(Ns6) of Murine Norovirus

IDENTIFICATION AND BINDING CHARACTERISATION OF PEPTIDE-BEARING PHAGES TOWARDS RECOMBINANT PROTEASE (NS6) OF MURINE NOROVIRUS

NUR SAKINAH BINTI SOID

UNIVERSITI SAINS MALAYSIA

2019 IDENTIFICATION AND BINDING CHARACTERISATION OF PEPTIDE-BEARING PHAGES TOWARDS RECOMBINANT PROTEASE (NS6) OF MURINE NOROVIRUS

NUR SAKINAH BINTI SOID

Thesis submitted in fulfilment of the requirements for the degree of Master of Science

March 2019

I dedicate this thesis to those who are supporting me mainly

my beloved father and mother

Soid bin Mansor

and

Shahrozat binti Abdul

ACKNOWLEDGEMENT

In the name of Allah, the Most Gracious and the Most Merciful.

Acknowledging the dedicated efforts of those who gave me support and made my postgraduate studies a great experience, is a great pleasure for me. Alhamdulillah, all praises and grateful to Allah, God the Almighty for giving me strength, patience, inspiration and opportunity to complete my Msc project. A lot of experiences and knowledge were gained along the way. Throughout the most exciting in learning the practical aspect of this course, I would like to thank those who support me along the way this project being done. My greatest gratitude goes to my family especially my parents, husband and siblings for their moral support. To my family, I love all of you. I also would like to express my sincere appreciation to my supervisor, Dr Muhammad Amir bin Yunus for his knowledge, guidance and encouragement towards the completion of my thesis. Special thanks to my co-supervisor Dr Ida Shazrina and Dr Kumitaa for their endless supervision throughout my study. In addition, great thanks for my lab mates Nalini, Nurul, Nithya, Khirun, Laina, Adam, Azali and Din who are together with me in the hardness and happiness throughout this project. Not to be forgotten, to all IPPT staffs especially laboratory staffs, En Zul and Pn Hikmah whom their assistant, direction and knowledge have directly contributed towards completing my thesis project. Apart from that, I dedicate my appreciation to my beloved housemate (Fatin, Aida and Athirah) for always being with me during ups and downs and a very special thanks to all my postgraduate friends, for their friendship and useful discussions over the last three years. Thanks a lot from the deepest of my heart for those who have contributed directly or indirectly for my project. Last but not least, I would like to acknowledge Yayasan Khazanah who has supported me financially throughout my study year.

May Allah s.w.t bless all of you. Wassalam.

TABLE OF CONTENT

ACKNOWLEDGEMENT……………………………………………...... ii TABLE OF CONTENT………………………………………………...... iii LIST OF TABLES…………………………………………………………. viii LIST OF FIGURES………………………………………………………... x LIST OF ABBREVIATIONS……………………………………………... xii LIST OF SYMBOLS………..……………………………………………... xv ABSTRAK………………………………………………………………….. xvi ABSTRACT……………………………………………………………...... xviii

CHAPTER 1 : INTRODUCTION………………………………………... 1 1.1 Research Overview…………………………………………………… 1 1.2 Objectives of study………………………………………………...... 3 1.2.1 General objective……………………………………………. 3 1.2.2 Specific objectives…………………………………………... 3

CHAPTER 2 : LITERATURE REVIEW………………………………... 4 2.1 Gastroenteritis……………………………………………………...... 4 2.2 Caliciviruses…………………………………………………………... 5 2.2.1 Classification and Taxonomy……………………………….. 6 2.2.2 Morphology and Genome Organisation of Caliciviruses…… 7 2.3 Norovirus…………………………………………………………...... 8 2.3.1 Norovirus Genome Structure………………………………... 11 2.3.1(a) Norovirus Non-Structural Proteins…………...... 14 2.3.1(b) Norovirus Structural Proteins………………...... 17 2.3.2 Virus Life Cycle……………………………………………... 19 2.3.2(a) Attachment and Entry………………………...... 19 2.3.2(b) Translation of Viral Proteins……………………... 20 2.3.2(c) Genome Replication…………………………...... 23 2.3.2(d) Capsid Assembly and Virus Release……………... 25 2.3.3 Murine Norovirus…………………………………………… 26 2.3.4 NS6 Protease……………………………………………...... 28

iii

2.4 Antivirus………………………………………………………………. 33 2.5 Phage Display………………………………………………………… 36 2.6 Secondary and Tertiary Structure of Protein………………………….. 38 2.7 Bioinformatics of Protein Ligand Interaction………………………… 40

CHAPTER 3 : MATERIALS AND METHODS……………………...... 45 3.1 List of Reagents/Chemicals………………………………………...... 45 3.2 Plasmid Construction…………………………………………………. 52 3.2.1 Bioinformatics Tools……………………………………...... 52 3.2.2 Polymerase Chain Reaction (PCR) …………………………. 52 3.2.3 Site-Directed Mutagenesis…………………………………... 53 3.2.4 Agarose Gel Electrophoresis……………………………...... 54 3.2.5 Cloning………………………………………………………. 54 3.2.6 Restriction Enzyme Reaction………………………………... 54 3.2.7 Gel and PCR Purification…………………………………… 55 3.2.8 DNA Quantification…………………………………………. 56 3.2.9 Ligation Reaction……………………………………………. 56 3.2.10 Transformation………………………………………………. 56 3.2.11 Screening Positive Colonies………………………………… 57 3.2.12 Medium Scale Plasmid Preparation…………………………. 58 3.2.13 Analysis of Plasmid Preparation…………………………….. 59 3.3 Protein Expression and Purification…………………………………... 60 3.3.1 Preparation of BL21 (DE3) Competent Cell……………...... 60 3.3.2 Transformation into BL21 (DE3) ………………………...... 60 3.3.3 Growth Curve of Transformed BL21 DE3 (+pCG1) ……….. 61 3.3.4 IPTG Induction……………………………………………… 61 3.3.5 Sodium Dedocyl Sulfate (SDS) polyacrylamide gel 62 electrophoresis (PAGE) …………………………………….. 3.3.6 Cell Lysis……………………………………………………. 63 3.3.7 Protein Purification………………………………………….. 63 3.3.7(a) Equilibration……………………..……………….. 63 3.3.7(b) Sample Application……………..………………... 63 3.3.7(c) Washing………………………..…………………. 64

3.3.7(d) Elution…………………………..………………... 64 3.3.8 Determination of Protein Concentration…………………….. 64 3.3.9 Western Blot………………………………………………… 65 3.4 Preparation and Optimisation for Peptide Phage Selection………...... 66 3.4.1 Quantification of Phage’s Host Bacterial Cell………………. 66 3.4.1(a) E. coli ER2738 Culture………...………………… 66 3.4.1(b) E. coli ER2738 Growth Curve and 66 Quantification…………..………..……………….. 3.4.2 Phage Titration………………………………………………. 67 3.4.3 Determination of Optimum Concentration of Protein to be 68 Coated on Plate…………………………………………...... 3.5 Subtractive Screening of Peptide Phage Display Library against 69 Target Protein……………………………………………...... 3.5.1 Panning against Streptavidin……………………………...... 69 3.5.2 Panning against C139A NS6 Protease…………………….. 71 3.5.3 Phage Titration………………………………………………. 71 3.5.4 Phage Amplification, Precipitation and Purification………... 71 3.6 Isolation of Phage DNA………………………………………………. 72 3.6.1 Plaque Isolation and Amplification…………………………. 72 3.6.2 Phage DNA Extraction……………………………………… 72 3.6.3 PCR Amplification of DNA Insert from Phage Genome…… 73 3.6.4 Purification of PCR Amplicon………………………………. 73 3.6.5 DNA Sequencing Analysis………………………………….. 74 3.7 Binding Affinity Assessment of Phage……………………………….. 74 3.8 Bioinformatics Study…………………………………………………. 75 3.8.1 Prediction of Protein Structure……………………………… 75 3.8.2 Prediction of Selected Peptide Phage Binding Sites on 75 C139A NS6 Protease……………………………………….

CHAPTER 4 : RESULTS……………………………...... 77 4.1 Recombinant MNV-1 C139A NS6 Production and Purification…… 77 4.1.1 Cloning of C139A NS6 Gene into Expression Plasmid..….. 77 4.1.2 Overexpression and Purification of MNV-1 C139A NS6…. 84

4.2 Identification of Peptide Phage with Binding Capacity towards the 87 Target Proteins ……………..……………...... ……………. 4.2.1 Parameters Optimisation for Peptide Phage Selection …...... 87 4.2.1(a) Optimum Growth Time for E. coli ER2738 87 Culture …………………………….….………….. 4.2.1(b) Peptide Phage Concentration in the Ph.D-7 Phage 89 Display Peptide Library Kit…..………………….. 4.2.1(c) Determination of Optimum Concentration of 89 Streptavidin to be Coated on Plate…..………...... 4.3 Peptide-phage with Binding Activity towards Streptavidin………….. 90 4.3.1 Panning of Peptide Phage Library against Streptavidin…….. 90 4.3.2 Identity of Peptide Sequence from Streptavidin-Binding 92 Peptide Phage... ……………………………………...... 4.3.3 Binding Kinetics of Selected Peptide Phage towards 94 Streptavidin……………………………………………...... 4.4 Peptide-phage with Binding Activity towards MNV-1 C139A NS6.. 95 4.4.1 Determination of Optimum Concentration of C139A NS6 95 to be Coated on Plate………………………………………... 4.4.2 Panning of Peptide Phage Library against C139A NS6…… 96 4.4.3 Identity of Peptide Sequence from C139A NS6-Binding 97 Peptide Phage……………………………………...……….... 4.4.4 Binding Kinetics of Selected Peptide Phage towards 103 C139A NS6……..……………………………………...... 4.5 Structure Prediction C139A NS6 Protease ……………………...... 106 4.6 Binding Simulation (Docking) of Selected Peptide Sequences 108 towards C139A NS6……………………………………………...... 4.6.1 Binding Simulation of 7-mer Peptide ADARYKS on 108 C139A NS6…….……………………………………...... 4.6.2 Binding Simulation of 7-mer Peptide QTEKNPL on 110 C139A NS6……..……………………………………...... 4.6.3 Binding Simulation of 7-mer Peptide NSLKVLG on 112 C139A NS6…….……………………………………......

CHAPTER 5 : DISCUSSION……………………………………...... 114 5.1 Production of Recombinant C139A NS6…………………………… 114 5.2 Parameters Optimisation for Peptide Phage Selection………………... 117 5.2.1 Optimum Growth Time for E. coli ER2738 Culture……...... 117 5.2.2 Peptide Phage Concentration in the Ph.D-7 Phage Display 118 Peptide Library Kit………………………………………….. 5.2.3 Optimum Concentration of Target Proteins on ELISA Plate.. 119 5.3 Peptide Phage Library Panning against Streptavidin…………………. 119 5.4 Binding Kinetics of Selected Peptide Phage towards Streptavidin...... 123 5.5 Peptide Phage Library Panning against C139A NS6 Protein……….. 124 5.6 Binding Kinetics of Selected Peptide Phage towards C139A NS6…. 128 5.7 Prediction of Protein Interaction between Selected 7-mer Peptide 130 Sequences and C139A NS6 Protein………………………………… 5.7.1 Structure Prediction of C139A NS6 using I-TASSER…….. 130 5.7.2 Binding Simulation of Selected 7-mer Peptide Sequences on 130 C139A NS6………………………………………………...

