0.020807 GN=PELO PE=1 SV=2 Medium Q05516 Zinc finger and BTB domain-containing protein 0.022025 16 OS=Homo sapiens GN=ZBTB16 PE=1 SV=2 Medium Q08554 Desmocollin-1 OS=Homo sapiens GN=DSC1 PE=1 0.022025 SV=2 Medium Q8WWY7 WAP four-disulfide core domain protein 12 0.022025 OS=Homo sapiens GN=WFDC12 PE=2 SV=1 Medium Q13492 Phosphatidylinositol-binding clathrin assembly 0.022025 protein OS=Homo sapiens GN=PICALM PE=1 SV=2 Medium O75762 Transient receptor potential cation channel 0.022025 subfamily A member 1 OS=Homo sapiens GN=TRPA1 PE=1 SV=3 Medium Q9UJS0 Calcium-binding mitochondrial carrier protein 0.02225 Aralar2 OS=Homo sapiens GN=SLC25A13 PE=1 SV=2 Medium H0Y8C6 Importin-5 (Fragment) OS=Homo sapiens 0.022603 GN=IPO5 PE=1 SV=1 Medium B3VAK0 Core protein OS=Hepatitis B virus PE=4 SV=1 0.022603 Medium Q6IQ22 Ras-related protein Rab-12 OS=Homo sapiens 0.022603 GN=RAB12 PE=1 SV=3 Medium P20062 Transcobalamin-2 OS=Homo sapiens GN=TCN2 0.022603 PE=1 SV=3 Medium Q8NAT2 Tudor domain-containing protein 5 OS=Homo 0.022603 sapiens GN=TDRD5 PE=1 SV=3 Medium Q9H1K1 Iron-sulfur cluster assembly enzyme ISCU, 0.022603 mitochondrial OS=Homo sapiens GN=ISCU PE=1 SV=2 Medium Q16613 Serotonin N-acetyltransferase OS=Homo sapiens 0.023543 GN=AANAT PE=1 SV=1 Medium P51636 Caveolin-2 OS=Homo sapiens GN=CAV2 PE=1 0.02377 SV=2 Medium Q96B01 RAD51-associated protein 1 OS=Homo sapiens 0.027193 GN=RAD51AP1 PE=1 SV=1 Medium A0A087WYC5 Ig gamma-1 chain C region OS=Homo sapiens 0.027193 GN=IGHG1 PE=1 SV=1 Medium C9JVQ0 Small nuclear ribonucleoprotein G OS=Homo 0.028571 sapiens GN=SNRPG PE=4 SV=1

152

Medium P27797 Calreticulin OS=Homo sapiens GN=CALR PE=1 0.028571 SV=1 Medium K7ELL7 Glucosidase 2 subunit beta OS=Homo sapiens 0.029946 GN=PRKCSH PE=1 SV=1 Medium Q01484 Ankyrin-2 OS=Homo sapiens GN=ANK2 PE=1 0.030867 SV=4 Medium O00483 Cytochrome c oxidase subunit NDUFA4 0.030867 OS=Homo sapiens GN=NDUFA4 PE=1 SV=1 Medium P53355 Death-associated protein kinase 1 OS=Homo 0.031272 sapiens GN=DAPK1 PE=1 SV=6 Medium P17096 High mobility group protein HMG-I/HMG-Y 0.031272 OS=Homo sapiens GN=HMGA1 PE=1 SV=3 Medium A0A0A0MS20 Leukocyte immunoglobulin-like receptor 0.031272 subfamily B member 4 OS=Homo sapiens GN=LILRB4 PE=4 SV=1 Medium Q5VTL8 Pre-mRNA-splicing factor 38B OS=Homo sapiens 0.031705 GN=PRPF38B PE=1 SV=1 Medium P30793 GTP cyclohydrolase 1 OS=Homo sapiens 0.031705 GN=GCH1 PE=1 SV=1 Medium Q8TAP9 M-phase-specific PLK1-interacting protein 0.031705 OS=Homo sapiens GN=MPLKIP PE=1 SV=1 Medium Q9Y2G3 Probable phospholipid-transporting ATPase IF 0.031705 OS=Homo sapiens GN=ATP11B PE=1 SV=2 Medium O96000 NADH dehydrogenase [ubiquinone] 1 beta 0.032084 subcomplex subunit 10 OS=Homo sapiens GN=NDUFB10 PE=1 SV=3 Medium E7EX17 Eukaryotic translation initiation factor 4B 0.032084 OS=Homo sapiens GN=EIF4B PE=1 SV=1 Medium Q05823 2-5A-dependent ribonuclease OS=Homo sapiens 0.032281 GN=RNASEL PE=1 SV=2 Medium Q14257 Reticulocalbin-2 OS=Homo sapiens GN=RCN2 0.032581 PE=1 SV=1 Medium Q5JU67 Uncharacterized protein C9orf117 OS=Homo 0.032581 sapiens GN=C9orf117 PE=2 SV=1 Medium Q9HCD5 Nuclear receptor coactivator 5 OS=Homo 0.032581 sapiens GN=NCOA5 PE=1 SV=2 Medium Q5SVJ8 Calcium-activated potassium channel subunit 0.032581 alpha-1 OS=Homo sapiens GN=KCNMA1 PE=4 SV=2 Medium Q6ZVX7 F-box only protein 50 OS=Homo sapiens 0.032581 GN=NCCRP1 PE=1 SV=1 Medium Q9Y490 Talin-1 OS=Homo sapiens GN=TLN1 PE=1 SV=3 0.032806 Medium Q9GZW8 Membrane-spanning 4-domains subfamily A 0.035267 member 7 OS=Homo sapiens GN=MS4A7 PE=1 SV=1