CHAPTER 6 : CONCLUSION & FUTURE RECOMMENDATIONS.. 134 6.1 Conclusion Future Direction …………………………………………. 134 6.2 Recommendations for Future Research….…………………………… 134

REFERENCES…………………………………………………………….. 136

APPENDICES

LIST OF AWARDS, CONFERENCE AND PUBLICATION

vii

LIST OF TABLES

Page

Table 3.1 Bacterial strain and plasmid 45

Table 3.2 Antibodies 46

Table 3.3 Commercial kit 46

Table 3.4 Bacterial culture media and antibiotics 47

Table 3.5 Reagents 47

Table 3.6 Chemicals 48

Table 3.7 Consumables 49

Table 3.8 List of Equipments 49

Table 3.9 Recipe for preparation of reagents 50

Table 3.10 List of Primers 51

Table 3.11 PCR components 53

Table 3.12 PCR condition for NS6 protease gene amplification 53

Table 3.13 Components for GoTaq DNA polymerase 59

Table 3.14 Resolving gel 62

Table 3.15 Stacking gel 62

Table 3.16 Standard BCA 65

Table 3.17 PCR condition for amplification of DNA insert from 73 phage genome

Table 4.1 Number of recovered phages for every round of 91 panning

Table 4.2 The identified sequences from 20 single clone phages 94

Table 4.3 Total number input phage, recovered phage (output 97 phage), amplified phage and phage enrichment for each round of C139A NS6 panning

viii

Table 4.4 DNA sequences analysis of 98 samples of C139A 101 NS6 phage clones

Table 4.5 Maximum binding value, Bmax and dissociation 105 constant, Kd of selected clones

Table 4.6 Peptide-receptor interactions between ADARYKS 109 peptide and C139A NS6 receptor

Table 4.7 Peptide-receptor interactions between QTEKNPL 111 peptide and C139A NS6 receptor

Table 4.8 Peptide-receptor interactions between NSLKVLG 113 peptide and C139A NS6 receptor

LIST OF FIGURES

Page

Figure 2.1(A) Schematic representation of genome organization 13 of Norovirus

Figure 2.1(B) Schematic structure of capsid protein 13

Figure 2.2 Cartoon representation of the MNV NS6pro 30 structure

Figure 2.3 Schematic diagram of protease cleavage 31

Figure 3.1 Diagrammatic representation of the phage display 70 method

Figure 4.1(A) The schematic diagram of expression plasmid 79 (pET26Ub: MNV 3D-His)

Figure 4.1(B) The schematic diagram of pT7:MNV3’Rz plasmid 79 that contains the full genomic sequence of MNV-1

Figure 4.2 This schematic diagram of polymerase chain 80 reaction series to construct recombinant MNV-1 C139A NS6

Figure 4.3 The PCR amplicons from 1st pair of primer 81

Figure 4.4 The PCR amplicons from 2nd pair of primer 81

Figure 4.5 The final PCR amplicon from 3rd pair of primer 82

Figure 4.6 Product of restriction enzyme (SacII and BamHI) 82 reaction for expression plasmid pET26Ub: MNV 3D-His

Figure 4.7 Purified DNA products of restriction enzyme 83 reaction

Figure 4.8 Growth curve of C139A NS6-His 85

Figure 4.9 Expression of C139A NS6 protein 85

Figure 4.10 Western blot analysis of purified C139A NS6 86 protein

Figure 4.11 Purified C139A NS6 protein 86

Figure 4.12 Growth curve of E. coli ER 2738 88

Figure 4.13 Optimum concentration of streptavidin and BSA 90

Figure 4.14 Formation of blue plaques on LB/IPTG/XGAL 91 plate

Figure 4.15 PCR products from amplification using purified 93 phage genomic DNA of single clones

Figure 4.16 Library insert sequences fused to the pIII major coat 93 protein and located on the region between 2 restriction sites

Figure 4.17 Binding kinetics illustrating the specificity and 95 affinity of the peptide phage clone towards Streptavidin and BSA protein

Figure 4.18 Optimum Concentration of C139A NS6 to be 96 Coated on Plate

Figure 4.19 PCR products from amplification using purified 99 phage genomic DNA of single clones isolated from panning against C139A NS6

Figure 4.20 Percentage of hits of the identified peptides 102

Figure 4.21 Binding ability of selected peptide-bearing phage 104 clones at phage concentration 2 X 011 pfu/ml towards different target protein

Figure 4.22 Binding kinetics of selected phage clones towards 105 target protein C139A NS6

Figure 4.23 Schematic representation of protein structure 107

Figure 4.24 Predicted structure of ADARYKS peptide C139A 109 NS6 receptor complex as predicted by CABS-dock software

Figure 4.25 Predicted structure of QTEKNPL peptide C139A 111 NS6 receptor complex as predicted by CABS-dock software

Figure 4.26 Predicted structure of NSLKVLG peptide C139A 113 NS6 receptor complex as predicted by CABS-dock software

Figure 5.1 Schematic representation of the expression plasmid 115 pET26Ub:MNV C139A NS6 His

LIST OF ABBREVIATIONS

C139A NS6 active site mutant

BCA Bicinchoninic Acid

BSA Bovine serum albumin

CDC Centre for Disease Control & Prevention cDNA complementary Deoxyribonucleic Acid

CIP Calf alkaline phosphatase

DNA Deoxyribonucleic acid

ECL Enhanced Chemiluminescent

EDTA Ehtylenediaminetetraacetic Acid eIFs Eukaryotic initiation factors

ELISA Enzyme-linked Immnosorbent Assay

EtBr Ethidium Bromide

FCV Feline Calicivirus

FUT 2 α-(1,2)-fucosyltransferase gRNA genomic RNA

HBGAs histo-blood group antigens

HCV Hepatitis C virus

HPQ Histidine-Proline-Glutamine

HRP Horseradish Peroxidase

HuCv Human calicivirus

HuNv Human norovirus

IPTG Isopropyl β-D-1-Thiogalactopyranoside

xii

LB Luria Bertani

MNV Murine norovirus

NCBI National Center for Biotechnology Information

NV Norovirus

OD Optical density

ORF Open reading frame

PBS Phosphate Buffer Saline

PCR Polymerase Chain Reaction

PEC Porcine enteric calicivirus

PMO phosphorodiamidate morpholino oligomer

PPMO peptide conjugated phosphorodiamidate morpholino oligomers

PVDF polyvinylidene fluoride

RAG 2 recombination activation gene 2

RAW264.7 murine macrophage cell line

RdRp RNA dependent RNA polymerase

RNA Ribonucleic acid

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis sgRNA subgenomic RNA

STAT-1 signal transducer and activation of transcription 1

TBS tris buffered saline

TBST tris buffered saline containing Tween 20

TMB 3,3’5,5’-Tetramethylbenzidine

UTR Untranslated region

xiii

VF1 Virulence factor 1

VLP Viral-like particle

VPg Viral protein linked genome

WT Wild type

xiv

LIST OF SYMBOLS

% Percentage

> more than

° C degree Celsius

µl Microliter

µM Micromolar h Hour mg Milligram mins Minutes mM Millimolar nm Nanometer nt Nucleotides pfu plaque forming unit secs Seconds v/v volume per volume w/v weight per volume

IDENTIFIKASI DAN PENCIRIAN PENGIKATAN FAJ PEPTIDA

KE ATAS REKOMBINAN PROTEASE (NS6) MURIN NOROVIRUS

ABSTRAK

Jangkitan norovirus dianggap sebagai penyebab utama kes penyakit gastroenteritis akut yang berlaku secara epidemik dan berkala di seluruh dunia.

Sehingga kini, tiada langkah intervensi terapeutik berlesen sama ada dari segi vaksin atau ubat-ubatan yang tersedia untuk mengawal penularan patogen manusia ini.

Menyasarkan protein bukan struktural virus seperti protease adalah relevan kerana bahagian yang terjaga di samping fungsinya yang penting dalam sistem replikasi virus.

Dalam kajian ini, versi mutasi MNV NS6 yang aktif (C139A NS6) telah diklon, diekspres dan ditulenkan sebelum digunakan dalam pendulangan bio / pemilihan yang menggunakan kit Ph.D-7TM Phage Display Peptide Library (NEB). Selepas 6 pusingan pendulangan bio, beberapa peptida faj yang spesifik dengan C139A NS6 telah berjaya dikenal pasti dan diasingkan. Peptida faj yang telah dikenalpasti kemudiannya telah disaring menggunakan pangkalan data peptida dalam talian dan hasilnya menunjukkan bahawa semua motif adalah unik dan tidak mempunyai persamaan dengan mana-mana peptida yang pernah diterbitkan. Spesifikasi ikatan beberapa klon peptida faj dengan jumlah pengulangan tertinggi telah diuji menggunakan ELISA.

Keputusan ELISA menunjukkan klon yang membawa urutan peptida QTEKNPL mempunyai keupayaan mengikat yang tertinggi terhadap C139A NS6 berbanding klon lain dan juga klon kawalan. Dalam analisa silico, struktur tersier C139A NS6 dan interaksi peptida terpilih (ADARYKS, QTEKNPL dan NSKLVLG) terhadap reseptor C139A NS6 telah diramal masing-masing menggunakan perisian I-

TASSER dan CABS-dock. Analisis dok menunjukkan bahawa semua urutan peptida

xvi yang terpilih berinteraksi dengan reseptor C139A NS6 pada beberapa interaksi putatif. Menariknya, semua peptida yang dipilih mengikat reseptor C139A NS6 di tapak yang tiada dalam laporan penyelidikan sebelum ini. Analisis bioinformatik ini memberikan beberapa maklumat penting untuk penyelidikan pada masa hadapan berkaitan interaksi reseptor ligan yang melibatkan peptida terpilih dan protein

C139A NS6. Maklumat ini berpotensi untuk dimanipulasi dalam pembangunan perencat peptida kecil terhadap protease NS6 serta untuk kegunaan diagnostik dengan tujuan utama bagi mengawal replikasi norovirus.

xvii

IDENTIFICATION AND BINDING CHARACTERISATION OF PEPTIDE-

BEARING PHAGES TOWARDS RECOMBINANT PROTEASE (NS6) OF

MURINE NOROVIRUS

ABSTRACT

Norovirus infections are considered the most common causes of epidemic and sporadic cases of acute gastroenteritis worldwide. To date, there are no licensed therapeutic intervention measures either in terms of vaccines or drugs available for these highly contagious human pathogen. Targeting non-structural viral protein such as protease is relevant due to their well conserved regions along with its critical functions in viral replication. In this study, the active site mutant version of MNV NS6 protease (C139A NS6) was cloned, overexpressed and purified before being used as target in biopanning/selection employing the Ph. D-7TM Phage Display Peptide Library kit (NEB) which was validated in streptavidin panning. After six rounds of biopanning, several peptide phages that are specific towards C139A NS6 were isolated and identified successfully. Upon identification of peptide phage, the peptide sequences were screened using online peptide databases and results indicated that all the sequences were unique and did not possess common motif with any published peptides to date. Three peptide phage clones with highest number of hits were tested for their binding specificity towards purified C139A NS6 using ELISA. It was observed that clones carrying peptide sequence QTEKNPL had the highest binding affinity towards C139A NS6 as compared to other clones and irrelevant phage control. Tertiary structure of C139A NS6 and binding interaction of the selected peptides (ADARYKS, QTEKNPL and NSKLVLG) with C139A NS6 receptor were predicted using I-TASSER and CABS-dock software, respectively. Docking analysis

xviii showed that all the selected peptide sequences interact with C139A NS6 receptor at several contacts on the receptor binding sites that had never been published. This bioinformatics analysis uncovers some important information for future investigation in ligand-receptor interaction involving selected peptides and C139A NS6 protein which could potentially be manipulated in the development of small peptide inhibitor against NS6 protease as well as for the diagnostic purposes with ultimate purpose to control norovirus replication.

xix

CHAPTER 1

INTRODUCTION

1.1 Research overview

Virus is a small, infectious particle that can reproduce only by infecting a living cell and using its machinery to produce more viruses. They comprise of nucleic acid that can be either DNA or RNA which are enclosed in a protein shell called a capsid. Some viruses have an internal or external layer of membrane known as envelope. Virus are very diverse since they vary in their shapes, structures, kinds of genomes and type of hosts that they infect. Generally, viruses work by invading the host cells to make more copies of themselves and throughout the process, they often cause destruction to the invaded cell (Koonin, Senkevich, & Dolja, 2006).

Noroviruses belong to the family of Caliciviridae are positive-sense, single- stranded RNA virus. Due to their stability, low infectious dose, large host reservoir, short term immunity, multiple transmission routes and large diversity between strains, human norovirus particularly has been established as one of the leading causes that account for gastroenteritis cases worldwide with several clinical manifestations of acute diarrheal and vomiting, accompanied by several signs/symptoms like abdominal cramps, myalgia, malaise, headache, nausea and low-grade fever. This virus is thought to have a considerable economic impact due to the number of cases reported annually in developed countries.

Despite complexity in the progress of the vaccines due to the high degree of genetic variation of this virus, there are few vaccines for human calicivirus that are under pre-clinical and clinical trial (Lucero, Vidal, & O'Ryan, 2017; Tan & Jiang,

2014). Furthermore, immunity to human calicivirus is also short-lived since body are

1 not able to maintain immunity for a long time once a person is infected with natural infection (Mattison, 2011). For these reasons, antiviral therapy that is a class of treatment used specifically for treating viral infection seems to be an attractive strategy to allow the control of calicivirus outbreaks. An antiviral function by inhibiting the development of virus specifically by targeting the critical function of viral proteins, or parts of proteins which ultimately will affect the viral life cycle.

Norovirus NS6 protease is an important enzyme that enables the activation of its other non-structural proteins. This protease acts as a key point for the activation of norovirus replication. The importance of the enzymatic activity of the NS6 protease through its main function in cleaving the chain of non-structural large polyproteins into several active proteins has made it as an attractive target for developing antiviral therapies. This could be achieved by inhibiting the function of this protease which may result in the inhibition of the cleavage process of non-structural proteins which subsequently disrupt the replication of the virus in infected host.

Even though norovirus causes a huge lost to the western country and not widely spread in Malaysia, but due to current setting where immigration and emigration take place every single day, it is fair to take the possibilities of transmission of this virus into consideration. In this study, we aim to identify a peptide-bearing phages with the ability to bind the target (NS6) with high affinity and specificity using the phage display technique. After the identification of such peptide sequences, several commercial synthetic peptides could be synthesized and use to assess their inhibitory effects and efficiency to act as antiviral therapy. The benefit of developing peptide- based antiviral drugs is that the peptides can be designed to mimic or interact with conserved surface proteins and in case of strains variety, the peptide sequence could be modified to preserve therapeutic efficiency. As compared to other approach, the

2 strength of peptide-based approach is in their good efficacy, safe, selectivity and predictable biocompatibility and biodegradability and are easy to being scaled-up.

1.2 Objectives of Study

1.2.1 General Objective

This study was conducted to identify the peptide-bearing phages and characterise their binding ability towards recombinant protease (NS6) of murine norovirus (MNV).

1.2.2 Specific Objectives

1. To clone, express and purify mutant NS6 protease.

2. To select peptide-bearing phage that binds to the recombinant mutant NS6

protease.

3. To determine the ability of the selected peptide-bearing phage to bind to the

recombinant mutant NS6 protease.

4. To predict the tertiary structure of mutant NS6 protease and docking analysis of

the selected peptide sequences on mutant NS6 protease receptor.

CHAPTER 2

LITERATURE REVIEW

2.1 Gastroenteritis

Gastroenteritis is a condition of an inflammation of gastrointestinal tract that includes stomach and intestinal mucosal surface (Schlossberg, 2015). It is also known as infectious diarrhea whereby it is associated with symptoms such as diarrhea, vomiting, malaise, muscle pain, fatigue, dehydration and high-grade fever (Glass, Parashar, &

Estes, 2009; Karst, 2010). This condition can be caused by infectious agents such as viruses (rotavirus and norovirus), bacteria (Escherichia coli and

Campylobacter species), parasites (Giardia lamblia), fungus as well as non-infectious sources like medications and consumption of certain foods such as lactose and gluten

(Ciccarelli, Stolfi, & Caramia, 2013; Helms & Quan, 2006; Szajewska & Dziechciarz,

2010). However, the most common cause of gastroenteritis is due to infection by viruses; for example norovirus which causes 685 million cases and 213,515 death globally in developed and developing country (Pires et al., 2015).

Virus that are commonly known to cause viral gastroenteritis are rotavirus, norovirus, adenovirus and astrovirus (Oude Munnink & van der Hoek, 2016).

Rotavirus is the most known causes of gastroenteritis in children while norovirus has been identified as the major cause of epidemic gastroenteritis and common cause of diarrhea in adults (Glass et al., 2009; Hall et al., 2013). Initially in 1972, virus that was described as calicivirus was identified in the stool of patient with diarrhea during an outbreak of gastroenteritis in a school in Norwalk, Ohio (Kapikian, 2000; Kapikian et al., 1972). Currently, diarrheal disease is the top five causes of death worldwide with children are the most affected as compared to adult due to their lack of immunity development and relatively poor hygiene. In childhood gastroenteritis, norovirus have

4 been reported to be the second cause following rotavirus, but to date, the widespread use of vaccine against rotavirus has made norovirus to be more likely as the most common cause of gastroenteritis in childhood (Baehner, Bogaerts, & Goodwin, 2016;

Karst, 2010)

2.2 Caliciviruses

The Caliciviridae family consists of small RNA viruses that are important in both medical and veterinary area (Royall & Locker, 2016). It belongs to Class IV of the Baltimore scheme where positive single-stranded RNA viruses whose mRNA is identical in base sequence to virion RNA can be accessed by host ribosomes to directly form proteins. Viruses with this type of RNA genome can be divided into two groups where both reproduce in the cytoplasm. The first group is polycistronic mRNA type viruses where mRNA from the genomic RNA is translated into a polyprotein. This product will then subsequently cleave to form single mature proteins. In contrast, the second group is where viruses with complex transcription use different mechanisms to produce proteins from the same strand of RNA. It can be either using subgenomic mRNAs, ribosomal frameshifting and proteolytic processing of polyprotein

(Baltimore, 1971).

The name calicivirus derived from the Latin word calyx which means cup or chalice; and being referred to 32 cup-shaped depressions on the surface of the entire virus particle arranged in icosahedral symmetry (Green et al., 2000; Prasad, Matson,

& Smith, 1994). They are typically single-stranded and non-segmented, positive-sense

RNA genome with size ranging from 6.7–8.5 kb (Black, Burroughs, Harris, & Brown,

1978; Jiang, Wang, Wang, & Estes, 1993). The RNA genome of calicivirus were organised into two or three open reading frame (ORFs) flanked by two short

5 untranslated regions (Sosnovtsev et al., 2006). The genome structure in this family differs in the length of each ORF but they are all conserved. Major feature of

Calicivirus is the absence of a methylated cap at the 5’ terminus of the virion RNA.

Instead, a small protein (VPg) with size around 15 kDa was shown to be covalently linked to both 5’ end of the calicivirus genomic RNA (gRNA) and subgenomic RNA

(sgRNA), with polyadenylated at the 3’ end. This VPg protein drives the initiation of translation and define to be essential for the infectivity of the RNA (Black et al., 1978;

Burroughs & Brown, 1978). In addition, 3’ co-terminal sgRNA is also being transcribed during infection in all members of the Caliciviridae family (Royall &

Locker, 2016).

Caliciviruses infection are major cause of disease in humans and many animals as it can naturally infect organism such as human, cattle, cats and pigs (Hansman,

Jiang, & Green, 2010). Previously, this virus is not very well studied since there is no suitable animal model and lack of efficient cell culture system. However, advancement in modern genomic technologies has led to an increased in understandings of the virus family.

2.2.1 Classification and Taxonomy

Caliciviruses are classified into five established genera that are Norovirus, Lagovirus,

Vesivirus, Sapovirus and Nebovirus together with numbers of additional genera that have been proposed (Kitchen, Shackelton, & Holmes, 2011). They are Recovirus (eg,

Tulane virus) for a novel calicivirus detected in stool specimens from rhesus macaques, Valovirus that represent a novel group of swine caliciviruses known as the

St-Valérien-like viruses, Bavovirus that are found in chicken,

Nacovirus and Bacovirus that contain novel caliciviruses recovered from chickens and turkeys respectively, Minovirus that infect farmed fathead minnows (Pimephales

6 promelas), Salovirus that was identified in farmed Atlantic salmon (Salmo salar),

Sanovirus was found in goose and also Secalivirus that was acquired from a sewage sample which consist of partial genome of a highly divergent calicivirus (Farkas,

Sestak, Wei, & Jiang, 2008; L’Homme et al., 2009; Mikalsen et al., 2014; Mor et al.,

2017; Ng et al., 2012; Wang, Wang, Dong, Zhang, & Zhang, 2017; Wolf et al., 2012).

Members of caliciviruses that causes acute gastroenteritis infections in human are norovirus and sapovirus while other members like lagovirus, vesivirus and becovirus/nebovirus are not pathogenic to humans (Bailey & Goodfellow, 2009).

Initially, sapovirus was discovered in an outbreak of gastroenteritis in an orphanage in Sapporo, Japan (Chiba et al., 1979). This member of calicivirus is also known as

“Sapporo-like viruses” includes Manchester Virus and porcine enteric calicivirus

(PEC). The virus is transmitted through oral/fecal contact and most common symptoms are diarrhea and vomiting. The Vesivirus genus is circulating in swine, sea mammals, and felines as their natural hosts. This genus includes feline calicivirus and vesicular exanthema of swine virus; as well as San Miguel sea lion viruses that are found in marine and terrestrial mammals (Neill, Meyer, & Seal, 1995). Two main groups of Lagovirus are rabbit hemorrhagic disease virus that are highly fatal and

European brown hare syndrome virus that was observed in the livers of infected hares in 1982 (Green et al., 2000). The genus Nebovirus contains two virus species that infect cattle are Newbury-1 virus and bovine enteric calicivirus NB (Nebraska). These viruses cause diarrhea and intestinal disease in calves (Oliver, Asobayire, Dastjerdi, &

Bridger, 2006).