153

Medium H3BTQ9 Coiled-coil domain-containing protein C16orf93 0.035267 OS=Homo sapiens GN=C16orf93 PE=4 SV=1

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CHAPTER 6. CONCLUSION & FUTURE DIRECTIONS

As chronic HBV infection continues to pose a major health threat worldwide due to the lack of effective therapy regimens, more research efforts are geared towards finding novel and efficient therapeutics. This is why it is so critical to understand the basic molecular mechanisms that drives HBV biology and pathogenesis in order to develop targeted therapeutics. With this objective in mind, I undertook this study by combining a global approach to study HBc interactome as well as biochemical analysis to characterize the role of PLK1 in HBV infection.

Firstly, I was able to demonstrate that PLK1 is a positive effector of HBV replication using specific chemical inhibitors both in vitro and in vivo. This finding comes in agreement with previous work from our lab highlighting the positive role of PLK1 in cellular HBV replication models 1,2. Using biological gain of function –over-expression- as well as loss of function –siRNA-mediated- approaches, I further confirmed the positive role of PLK1 in HBV infection. Significantly, I also demonstrated that HBc served as phosphorylation substrate for PLK1 in vitro and I was able to map the phosphorylation residues to the serines 168, 176 and 178. Collectively, my studies suggest that PLK1 inhibitors could potentially serve as novel anti-HBV therapeutics.

It remains to be discerned, however, if PLK1 is the kinase responsible for HBc phosphorylation in vivo and at which specific residues. The development of phospho-

155 specific antibodies targeting the residues 168, 176 and 178 would help answer this question. While the importance of PLK1 for efficient HBV replication is evident, the mechanism by which PLK1 is involved remains an enigma. Direct phosphorylation of HBc by PLK1, thereby regulating capsid assembly or viral DNA synthesis, is a very attractive hypothesis given that HBc is a robust PLK1 in vitro substrate. My preliminary studies showed that alanine substitutions at the 3 identified in vitro PLK1 phosphorylation sites

(168, 176 and 178) in the 3DP background resulted in almost 50% decrease in cccDNA production in a trans-complementation assay for HBV replication. These preliminary findings support the hypothesis that PLK1 is acting directly by phosphorylating HBc. As

HBc contributes pleiotropically to HBV replication, the exact function of HBc affected by

PLK1 phosphorylation is yet to be determined. Functional analysis using the HBc phosphorylation mutants is a powerful tool to address such question.

Alternatively, PlK1 may be phosphorylating other/additional targets, either viral

(HBV polymerase) or host proteins, that in turn regulate viral replication. In a previous study, we have already shown that PLK1 phosphorylates chromatin modifying proteins

SUZ12 and ZNF198, targeting them for proteosomal degradation. Both proteins are components of chromatin modifying complexes that repress transcription3. Given that viral cccDNA is modified by host chromatin modifying enzymes4,5, it is reasonable to envision a mechanism by which down regulation of SUZ12 and ZNF198 by PLK1 phosphorylation results in activation of viral transcription. Indeed, siRNA mediated knockdown of PLK1 not only reduced viral replication (total viral DNA) but also viral transcription (total viral RNA) indicating a role of PLK1 in HBV transcription.