2.2.2 Morphology and Genome Organisation of Caliciviruses

In 1972, Albert Kapikian discovered a virion with 27 nm particle using immune- electron microscope (Kapikian, 2000). The icosahedral structure of virus is described 7 as the first calicivirus virion and was popularly known as Norwalk virus. For

Caliciviridae family, the viral genome acts as an mRNA template that can be readily translated, following entry into the infected cell. Members of these family have structure of gRNA that are conserved, however they differ in the length of each open reading frame (ORF). The Lagovirus, Sapovirus and Nebovirus genera have two ORFs while three distinct ORFs were observed for the Vesivirus and Norovirus genera

(Rohayem et al., 2010). Meanwhile, murine noroviruses was found to carry a fourth

ORF (McFadden et al., 2011)

There are differences in term of the expression of structural proteins that are the major capsid protein (VP1) and minor capsid protein (VP2) among the genera.

Within the Vesivirus and Norovirus genera, VP1 and VP2 are encoded by ORF2 and

ORF3 from the sgRNA while in the Sapovirus, Lagovirus, and Nebovirus genera, both structural proteins are encoded by ORF1 and ORF2. The position of the VP1 coding region of the later genera is at the 3’ end of ORF1. The VP1 termination sequence are typically overlap with VP2 start codons and they can be separated by a short stretch around 3–10 nucleotides. Thus, this condition allows the translation of VP2 to take place through reinitiation process even though they are originated from single strand of mRNA (Royall & Locker, 2016)

2.3 Norovirus

History of norovirus started in 1929, where common childhood illness that causes vomiting, diarrhea and fever was detected and named as “winter vomiting disease” by

Dr John Zahorsky who is a paediatrician (Zahorsky, 1929). In 1968, a virus is suspected when students and teachers of an elementary school in Norwalk, Ohio, experiences an outbreak of winter vomiting disease with symptoms of acute gastroenteritis, including vomiting and diarrhea (Adler & Zickl, 1969). Several

8 experiments that was conducted to identify an etiology for this infectious form of acute gastroenteritis failed, until in 1972 where Dr. Albert Kapikian and his group successfully described the Norovirus prototype Norwalk virus found in stool samples using immune electron microscopy (Kapikian et al., 1972). Following the visualisation of the Norwalk virus particle, its genome was then cloned in 1990; led to the development of molecular tools to study noroviruses in more detail (Xi, Graham,

Wang, & Estes, 1990)

Noroviruses infection has been identified in various host that includes humans, swine, cattle, sheep, mice, cats and dogs (de Graaf et al., 2017). Human caliciviruses can cause infection in a population even at low infectious dose (Teunis et al., 2008). It is also known as winter vomiting disease as the infection/epidemics illustrate that it occurred typically during cold weather (Robilotti, Deresinski, & Pinsky, 2015).

Norovirus outbreak mostly occur in semi-closed setting/environment communities that favour person-to-person transmission such as cruise ships, nursing homes, day care centres, schools, restaurants, hospitals, disaster relief/evacuation site, army barracks and even airplanes (Glass et al., 2009; Monroe, Ando, & Glass, 2000; Patel, Hall,

Vinjé, & Parashar, 2009; Robilotti et al., 2015; Widdowson, Monroe, & Glass, 2005).

The uncontrollable outbreak of norovirus infections is most likely due to high infectivity of norovirus particles, persistence of the virus in the environment, prolonged shedding of virus from both symptomatic and asymptomatic individuals and also short-term immunity (Kaufman, Green, & Korba, 2014). Norovirus is one of the genus under the Caliciviridae family that is considered as the most common cause of foodborne disease outbreak by Centers for Disesase Control and Prevention (CDC). It has been estimated that human pathogen within this genus cause at least 95% of nonbacterial gastroenteritis outbreaks and over 50% of all outbreaks worldwide (Karst,

2010). Thus, human norovirus (HuNv) is considered as a leading cause of gastroenteritis worldwide contributed to a severe economic burden to the health organisations of both the developed and developing world. This genus is highly transmissible primarily through exposure to contaminated food or water sources, person-to-person contact, an increased density of people, aerosolized vomitus particles and fomites (Karst, 2010; Lopman et al., 2012). They have been classified under

Category B biodefense agents due to their characteristics that are highly contagious, extremely stable in the environment, resistant to common disinfectants and associated with debilitating illness. These conditions provide fundamental towards a fast spreading and highly contagious disease (Karst, 2010).

It is reported that person of all age groups is vulnerable towards norovirus infection and secondary infection are also common (Estes, Prasad, & Atmar, 2006).

Norovirus replicate in gastrointestinal tracts of their host that result in acute or mild gastroenteritis characterised by symptoms such as vomiting and diarrhea coupled with or without nausea and abdominal cramp. Other symptoms include low-grade fever and malaise. There is also condition where the infected person is asymptomatic (Karst,

2010). On the contrary, infection that occur in an immunocompromized mice (lacking components of the innate system) indicates that a broad tissue tropism was involved as MNV causes encephalitis, vasculitis, pneumonia and hepatitis (Karst, Wobus, Lay,

Davidson, & Virgin, 2003).

Replication of norovirus RNA genome is dependent on virus-encoded RNA- dependent RNA polymerase, which has no proofreading activity that can correct the misincorporation of nucleotides during RNA synthesis. This condition results in high error-rate in genomic replication; causes the production of many progeny viruses with mutations. Therefore, norovirus is highly flexible and diversify as compared to other

RNA viruses (Estes et al., 2006; White, 2014). The Norovirus genus is classified based on phylogenetic clustering of the complete major capsid protein (VP1) amino acid sequence where members within a genogroup differ in their capsid genes by 45 -61%, while there are 14-44% and 0-14% different of members within genotype and strains within genotype respectively. These differences in term of intra-genus variation is high even compared to genera of other positive strand RNA virus families. High degree in genetic variation causes difficulty in developing protection against norovirus (Barclay et al., 2014; White, 2014; Zheng et al., 2006).

Currently there are 7 genogroups for norovirus and each of them is further characterised into few genotypes (Robilotti et al., 2015; Zheng et al., 2006). The norovirus strains that infect humans and causes acute gastroenteritis are found in 32 genotypes that are GI (n=9), GII (n=19) and GIV (n=1), whereas viruses in other genogroups infect other mammals. Noroviruses have also been isolated from other species including pigs (GII), bovine (GIII) and murine (GV) (Vinje, 2015). Recently proposed genogroups were GVI and GVII where norovirus was identified in domestic dogs with diarrhea (Mesquita, Barclay, Nascimento, & Vinjé, 2010). Among all the genogroups of norovirus, the GII.4 causes >80% of all human norovirus infections at any one time (Siebenga et al., 2009; White, 2014). This genotype virus is responsible for all six major norovirus pandemics of acute gastroenteritis in the last two decades

(White, 2014).

2.3.1 Norovirus Genome Structure

Norovirus virion is non-enveloped with icosahedral symmetry of proteinaceous capsid that packages the RNA genome. The capsid is made up of 180 copies of the ORF2 encoded protein, grouped as dimers in T=3 symmetry (Prasad, Hardy, Jiang, & Estes,

1996; Prasad et al., 1999). The linear single stranded positive sense RNA genome

11 with approximately 7.4–7.7 kb in size are typically organized into three open reading frames (ORF) (Figure 2.1A) (Jiang et al., 1993; Robilotti et al., 2015; Rocha-Pereira

& Nascimento, 2012; Thorne & Goodfellow, 2014). Murine noroviruses possess a fourth ORF that is overlapping with ORF2 but in an alternate reading frame; and only some sapovirus members display an alternative open reading frame at the equivalent position (Clarke & Lambden, 2000; Thackray et al., 2007). All six of the nonstructural proteins of the norovirus genome are encoded in a single ORF (ORF1) at the 5’ end region while the 3’ end region is transcribed into a subgenomic information. The subgenomic region encodes the major and minor structural proteins in two separate

ORFs that are ORF2 and ORF3 respectively (Karst, 2010).

A study conducted by Prasad et al (1999) using Norwalk VLP revealed that major capsid protein (VP1) which determines the antigenicity of the virus are divided into three parts that are N-terminal, shell domain (S) located at the base of the capsid and a protruding (P) domain that is further subdivided into the P1 and P2 regions

(Figure 2.1A and 2.1B). The P domain has ability to change shape that can result in its mutation due to its condition that is linked to the S domain by a flexible hinge region.

The S domain provides internal core of the capsid while P domain probably contain both immune and cellular recognition sites. The interaction of inner S domain subunits with their neighbouring S domains form a continuous internal shell structure to the capsid. Meanwhile, the cup-like structure was formed when P domain emerge from the S domain surface (Hardy, 2005; Prasad et al., 1999). Mutational analysis on VLP results described that even though shell domain consist of components that are sufficient to initiate the assembly of capsid, the entire P domain is also important to enhance the stability and regulate the size of the capsid structure (Bertolotti-Ciarlet,

White, Chen, Prasad, & Estes, 2002).

Figure 2.1 A) Schematic representation of genome organization of Norovirus. The non- structural protein is encoded in ORF1 while ORF2 and ORF3 encode for major and minor capsid protein respectively. There is presence of ORF4 that overlap with ORF2; only in murine norovirus. The VP1 (major capsid protein) is further characterised into domain. The small N-terminal domain (light blue) faces the interior of the particle. The shell domain (S) is colored blue. The P1 subdomains are colored red. The P2 domain (yellow) is an insertion in the P2 domain. The hypervariable region of VP1 is found in the P2 domain. B) The schematic structure of capsid protein from electron microscope based on description for ribbon in (A). The figure was adapted from (Lopman, 2015)

Norovirus RNA genome is covalently linked with a viral protein (VPg) at their

5’ ends and are polyadenlyated at their 3’ ends (Karst, 2010). Several conserved secondary structures that was important for the replication of a MNV was identified through bioinformatic analysis. These structures include two or more 5’ terminal stem- loops, a 3’ terminal hairpin and a stem-loop just upstream of the ORF1/2 junction in the antigenomic strand proposed to be a component of the subgenomic promoter

(Simmonds et al., 2008).

2.3.1(a) Norovirus Non-Structural Proteins

Norovirus positive sense ssRNA genome is typically arranged into three ORFs and the genome layout is almost identical even though they are from different genogroups of norovirus. The genomic and sgRNA of norovirus are uncapped and they are instead linked to a short viral protein (VPg) which act as a cap substitute at 5’ end followed by a short untranslated region (UTR) ranges from 3 and 5 nucleotides for human and murine norovirus respectively (Gutiérrez-Escolano, Brito, del Angel, & Jiang, 2000).

The secondary structures of these UTRs play important roles in viral protein translation

(McFadden et al., 2011).

The ORF1, encodes a large polyprotein that comprises of all six mature non- structural proteins (NS1-NS7), which is autocatalytically cleaved to produce individual non-structural proteins (Thorne & Goodfellow, 2014). The non-structural proteins were cleaved from polyprotein by the viral protease during and post- translation; whereby the viral protease itself is catalytically an active component of the polyprotein (Hardy, 2005). These non-structural proteins are important for the establishment of viral replication complexes and genome replication. ORF2 encodes the major capsid protein (VP1) in an alternate reading frame from ORF1 while ORF3 encodes for a minor structural protein (VP2). There are about 20 nucleotides overlapping between ORF1 and ORF2. Nevertheless, ORF2 is not co-translated with

ORF1, but it is translated from a sgRNA covalently linked VPg-capped and polyadenylated that is produced during viral replication. ORF3 encodes protein in different reading frame than ORF2 (Thorne & Goodfellow, 2014). The ORF3 is proceed with 3’ UTR (forms secondary structures which function during initiation of viral protein translation) as well as the genome is polyadenylated at the 3’ end

(McFadden et al., 2011; Rohayem, Robel, Jager, Scheffler, & Rudolph, 2006).

ORF1 that encode viral polyprotein which comprises of at least 6 individual protein subunits is the first protein translated following infection of a cell (Hardy,

2005). The first non-structural protein in norovirus is NS1/2 or known as N-term, p48 or ‘2B-like’ protein due to characteristic of this protein that have limited homology to picornavirus 2B protein. This protein carries similar function as picornavirus 2B that engage in rearrangement of membrane which lead to alteration of the membrane permeability (Fernandez-Vega et al., 2004). In addition, NS1/2 also interacts with a host protein necessitate in regulating vesicle transport and can inhibit cellular protein trafficking of cell surface expression (Ettayebi & Hardy, 2003). Other than that, expression of this 45 kDa protein using Norwalk virus showed that this protein is associated with Golgi disassembly suggesting their potential role in replication complex formation (Ettayebi & Hardy, 2003; Fernandez-Vega et al., 2004). According to Sosnovtsev et al. (2006) there is a presence of caspase cleavage site within the NS1/2 of MNV, thus suggesting some cleavage during infection that probably associates virus replication to induction of program cell death.

The second protein constituent of norovirus viral polyprotein is the nucleoside triphosphatase (NTPase), also referred to as p41. NS3 has been described to exhibit

NTPase activity that act to bind and hydrolyse nucleotide triphosphate (Pfister &

Wimmer, 2001). This protein shares sequence motifs with picornavirus protein 2C.

Both proteins do not have helicase activity but unlike 2C, p41 is able to hydrolyse all

NTPs instead of ATP only. According to a study conducted by Hyde et al. (2009) in

MNV infected cells, NTPase showed that it plays a critical role in the establishment of viral replication complexes.

The third protein component of the polyprotein is NS4, p22, or 3A-like protein as it is similar in gene order to the picornavirus 3A protein of picornavirus. VPg

15 recruitment to membranous replication complexes during replication is thought to be the function of NS4 (Wobus et al., 2004). Other than that, NS4 also may function in tissue culture adaptation of MNV since presence of sequence changes upon continuous passage of MNV-1 in RAW264.7 cells give rise to attenuated viruses (Bailey,

Thackray, & Goodfellow, 2008).

The NS5 protein or VPg is covalently linked to the 5’ ends of viral gRNA and sgRNA of caliciviruses in place of a typical 5’ cap structure (Rohayem et al., 2006). It acts in a few roles in the viral life cycle but mainly it functions in translation initiation

(Karst, 2010; Thorne & Goodfellow, 2014). During viral genome replication, the association of NS5 and RNA is thought to occur when NS5 is used as a peptide primer for genome replication by viral RNA-dependent RNA polymerase or RdRp (NS7). The uridylylation of VPg made it possible to act as primer during viral replication (Belliot,

Sosnovtsev, Chang, McPhie, & Green, 2008; Rohayem et al., 2006). Biochemical analysis done in vitro performed by Rohayem et al (2006) confirmed the ability of norovirus NS7 to transfer nucleotide to NS5. Plus, there is study carried out by Guix et al (2007) stated that VPg that is covalently linked to 5’ end of human Norwalk virus

RNA is important for virus infectivity. It is proven when MNV RNA generated from cDNA clone that is capped in vitro were found to be infectious when transfected into cell (Yunus, Chung, Chaudhry, Bailey, & Goodfellow, 2010). Other than that, there is also an interaction between VPg and host translation initiation factors take place; probably to recruit them to the 5’ end of the RNA genome for translation initiation of virus (Karst, 2010).

Norovirus ORF1 encodes for a large polyprotein that is post-translationally cleaved by the virus-encoded 3C-like protease (NS6) (Blakeney, Cahill, & Reilly,

2003; Sosnovtsev et al., 2006). Even though it is part of the ORF1 polyprotein, the

NS6 is able to be released first from the polyprotein by autocatalytic cleavage then followed by specific cleavage of other proteins from ORF1. This 19 kDa protein is thought to play a role in inhibition of cellular protein synthesis in infected cell. NS6 also able to cleave poly A-binding protein and host cell protein named as elF4G; both were necessary for mRNA translation of host cell (Kuyumcu-Martinez et al., 2004;

Willcocks, Carter, & Roberts, 2004). This protein will be discussed in more detail in later Section 2.3.4.

The NS7 protein or can be referred as 3Dpol encodes for viral RNA-dependent

RNA polymerase (RdRp) or also known as RNA replicase is an important enzyme for gRNA and sgRNA viral replicaton as it functions in catalysing the synthesis of RNA from RNA template. This protein that is positioned at C-terminus of norovirus non- structural polyprotein also possess a typical right-handed conformation (palm, fingers and thumb) shared by all positive-sense RNA viruses (Ferrer-Orta, Arias, Escarmís, &

Verdaguer, 2006). There are four main mechanisms in which recombinant calicivirus

RdRp have been demonstrated to initiate RNA synthesis. The mechanisms include a de novo initiation and primer-independent initiation, back-priming base initiation and a protein-primed initiation via VPg nucleotidylylation (Rohayem et al., 2006).

2.3.1(b) Norovirus Structural Proteins

Norovirus ORF2 and ORF3 encodes the major and minor structural protein respectively. Both structural proteins are expressed from the viral VPg-linked sgRNA that is 3’ co-terminal with the gRNA and polyadenylated.

Norovirus ORF2 contain 58.9 kDa major structural protein VP1 or capsid.

Independent expression of VP1 from other viral components results in formation of dimer that can lead to the self-assembly of VLPs (Jiang, Wang, Graham, & Estes,

1992). These structures are morphologically and antigenically exhibit the same properties of native norovirus virions and have been remarkably useful to study a variety of virus-host interactions in the absence of a cell culture system (Jiang et al.,

2000). The P2 domain of VP1 consist of a hypervariable region that is suspected to have receptor binding and antigenic sites; consistent with its exposed location on the virion surface. Study conducted by Bailey et al (2008) showed that in vivo attenuation is observed when a single amino acid is changed in the P2 domain of MNV (Bailey et al., 2008; Wobus et al., 2004).

Minor structural protein or VP2 is encoded by norovirus ORF3 and it is present in only 1–2 copies per virion (Glass et al., 2000; Karst, 2010). Among the virus within

Caliciviridae family, VP2 is small, basic, and quite divergent in both size and sequence. VP2 is thought to increases the expression level of VP1 and also stabilisation of capsid (Bertolotti-Ciarlet, Crawford, Hutson, & Estes, 2003). Its role in viral replication is currently undefined but there is evidence that VP2 is essential for production of infectious particle and for viral replication in feline calicivirus

(Sosnovtsev, Belliot, Chang, Onwudiwe, & Green, 2005). Moreover, the basic charge of VP2 indicates that it may be responsible in encapsidation of the viral genome. The expression of VP1 is speculating to occur via VPg-directed translation initiation which is similar to expression of non-structural proteins from gRNA while VP2 proceeds via a translation termination re-initiation (TTR) mechanism (Karst, 2010).

Fourth ORF was found in MNV where it overlapped with VP1 coding region and encodes for virulence factor 1 (VF1) (McFadden et al., 2011).

2.3.2 Virus Life Cycle

Previously, knowledge on norovirus pathogenesis are limited and primarily based on physical, histological and biochemical studies conducted on infected human volunteer.

Investigation and progression study on its biological aspect has been very slow due to lack of efficient cell culture system and absence of animal model system. However, in

2003, MNV has been discovered as biological model system to study norovirus since it was able to replicate efficiently in tissue culture and also due to establishment of reverse genetics system based on the first isolated strain from MNV (Chaudhry,

Skinner, & Goodfellow, 2007; Karst et al., 2003; Ward et al., 2007; Wobus et al., 2004;

Yunus et al., 2010). The discovery of excellent model system allowed investigation and analysis of the norovirus’ infectivity and pathogenesis in vivo. Even though some aspects of MNV such as the disease manifestation does not apply to human norovirus, but this model is the only readily available system which could contribute to understand norovirus translation, replication and its pathogenesis.

2.3.2(a) Attachment and Entry

The life cycle of norovirus begins when there is interaction of virus with its receptor on the cell surface of the host which is known to involve carbohydrate structures such as histo blood group antigen (HBGAs) in human norovirus cases (Donaldson,

Lindesmith, LoBue, & Baric, 2010). The susceptibility of individual towards norovirus infection is determined by the secretor status of the H antigen (Hutson, Airaud,

LePendu, Estes, & Atmar, 2005). Study conducted by Thorven et al. (2005) showed that individuals who are non-secretors of H-type 1 were found to be resistant to norovirus infection. The absent of H type secretion is due to mutation in the α-(1,2)- fucosyltransferace (FUT2) gene that is involve in the production of H-type 1 antigen in saliva and mucosa. On the other hand, study performed on MNV indicated that the

19 attachment receptor that is bind in the strain dependent manner of the host cells is through ganglioside-linked terminal sialic acid moieties, glycolipids and glycoproteins

(Taube et al., 2012; Taube et al., 2009). Once bound to its receptor, MNV enters cell by which the mechanism remains unclear. However, it is found out that the method of

MNV entry into the host cell is dependent on dynamin and cholesterol instead of clathrin and caveolin like feline calicivirus (FCV) (Gerondopoulos, Jackson,

Monaghan, Doyle, & Roberts, 2010; Stuart & Brown, 2006). This is because study conducted for MNV is by using RAW264.7 where it did not express caveolin and also there is no reduction in MNV infectivity when there is interfering in the clathrin pathway. Unlike FCV, flotillin depletion and endosomal acidification blockers also did not cause reduction in MNV infectivity. Cholesterol and dynamin pathways is thought to be the method of virus entry due to reduction in MNV infectivity post treatment with methyl-beta-cyclodextrin and dynamin inhibitor; even though the reduction is observed only when the cells is treated before and not after infection

(Gerondopoulos et al., 2010).

2.3.2(b) Translation of Viral Proteins

After entry on the positive sense VPg-linked RNA genome into the cytoplasm of the permissive cell, it can immediately act as an mRNA template for initial round of viral

RNA translation. Protein synthesis takes place upon recognition of viral RNA by cellular translation initiation factors first, before they are being translated into protein by cellular translation apparatus. Translation of viral gRNA typically use novel mechanism involving VPg to initiate translation and to increase coding capacity of their relatively short genome (Firth & Brierley, 2012; Thorne & Goodfellow, 2014).