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In addition to characterizing the role of PLK1 as a positive effector of HBV replication possibly via HBc phosphorylation, I sought to identify new host molecules usurped by

HBc. Our understanding of HBc as a multi-functional protein in HBV replication is expanding. Aside from the structural role as the building block of the viral capsids, HBc plays a role in encapsidation of viral pgRNA, reverse transcription, subcellular localization and potentially cccDNA regulation5,6. HBc also interacts with a wide array of host proteins enabling HBV to replicate efficiently as well as to suppress/evade host immunity. To gain more insights into HBc biology, I undertook a global mass spectrometry-based approach to identify novel HBc interacting partners. Of marked interest, I identified host nuclear speckle and paraspeckle proteins as potential HBc interacting partners. Furthermore, I established a novel link between HBc and lncRNAs

NEAT1 and NEAT2; a link that merits further investigation as it promises to expand our understanding of how lncRNAs NEAT1 and NEAT2 are involved in host immune response to viral insults. These findings, albeit preliminary in nature, open the floor for future investigation. For example, it is intriguing to determine if nuclear speckle or paraspeckle proteins regulate HBV replication. Specifically, the question whether HBV cccDNA localizes to these nuclear loci is an interesting one. More broadly, does HBc phosphorylation status and more specifically PLK1 mediated phosphorylation regulate such a process. All such questions promise to bridge many gaps of our understanding of

HBV pathogenesis, host immunity and lncRNA biology. The potential clinical significance for such studies cannot be over-estimated.

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6.1 References

1. Studach L, Wang W-H, Weber G, et al. Polo-like kinase 1 activated by the hepatitis

B virus X protein attenuates both the DNA damage checkpoint and DNA repair

resulting in partial polyploidy. J. Biol. Chem. 2010;285:30282–93. Available at:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2943266&tool=pmc

entrez&rendertype=abstract [Accessed March 14, 2012].

2. Studach LL, Rakotomalala L, Wang W-H, et al. Polo-like kinase 1 inhibition

suppresses hepatitis B virus X protein-induced transformation in an in vitro model

of liver cancer progression. Hepatology 2009;50:414–423. Available at:

http://doi.wiley.com/10.1002/hep.22996.

3. Zhang H, Diab A, Fan H, et al. PLK1 and HOTAIR Accelerate Proteasomal

Degradation of SUZ12 and ZNF198 during Hepatitis B Virus-Induced Liver

Carcinogenesis. Cancer Res. 2015;75:2363–2374.

4. Pollicino T, Belloni L, Raffa G, et al. Hepatitis B virus replication is regulated by the

acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones.

Gastroenterology 2006;130:823–837.

5. Lucifora J, Protzer U. Attacking hepatitis B virus cccDNA - The holy grail to

hepatitis B cure. J. Hepatol. 2016;64:S41-8.

6. Zlotnick A, Venkatakrishnan B, Tan Z, et al. Core protein: A pleiotropic keystone in

the HBV lifecycle. Antiviral Res. 2015;121:82–93.

VITA

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Ahmed Diab

Education PhD, Cancer Biology, Department of Basic Medical Sciences, West Lafayette, IN, USA. Title: Molecular Regulatory Mechanisms In Hepatitis B Virus Pathogenesis Co-supervisors: Ourania Andrisani, Fabien Zoulim 08/2011-12-2016 Master of Science in Biosciences, King Abdullah University of Science and Technology (KAUST), Kingdom of Saudi Arabia. GPA: 3.79/4.00 08/2010-12/2011 Bachelor of Science with High Honors in Biology (Magna Cum Laude), The American University in Cairo (AUC), Egypt. Minor: Chemistry 09/2006-06/2010

Research experience:

Postdoctoal Fellow, Mendez Lab. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA. 09/2016-present  Studying the role of HPV16 E6 in DNA Repair in head and neck squamous cell carcinoma. PhD Candidate, Andrisani Lab. Department of Basic Medical Sciences, Purdue University, IN, USA 06/2012-08/2016  Studying the role of PLK1 in HBV mediated HCC and in viral replication. Chateaubriand Visiting Fellow, Zoulim/Durantel Group, Department: Immunity, Microenvironment and Virus, Center for Cancer Research of Lyon, INSERM 1052, France,  Studying the proviral role of PLK1 in HBV replication. 12/2013-08-2014 Visiting Research Scholar, Plant Physiology Lab, Department of Applied Biology, University of Perugia, Italy. 06/2011-07/2011  Assessing the expression level of atDGK4 in different plant tissues and under different biotic and abiotic stress stimuli. Graduate Research Assistant, Biomolecular Lab, Bioscience Division, KAUST.  Assessing the potential adenylyl cyclase activity of AtDGK4 & the guanylyl cyclase activity of K+ transporter AtKUP5. 01/2011-05/2011 Undergraduate Thesis Project, Biology Department-AUC & Theodor Bilharz Research Institute, Cairo Egypt. 09/2009- 05/2010  Examined the efficacy of Praziquantel, Furoxan & Auranofin as antischistosomal drugs on fresh S. mansoni worms.  Conducted an enzyme assay to examine the inhibitory activity of these drugs on the S. mansoni Redox Cascade.

159 Summer Intern, Department of Microbiology & Immunology, Rush University Medical Center, Chicago, IL. 06/2009-08/2009  Cloned, expressed and purified Schistosomal peroxiredoxins (prx I, II) -potential drug targets for Schistosomiasis. Trainee, Gene Expression Lab, Agricultural Genetic Engineering Research Institute (AGERI), Cairo, Egypt. 07/2007-08/2007

Academic Honors/ Awards:

Best Poster Award, 2nd place, Purdue Health and Disease Poster Competition Spring 16 Young Scientist Travel Grant, Hepatitis B Foundation Fall 15 Purdue Graduate Student Government Travel gran, Purdue Graduate School Fall 15 Miles Graduate Scholarship Award, Purdue Center for Cancer Research Summer 15 Young Investigator Bursary, European Association for the Study of the Liver Spring 15 Graduate Student Travel Award, Purdue Center for Cancer Research Spring 15 Chateaubriand Fellowship, French Embassy in Washington, USA Summer 13 PULSe Graduate Assistantship- PhD in Interdisciplinary Life Sciences, Purdue Spring 11 KAUST Fellowship- for academic excellence. M. S in Biosciences- KAUST, KSA Summer10 Plate for highest GPA- Junior Class, Biology Department, AUC, Egypt Spring 09 KAUST Discovery Scholarship- Full tuition merit scholarship with stipend, AUC, Fall 08 Public School Scholarship- Full tuition merit scholarship, AUC, Egypt Fall 06

Publications:

Zhang, H; Diab, A; Fan, H; Mani, S; Hullinger, R; Merle P & Andrisani O. PLK1 and HOTAIR accelerate proteasomal degradation of SUZ12 and ZNF198 during hepatitis B virus- induced liver carcinogenesis. Cancer Research, 2015 doi: 10.1158/0008-5472. Mani, S; Zhang, H; Diab, A; Pascuzzi, P; Fares, N; Bancel B; Merle P & Andrisani O. EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes. Journal of Hepatology, May 2016. doi: 10.1016/j.jhep.2016.05.022. Fan, H; Cui, Zhibin; Zhang, H; Mani, S; Diab, A; Lefrancois, L; Fares, N; Merle P & Andrisani O. DNA demethylation induces SALL4 gene re-expression in subgroups of hepatocellular carcinoma associated with Hepatitis B or C virus infection. Oncogene, in press. Diab, A; Foca, A; Jalaguier, P; N’Guyen, L; Isorce, N; Zoulim, F; Andrisani O & Durantel, D. Polo-like-kinase 1, a positive effector in hepatitis B virus replication: therapeutic implications. In preparation. Diab, A; Zoulim, F; Durantel, D & Andrisani O. Hepatitis B virus (HBV) core antigen (HBc): function in virus biosynthesis and host gene regulation. In preparation.