The VPg which is the viral protein genome linked is a non-structural protein that is covalently linked to the 5’ end of the gRNA and sgRNA where they mediate translation

20 process to produce viral protein. It also serves as a cap substitute to ensure that its

RNA is translated instead of host cell mRNA which constitute of classical 5’ cap structure (Bailey & Goodfellow, 2009; Thorne & Goodfellow, 2014). Recently, study conducted by Leen et al. (2013) provided information that virus-encoded VPg protein possessed a compact helical core flanked by intrinsically flexible disordered N- and

C- terminus. These flexibilities might aid to the numerous roles carried by VPg in norovirus life cycle (Goodfellow, 2011).

The VPg protein also play a role in the interaction with the component of elF4F host cell translation initiation factor complex. The translation initiation machinery includes cap binding protein (elF4E), a scaffold protein (elF4G) that link the elF4F complex to the elF3 and RNA helicase (elF4A). The Vpg protein bind to the cap binding of elF4F component and this interaction result in recruitment of elF4G scaffold. Recruitment of RNA helicase component elF4A and elF3 complex is enabled following binding of elF4G and elF4E. After that, 43S ribosomal pre-initiation complex is recruited to the complex by elF3 prior to translation initiation (Chaudhry et al., 2006; Daughenbaugh, Fraser, Hershey, & Hardy, 2003; Goodfellow et al., 2005).

The 43S pre-initiation complex is formed upon the interaction of the Met-tRNAiMet- elF2-GTP and 40S ribosomal subunit is stabilized by elF3 (Putics, Vashist, Bailey, &

Goodfellow, 2010). Although Vpg is observed to bind with elF4E of both MNV and

FCV, but its interaction is crucial only in FCV. This is because inhibition in the elF4E activity of FCV show that it severey affect the Vpg linked RNA of FCV but not the in vitro translation of MNV Vpg linked RNA (Chaudhry et al., 2006).

The viral genome translation process will result in the production of proteins where each carry an essential role for viral replication. The first open reading frame encode for non-structural protein that is translated in a form of large polyprotein. This

21 polyprotein is then co- and post-translationally cleaved by the viral encoded protease at five specific junctions; releasing six mature forms of non-structural proteins

(Blakeney et al., 2003; Liu, Viljoen, Clarke, & Lambden, 1999). Research performed by Sosnovtsev et al. (2006, 2002) stated that these proteins able to carry out their function in their separate form as compared to FCV in which their protease and polymerase is in the fusion form named p76. On the other hand, ORF2 and ORF3 encode for VP1 (major) and VP2 (minor) capsid protein respectively. These proteins are part of sgRNA where in the infected cell, they are found to be in higher levels as compared to gRNA (Prasad, Rothnagel, Jiang, & Estes, 1994). Moreover, it is common for positive sense RNA viruses to produce sgRNA during the viral life cycle in order for them to control the viral protein expression. In addition to that, norovirus exhibit a polycistronic subgenomic that require a process to ensure that ribosomes are able to bind and translate the open reading frames that are located downstream. Hence, termination-reinitiation process is used with the aid of overlapping start and stop codon in between the ORF segment to allow expression of products from adjacent ORFs. In this process, the upstream ORF is translated by ribosome until it reaches stop codon at the end of its ORF. After that, the proportion of the ribosomal subunit that remain associated to the mRNA was reinitiated to translate downstream ORF at the start codon of the respective ORF (Napthine et al., 2009).

As mentioned previously, MNV exhibit the fourth ORF that is overlapped with

VP1, known as virulence factor 1 (VF1). This VF1 is antagonist to the innate immune response since protein express from ORF4 slow down the upregulation gene activated by the innate pathway. In comparison to the position of ORF2, this sgRNA VF1 started

13 bases downstream and it is possibly initiated by leaky scanning and two slips in the ribosome. Reverse genetic study indicated that this protein is not required for

22 replication in tissue culture and indeed only function as a viral accessory protein

(McFadden et al., 2011).

2.3.2(c) Genome Replication

The expression of viral non-structural protein lead to the formation of cytoplasmic membrane-bound replication complexes; thus allowing the replication process of viral genome (Bailey & Goodfellow, 2009). The replication complex is thought to act as the surface for the assembly of both viral and cellular replication factor where it also comprises of a few components that are essential for viral replication such as viral

RdRp, viral RNA (single and double stranded intermediates), other viral enzymes as well as host cell factors. The replication for all positive strand RNA viruses requires genome to function as mRNA for production of viral protein needed for genome replication and as a template for negative strand RNA synthesis (Thorne &

Goodfellow, 2014).

The RNA synthesis takes place following the interaction of RdRp with other viral and cellular factors which enable it to bind at the promoter sequence located 3’ end of both strand of RNA. Since norovirus viral genome RNA is in the form of positive strand, it can directly be used as the template to generate negative strand RNA by de novo initiation. The produced negative sense RNA is then subsequently used for the production of higher levels of positive strand gRNA and sgRNA. The production of sgRNA is the unique characteristic possess by all caliciviruses during their replication in infected cells. The sgRNA is attach to Vpg at the 5’end and polyadenylated at the

3’ end. They are expressed at the 3’ co-terminal of the full-length gRNA. The sgRNA of caliciviruses consist of ORF2 and ORF3 (and ORF4 for MNV) which encode for viral structural proteins. There are two proposed mechanism for production of sgRNA where both use negative sense RNA intermediate and RdRp encoded by NS7 gene in

ORF1 (Karst, 2010; Putics et al., 2010; Royall & Locker, 2016). Plus, norovirus RdRp exhibit an active site residues that is structurally and functionally conserved as other positive strand RNA viruses RdRp (Högbom, Jäger, Robel, Unge, & Rohayem, 2009).

The first mechanism involves premature termination during the synthesis of negative-sense gRNA. The NS7 RdRp terminates during elongation upon reaching termination signal that is located upstream of VP1 coding region, generating negative sense of subgenomic strand. The negative strand is then used as a template for VPg- dependent RNA synthesis to produce positive sense sgRNA; arising from de novo initiation. The replication of sgRNA will be repeated and eventually packed using VP1 capsid protein to form new virus particle. The model also proposed that higher production of positive sense viral RNA as indicated by increasing level of VP1 would lead to the formation of multimeric complexes by VP1 in order to prevent its interaction with RdRp. These result in the capsid assembly and consequently the formation of new infectious viral particles (Putics et al., 2010; Thorne & Goodfellow,

2014).

The second mechanism for sgRNA synthesis known as internal initiation involved a presence of secondary structure upstream of ORF2 in the negative sense gRNA where it serves as the promoter to generate positive-sense sgRNA. The RdRp will initiate RNA synthesis following VPg-dependent fashion to produce new strand of sgRNA strand. Sufficient sgRNA is further encapsidated to produce new viral particle

(Thorne & Goodfellow, 2014). Bioinformatic study conducted by Simmonds et al.

(2008) in among all the members of Caliciviridae family found out the presence of highly conserved RNA stem loop structure located downstream of VP1 coding region on the negative sense gRNA. The structure that is positioned 6 nucleotide (nt) from the start of the sgRNA is confirmed for its accuracy by the genetic and biochemical

24 analysis carry out by Yunus et al. (2015) where it plays an important role in the replication process as well as providing a geno-specific interaction with RdRp.

2.3.2(d) Capsid Assembly and Virus Release

The mechanism of viral assembly, encapsidation and the exit of viral particles are not fully elucidated yet. However, there is observation by Bertolotti-Ciarlet et al. (2002) on the ability of VP1 to direct a self-assemble to form VLP. Even though VLP assembly does not require VP2, its role in promoting the stability of VP1 and synthesis of infectious virion is important (Bertolotti-Ciarlet et al., 2002; Sosnovtsev et al.,

2005). In addition, the interaction of basic nature VP2 with acidic viral RNA has linked it to encapsidation (Sosnovtsev et al., 2005). Also, there is a study that support the interaction of VP1 and VP2 where there is a binding of VP2 mapped to a conserved motif in the VP1 shell domain (Vongpunsawad, Prasad, & Estes, 2013). The observation indicated the function of VP2 in the encapsidation as it is placed in the interior part of the capsid. However, the interaction between VP2 and gRNA remains unclear.

The mechanism of viral release from the host cell has yet to be studied in detail.

Since many calicivirus infection induce apoptosis, it is postulated that the mechanism of viral release is through apoptosis-induced membrane collapse (Bok, Prikhodko,

Green, & Sosnovtsev, 2009; Thorne & Goodfellow, 2014). There is a study conducted through intestinal biopsies which found that there is an accumulation of apoptotic epithelial cells observed in the infected immunocompromised patients (Bok et al.,

2009; Furman et al., 2009; Thorne & Goodfellow, 2014)

2.3.3 Murine Norovirus

MNV is one of the Caliciviridae member that has been discovered to infect mice and is closely related to human norovirus. In 2003, the prototype strain of MNV known as

MNV-1 was first isolated intracerebrally and characterised in immunocompromised mice with genetic defect in a specific signalling pathway that is important for initial innate immune system (Henderson, 2008; Karst et al., 2003; Wobus, Thackray, &

Virgin, 2006).

The knockout mice are lack in recombination-activating gene 2 (RAG2) and signal transducer and activator of transcription 1 (STAT-1). Other than that, mice that are lacking in both interferon alpha/beta and gamma receptor is found to be susceptible to the pathogen (Karst et al., 2003; Wobus et al., 2006). Lacking both these receptors or knockout mice (STAT-1-) infected with MNV-1 can lead to fatal systemic disease as compared to RAG2 knockout mice that is not lethal, but it causes persistent infection with continuous virus shedding (Karst et al., 2003; Mumphrey et al., 2007; Wobus et al., 2006). However, it is observed that infected mice lacking in either interferon receptors do not lead to fatal disease (Karst et al., 2003). Both types of interferon responses use STAT-1 protein that is important for innate immune responses against pathogen as their direct intracellular mediator (Wesoly, Szweykowska-Kulinska, &

Bluyssen, 2007).

In nature, MNV is an enteric virus where most of the strain were originally isolated from faeces samples or mesenteric lymph node, can be orally infectious as well as can be detected in mucosal sites particularly intestines and mesenteric lymph node. Even though the mode of transmission is similar to human norovirus, but the symptoms of gastroenteritis exhibit by MNV infected immunocompromised mice is differ than human calicivirus. In MNV infected immunocompromised mice, the

26 symptoms include high rates of encephalitis, meningitis, vasculitis of cerebral vessels, hepatitis and pneumonia, which frequently lead to death (Karst et al., 2003; Mumphrey et al., 2007) .

Other than MNV-1, three new strains have been isolated and identified in 2006 namely MNV-2, MNV-3 and MNV-4 respectively. These strains are highly conserved with about 86% to 91% identical in the nucleotide sequence. Although they all are genetically similar, but the later three identified strains are observed to cause persistent infections and prolonged fecal shedding in immunocompetent mice as compared to

MNV-1 that only cause short subclinical infection with less than 1 week fecal shedding

(Hsu, Riley, & Livingston, 2007; Hsu, Riley, Wills, & Livingston, 2006). In addition,

MNV is also a widespread pathogen since study conducted by Muller et al (2007) showed that 67.5 % mice in a research colony contain MNV reactive antibodies.

MNV is the first norovirus that is able to grow in cell culture as it can infect and replicate efficiently in vitro in macrophages and dendritic cells (Wobus et al.,

2004). Thus, it serves as the most accessible model to study the mechanism of norovirus translation and replication since it is the first norovirus that is capable of full infectious cycle in tissue culture (Karst et al., 2003; Wobus et al., 2004). This strain is routinely propagated in RAW264.7 cells (mouse macrophage cell line) to elucidate the mechanism of infection and replication of human norovirus. Other than that, the availability of small animal model that are versatile and inexpensive enable MNV to be used as an excellent model to study the viral pathogenesis in a natural host. Even though the clinical manifestation of MNV and human norovirus is typically not similar, but MNV can act as a model for human norovirus since both have high degree of genetic identity (Wobus et al., 2006).

Furthermore, the availability of robust norovirus system has provided an insight towards establishment of reverse genetic system to study the molecular biology of norovirus. The system that is based on the first isolated strain of MNV, the MNV-1

(CW1) serve an opportunity to study the effect of genetic changes on the phenotype and virulence of the virus. Thus, the MNV model system has been an authentic model for studying the intracellular life cycle of noroviruses that cannot be explored without a cell culture system or genetic manipulability host (Thackray et al., 2007; Wobus et al., 2006).

2.3.4 NS6 Protease

Viral genome that comprises particularly those of small RNA viruses is commonly known to have a limited coding capacity due to the error prone nature of the viral replication machinery and the dimensions of the viral capsid. Therefore, a number of strategies has been employed by almost all positive sense single-stranded RNA viruses in order to maximize their protein coding capacity (Firth & Brierley, 2012). Formation of a single large polyprotein is one of the mechanism that is commonly incorporated to encode many of the viral proteins responsible for replication of the viral genome.

This polyprotein is then being cleaved into at least 6 proteins, plus several stable intermediates by the virally encoded protease NS6 which play a central role in the maturation of functional viral proteins, and hence in its genome replication and the formation of virus particles (Emmott, Sweeney, & Goodfellow, 2015; Thorne &

Goodfellow, 2014). Studies on the cleavage of the norovirus polyprotein by the viral protease have been performed using several approaches including in vitro assays using

FRET peptides, western blotting of infected cell lysates and luciferase based in cell assay (Emmott et al., 2015; May, Korba, Medvedev, & Viswanathan, 2013; Qu,

Vongpunsawad, Atmar, Prasad, & Estes, 2014; Sosnovtsev et al., 2006; Takahashi et al., 2013).

Calicivirus NS6 is related in term of sequence and structure of picornavirus 3C proteases that also have the same role in polyprotein processing of single-stranded

RNA viruses (Leen, Baeza, & Curry, 2012). X-ray crystallographic analysis reveal that norovirus NS6 protease is a cysteine protease that adopts a chymotrypsin-like fold similar to the 3C proteases from picornaviruses (Allaire, Chernaia, Malcolm, & James,

1994; Leen et al., 2012). The crystal structure of the NS6 protease from murine norovirus 1 is adapted from Leen et al. (2012) as shown in Figure 2.2. The NS6 protease fold is composed of two well-defined β-barrel domains that is tightly packed against each other; a β-sheet domain 1 and a β-barrel domain 2, joined by a linking loop. The interface between these two domains forms the peptide-binding cleft of which is the protease active site at the centre, consisting of a catalytic triad of a cysteine

(Cys139) as the nucleophile, histidine (His30) residue as the general base catalyst and either a glutamic acid (Glu54) or aspartic acid (Asp54) residue in HuNv and MNV respectively that function to stabilize the imidazole ring of the histidine (E Zeitler, K

Estes, & Prasad, 2006; Herod et al., 2014; Leen et al., 2012; Rocha-Pereira, Neyts, &

Jochmans, 2014; Someya, Takeda, & Miyamura, 2005). All three amino acids of the triad have been shown to involve in the catalytic mechanism even though they are not fully defined (E Hardy, J Crone, E Brower, & Ettayebi, 2002; E Zeitler et al., 2006;

Leen et al., 2012). Probably, the active site of these chymotrypsin-like viral proteases have a cysteine, rather than a serine (Nakamura et al., 2005). However, they are likely to resemble the mechanism of the serine proteases since the catalytic triad is similar in term of arrangement to the Ser-His-Asp triad characteristic of serine proteases (Allaire et al., 1994; Leen et al., 2012).

Figure 2.2 Cartoon representation of the MNV NS6pro structure. The N and C-terminal domains are coloured as green and orange respectively. The side-chains of the amino acids that make up the catalytic triad, A139 (mutated from Cys), H30 and D54, are shown as sticks. The figure was adapted from (Leen et al., 2012)

The MNV genome consist of four ORFs. ORF1 encodes for approximately 190 kDa single large non-structural polyprotein that is cleaved by the viral encoded protease at five specific sites to release the six functional units (NS1/2 – NS7), whereas

ORF2, ORF3 and ORF4 each encode single proteins (Belliot et al., 2003; Hussey et al., 2011; Muhaxhiri et al., 2013; Sosnovtsev et al., 2006). The five non-structural boundaries are defined by Q-G, E-G or E-A at the cleavage junctions and also there is an addition of Q-N in the case of the MNV (Belliot et al., 2003; Herod et al., 2014).

Initially, upon reaching of VPg-conjugated gRNA to the cytoplasm of the host cell, ribosome is recruited to the VPg to translate the viral polyprotein using the host machinery. Upon translation of the viral polyprotein, the viral NS6 protease present within the C-terminal half of the polyprotein performs all the processing, including its own autocatalytic release from the precursor ((Belliot et al., 2003; Liu, Clarke, &

Lambden, 1996; Scheffler, Rudolph, Gebhardt, & Rohayem, 2007; Sosnovtsev et al.,

2006). The cleavage events take place through two distinct cleavage steps that are early and late cleavage. During the early stage, the cleavage take place between QG dipeptide; releasing p48 and p41 NTPase proteins. Meanwhile the late stage follows

30 two predicted pathways where the first pathway involves the cleavage at the E-A dipeptide position between VPg and Pro. This cleavage will result in the release of p22

VPg and ProPol products. The second pathway of cleavage occurs at the E-G dipeptide downstream of Pro to release Pol and followed by subsequent cleavage to release p22 at the E-G dipeptide downstream p22. Finally, all six viral proteins were release from the viral polyprotein following cleavage between VPg and Pro at the E-A dipeptide

(Belliot et al., 2003). The cleavage products were detected in vitro and in vivo in various animal calicivirus models. The summary of protease cleavage is shown in

Figure 2.3. Although there are some variations in the sequence of the five cleavage sites recognised by norovirus NS6, common features are identifiable.

Figure 2.3 Schematic diagram of protease cleavage to release individual non-structural proteins. The diagram is adapted from (Belliot et al., 2003) with slight modification.

Structural data suggests that domain 2 of the protease possesses specific subsites or ‘pockets’, which interact with non-structural boundaries are well defined and named S5–S1 according to the standard nomenclature for proteases (Schechter &

Berger, 1968). The substrate binding pocket largely dictate boundary specificity and cleavage (Herod et al., 2014). A preferred temporal order that is partially dictated by the amino acids in the P5–P2 and P2’ positions flanking the scissile bond dipeptide

(P1–P1’) is followed for cleavage of the five non-structural boundaries (Belliot et al.,

2003; Blakeney et al., 2003; E Hardy et al., 2002; May, Viswanathan, Ng, Medvedev,

& Korba, 2014; Scheffler et al., 2007; Someya & Takeda, 2009). Amino acids of glutamine and glutamic acid are on the P1 position at the N-terminal side of the cleaved peptide bond while at position P1’ of C-terminal side the amino acid is usually glycine or alanine. The properties of the residues at position P2 and P4 tend to be large and hydrophobic in nature (Belliot et al., 2003; Blakeney et al., 2003).

Outside the catalytic triad, the crystal structures of the protease in complex with boundary substrates showed that, most of the substrate binding interactions take place within domain 2 of the protease. The interaction particularly within the S2 and S4 pockets, that interact with the P2 and P4 boundary residues respectively (Hussey et al.,

2011; Leen et al., 2012; Muhaxhiri et al., 2013). It is suggested that mechanism on recognition of norovirus protease towards cleavage boundaries with different affinities upon substrate binding are through conformational change in the S2 and S4 pockets to accommodate variations in the P2–P5 boundary residues (Muhaxhiri et al., 2013).

However, there is less information on the prime-side binding pockets that interact with the P’ residues downstream of the cleavage junction, in comparison with the S5–S1 pockets. Plus, residues with smaller side-chain groups are preferred according to analysis of the residues which occupy the boundary P1’ position; suggesting

32 requirement of only a small S1’ pocket. Biochemical studies also suggested that is only minor effects on cleavage efficiency of P2’ residue (May et al., 2014). However, little structural information on the prime-side interactions and the available crystal structures show that beyond the S1’ pocket, the enzyme does not appear to have an extended major binding groove (Herod et al., 2014).

2.4 Antivirus

Once a virus infection occurs, an antiviral therapy seems to be one of the option to control the viral infection. The antiviral drugs are a class of medication used specifically for treating viral infections where they molecularly target all the steps in the virus life cycle starting from viral entry towards the virus release from the host cell. This type of drug does not work by destroying their target pathogen, but they do cause inhibition in the viral development. Thus, the general idea behind the development of antiviral is by identifying and then targeting the viral protein or part of it that can be disabled. In order to produce a single class of antiviral drugs that have broad effectiveness, the targets should be common across many strains of a virus or even among different species of virus in the same family (Vardanyan & Hruby, 2016).

There are many difficulties encountered to develop a safe and effective antiviral drug. The obstacles are due to viral variation and the nature of virus that use the host's cells to replicate. Thus, an efficient antiviral drug is the one that can interfere the target without causes harmful towards the cell of host organism. Over the past decade, the development of norovirus vaccines has been the focus of much research and it still attracts great attention in the scientific field. However, progress on the identification of antiviral therapies for human noroviruses is hampered due to the high degree of genetic diversity of these viruses, the lack of long-lasting protective immunity and the epidemiology of outbreaks (Iturriza-Gómara & Lopman, 2014;

Karst, Wobus, Goodfellow, Green, & Virgin, 2014; Melhem, 2016; Vashist, Bailey,

Putics, & Goodfellow, 2009). Currently, the only available treatment for norovirus infections are by quarantining the infected individual to prevent further spread. The infected person is then subjected to the treatment of the main symptoms typically using orally antiemetic medication or intravenous rehydration if the case is severe (Bucardo et al., 2008; Vashist et al., 2009). To date, the identification of small molecules capable of inhibiting norovirus replication has been limited. However, the use of antisense technology and small molecule inhibitors holds much promise as an antiviral therapy to control the norovirus. This small molecule inhibitors of norovirus infection would resolve the condition where close person to person contact cannot be avoided and quarantine is impractical or difficult such as in cruise ships, military camps and hospitals (Vashist et al., 2009).