160

Presentations: Invited talks: Molecular Regulatory Mechanisms in Hepatitis B Virus Pathogenesis and liver transformation. Fred Hutchinson Cancer Center, Seattle May 2016 Targeting Plk1 for Hepatitis B Virus Treatment. Department of Basic Medical Science,

Purdue University April 2015 Understanding the Role of PLK1 Phosphorylation of Chromatin Regulators in pX- Mediated Liver Cancer. INSERM, Lyon February 2014

Poster presentations:

Diab, A; Durantel, D; Zoulim, F & Andrisani, O. M. Targeting Plk1 for Hepatitis B Virus Treatment. Purdue Health & Disease Poster Competition. March 2016 Diab, A; Foca, A; Jalaguier, P; N’Guyen, L; Isorce, N; Zoulim, F; Andrisani, O. M & Durantel, D. Polo-like-kinase 1, a positive effector in hepatitis B virus replication. Hepatitis B Meeting, Poster, Bad Nauheim, German October 2015 Diab, A; Foca, A; Jalaguier, P; N’Guyen, L; Isorce, N; Zoulim, F; Andrisani, O. M & Durantel, D. Polo-like-kinase 1, a positive effector in hepatitis B virus replication. The International Liver Congress, E-Poster, Vienna, Austria. April 2015 Diab, A; Foca, A; Jalaguier, P; N’Guyen, L; Isorce, N; Zoulim, F; Andrisani, O. M & Durantel, D. Oral Presentation. Polo-like-kinase 1, a positive effector in hepatitis B virus replication: therapeutic implications. Oral Presentation. ANRS Hepatitis meeting. Paris, France. January 2015 Diab, A & Andrisani, O. M. Targeting Plk1 for Hepatitis B Virus Treatment. Purdue University Center for Cancer Research. Poster, West Lafayette, IN. November 2014 Diab, A & Andrisani, O. M. The nuclear proteins SUZ12 and ZNF198 in Hepatitis B Virus (HBV) replication. Phi Zeta Poster Session, College of Veterinary Medicine, Purdue University West Lafayette, IN. April 2013 Diab, A & Andrisani, O. M. Understanding the Role of PLK1 Phosphorylation of Chromatin Regulators in pX-Mediated Liver Cancer. Purdue University Center for Cancer Research, Poster, West Lafayette, IN. September 2012 Diab, A & Andrisani, O. M. Understanding the Role of PLK1 Phosphorylation of Chromatin Regulators in pX-Mediated Liver Cancer. Purdue Interdisciplinary Graduate Program Spring Reception, Poster, West Lafayette, IN. April 2012 Diab, A & Andrisani, O. M. Understanding the Role of PLK1 Phosphorylation of Chromatin Regulators in pX-Mediated Liver Cancer. Chronic Disease Research Poster Session, Purdue University College of Human and Health Sciences March 2012 Diab, A & Zada S. The efficacy of Praziquantel, Furoxan & Auranofin as antischistosomal drugs on fresh S. mansoni worms. Poster and Oral Presentation. Sixth Annual Biology Senior Thesis Conference, AUC, Cairo, Egypt. May 2010

Language Skills: Languages: Arabic (mother tongue), English (native speaker), French (working proficiency), Farsi and German (Basic Knowledge).

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Extracurricular Activities:

Model United Nations Coach for high-school students in Algeria, Dec 2009- Jan 2010  Selected by the U.S. Embassy of Algiers- Cultural Affairs section to train and coach a delegation of 6 MUN participants. Human Resources Director and Upper Board Member, AYB (Alashanek Ya Balady), AUC Egypt. Sep 2009- May 2012  Alashanek Ya Balady is and NGO that aims at sustainable development of the under-privileged community of Ain El Seera in Cairo. English Teacher for youth in the underprivileged Old Cairo area, Help Club, Egypt 2009 Co-organizer. Youth Leadership Initiative, Vodafone HR Directory. 3, 10, 17, 24 Oct 2009 • Recruited 30 AUC students to attend the sessions on Leadership. • Attended the leadership sessions as a participant.

Delegate. 1st Lebanon International Model United Nations (LEBIMUN 2009), Beirut, Lebanon, 26-31 July 2009

Participant. Middle East Partnership Initiative (MEPI) Student Leaders Alumni Conference. Cairo, Egypt, organized by US Department of State. 3-7 March 2009 Volunteer, children program- HSC pediatric center- Washington DC. USA, July 2008 • Organized and coached soccer trainings and tournaments for patients.

MEPI Leadership Training, Georgetown University, Washington DC., USA, July-August 08  Nominated by the American Embassy to represent Egypt in a youth leadership program including political, community service and leadership aspects.  Enrolled in summer courses at Georgetown studying Middle East and American politics.  Interacted with young Arab leaders from 20 Middle East countries and increased my multi-culturally- diverse exposure.