Anti-sense morpholino oligonucleotides is one of the anti-sense technology that is use for inhibiting virus replication in tissue culture. However, the uses are limited due to poor stability of unmodified antisense oligonucleotides. Thus, the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) has resolve the limitation since they are nuclease resistant as well as readily enter cells. In case of treatment for calicivirus by using non-conjugated peptide of phosphorodiamidate morpholino oligomers (PMO), it has been reported that there is a clinical potential of the use of PMO for this family of viruses (Eisen & Smith, 2008;

Vashist et al., 2009). Plus, substantial reduction in norovirus gene expression at least in cell culture study has been observed upon usage of PPMOs for targeting the norovirus genome, although none inhibition of norovirus replication has been demonstrated yet in vivo (Bok et al., 2008). Another small molecule inhibitors that has been studied is nucleotide analogues which is capable of inhibiting RNA virus

34 replication by using their ability to be incorporated into newly synthesised viral RNA by the viral RdRp. The most well characterised and widely used nucleotide analogue is ribavirin. The in vitro analysis of ribavirin using HuNv replicon containing cell line showed that it has anti-norovirus activity even though its clinical use has yet to be examined (Chang & George, 2007).

Other than PPMO/PMO and nucleotide analogues, there have been several other potential targets for anti-norovirus antivirals. Since the viral protease that encoded as part of a large polyprotein from ORF1 is an enzyme involved in virus replication, it represented as a key drug target for development of antiviral therapeutics. Cleavage of the viral polyprotein by protease lead to production of several non-structural proteins that play an essential role in viral replication.

Therefore, targeting protease activity may provide mechanism to inhibit norovirus replication. Several classes of compounds such as acyclic-sulfamide based compound, piperazine derivatives and pyranobenopyrone compounds are reported to have activity against protease has been observed from the numerous studies, but the knowledge on the function and specificity of norovirus are limited (Kaufman et al., 2014). In addition to polyprotein processing, the antiviral intervention also can be developed through targeting the unique mechanism used by norovirus for protein synthesis. The protein synthesis of norovirus takes place upon interacting of host cell protein that include components of the eIF4F cap-binding complex such as RNA helicase eIF4A. It is found that the small molecule known as hippuristanol cause an effect towards in vitro translation of MNV as it was able to inhibit the RNA helicase activity of eIF4A

(Daughenbaugh et al., 2003).

2.5 Phage Display

Over the past years, phage display technique has been employed for the identification of wide range of biological agents including peptides, antibodies, receptors and enzymes with high affinity and specificity to almost any targets. The identified sequence can be engineered to produce successful drugs with attribution in term of potency, specificity, cross-reactivity and stability (Nixon, Sexton, & Ladner, 2014).

This technology has a lot of contribution and influence in the fields of pharmacology, immunology, cell biology and drug discovery (Arap, 2005). The history of this powerful tool started in 1985 when George P. Smith discovered the phage display technology where it was proven that foreign DNA fragments can be inserted into a location in the genome of the phage capsid proteins. The encoded peptides are expressed or “displayed” on the surface of filamentous phage as a fusion product with one of the phage coat proteins (Bazan, Calkosinski, & Gamian, 2012; Molek, Strukelj,

& Bratkovic, 2011).

Phage display technology provides a direct physical linkage between phenotype (the displayed peptide) and genotype (the encoding DNA). Phages containing such binding proteins can then be extracted by affinity selection. The phage library is screened with one or several immobilized targets in a process called

“panning” or “biopanning” in which only binding phages are captured. The process is followed by an extensive washing to remove unspecific phages. Since phage particles can withstand harsh conditions such as low pH and low temperatures without losing bacterial infectivity, the bound phages are eluted using acid, high salt or specific target ligand to dissociate bound phage from a target (Pande, Szewczyk, & Grover, 2010).

The phage population is enriched by re-infection of bacterial cells before the next round of panning. This “biopanning” process can be repeated a few times until a

36 population of best binders is enriched. The genotype-phenotype coupling allows the amino acid sequence of the specific binding proteins to be rapidly determined by sequencing the DNA of the phage vector. Sequencing analysis will enable synthesis of the sequence as recombinant or synthetic peptide and eventually enhance the determination of its specificity and selective ligands to target receptors (Koivunen,

Arap, Rajotte, Lahdenranta, & Pasqualini, 1999; Wu, Liu, Lu, & Wu, 2016))

Phage display has been used widely in numerous applications such as in protein-protein interaction, vaccine development, epitopes and mimotopes mapping and enzyme specificity assay. Phage display is an excellent method to study protein- protein interaction (Hertveldt, Beliën, & Volckaert, 2009; Sidhu, Fairbrother, &

Deshayes, 2003). It is applicable to several protein interaction partners and used in applications like mapping intracellular interactions of the distinct protein domain. Src homology-3 (SH-3) domain is one of the examples of protein interaction partners

(Kärkkäinen et al., 2006). Other application of phage display is in vaccine development where designing and developing vaccines using this method has advantages over other methods. Phage display is more suitable for vaccine development due to the characters of bacteriophages that are non-virulent viruses and non-infectious to human; enable it to be considered safer than viral vaccines (Bazan,

Całkosiński, & Gamian, 2012). In addition to that, differ than normal vaccines that only induce humoral immune response, phage derived vaccines are able to induce both humoral and cellular immune response (Liljeqvist & Ståhl, 1999).

Phage display technology is a cheap and rapid method that can be applied in the epitope mapping. It involves mapping the epitope of the antigen that has specific interaction with the antibody. Epitope refers to the binding site of antibody on the antigen surface while mimotope describes peptides that mimic epitopes. Generally,

37 epitope mapping is related to the study of antibody and antigen interaction and their binding sites. The identification of epitopes is essential in diagnostic vaccine development, therapeutic antibodies and immunotherapy (Pande et al., 2010). On the other hand, mimotopes together with carrier proteins or presented as polymers have been developed for cancer and anti-allergic (Knittelfelder, Riemer, & Jensen-Jarolim,

2009). Analysis of enzyme activity and specificity are facilitated by the application of phage display tools. It has been used to develop modulators of both the active and allosteric sites of the enzyme. The enzyme-substrate interactions that may be responsible for enzyme specificity can be studied in detail following the application of this technology. Furthermore, there were studies conducted using phage display to develop peptide inhibitors that bind either anthrax toxin or its cell surface receptors

(Pande et al., 2010)

Thus, the advancement of phage display technique and better understanding of the principles underlying protein-protein interactions, have led to the emergence of various phage display projects based on a range of protein scaffolds, including protease inhibitors.

2.6 Secondary and Tertiary Structure Prediction of Protein

There are several protein prediction software that are available online such as HHpred

(Söding, Biegert, & Lupas, 2005), RaptorX (Wang, Li, Liu, & Xu, 2016) and Iterative

Threading ASSEmbly Refinement (I-TASSER). However, I-TASSER with the group name ‘Zhang-Server’ is the most successful method measured by the community-wide blind Critical Assessment of Structure Prediction (CASP) experiments and ranked as the top server for automated protein structure prediction in the 7th-11th CASP competitions (Kryshtafovych, Fidelis, & Moult, 2014; Moult, 2005) for predicting the art in protein structure and function (Kryshtafovych et al., 2014; Moult, 2005; Roy,

Kucukural, & Zhang, 2010; Yang et al., 2015; Zhang, 2008). In comparison with other online server, I-TASSER server is unique in term of their significant accuracy and reliability of full-length structure prediction for protein targets of varying difficulty as well as comprehensive structure-based function predictions (Roy et al., 2010).

I-TASSER methods involved three steps which are structural template identification, iterative structure assembly and structure-based function annotation.

Initially, I-TASSER identifies homologous structure templates from the PDB library based on the query sequence using LOMETS which is a meta-threading algorithm that consists of multiple individual threading programs (Wu & Zhang, 2007). The models are then constructed by reassembling the threading templates based on replica- exchange Monte Carlo simulations (Zhang et al., 2003). The significance and quality of templates were selected based on the Z-score of each treading program, which measures the difference between raw alignment score and the mean. I-TASSER will normalises the Z-score between each threading program and selects the best threading templates. For each template, I-TASSER generates a high number of structure called decoys. Normalised Z-score higher than 1 indicates confident alignment (Yang &

Zhang, 2015).

After that, SPICKER identifies the lowest-free energy states from the trajectory structure of Monte Carlo simulation and subsequently generates five final models which corresponds to five largest structure clusters to identify near-native protein folds. Among the five models, one of them represents the best model that was identified based on quantitative measurement, C-score that indicates the confidence of each models based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations (Zhang & Skolnick,

2004). Thus, the best model of the predicted structure that has been suggested through

I-TASSER server was used as the input in a docking program together with the sequence of the selected peptide clones.

2.7 Bioinformatics of Protein Ligand Interaction

In living organisms, all the metabolics, reactions and molecular pathways involve the interaction between proteins and ligands in order to ensure succesful function of certain reaction. This process that known as molecular recognition happen upon interaction among biological macromolecules or between this molecules and various small molecules through weak bonding (non-covalent) in order to form a specific complex (Du et al., 2016; Janin, 1995). The signal transmission based on ligand mediated is essential through molecular complementary. These chemical interactions basically comprise of biological recognition at the molecular level (Dunn, 2001). The function of protein evolves on the concept of development of specific sites that are designed to bind the molecule of ligand. The interaction between protein and ligand takes place through the conformational changes among two important characteristics that are specificity and affinity (Du et al., 2016). Specificity differentiate between high and less specific partners while affinity regulates the fact that weak interacting partners with high concentration cannot replace the effect of interaction between specific partner with low concentration that are interacting with high affinity (Demchenko,

2001). Upon the binding of ligand interactions, it results in the changes of the protein state; thus contributing towards the protein function (Dunn, 2001).

The information on the recognition between protein and ligand helps to aid towards the discovery, design and development of drugs. Basically, there are three important parts that need to be considered with regards to protein ligand interactions.

These include physicochemical mechanism (binding kinetics, thermodynamic concepts and relationship and binding driving force), models of protein binding ligand

40 and methods that are available to explore the association between protein and ligand such as experimental and theoretical or computational approaches (Du et al., 2016).

Since protein plays variety of functions in the cells through its direct physical interaction with other molecules (to form complex), it is considered as an important class of macromolecules. In order to learn about its function, a thorough understanding about the mechanism associated with the formation of this complex is important.

The interaction between protein and ligand is guided by physicochemical mechanism. The primary factors that govern the interaction of the complex is in term of their binding kinetics that describes the rate at which protein and ligand bind to each other. Through this binding kinetics, it is described that a high binding affinity is a result of fast binding rate together with slow rate of dissociation (Du et al., 2016;

Steinbrecher & Labahn, 2010). On the other hand, a thermodynamic capability involves in a measurement of the capacity of a thermodynamic system (protein-ligand- solvent system) to carry a maximum or reversible work at constant pressure and temperature is known as Gibbs free energy. This energy is a quantity that is important to characterise the driving force in complex interaction and energy changes that dictate the association between protein and ligand. Negative value of changes in Gibbs free energy (∆G) or binding free energy of the system when the system reach equilibrium state at constant pressure and temperature will lead to occurrence of protein-ligand binding in any spontaneous process (Du et al., 2016; Gilson & Zhou, 2007). The ∆G can also be parsed into its enthalpic and entropic contribution upon ligand binding where measurement of the total energy of a thermodynamic system is enthalpy and the distribution of how evenly heat energy is over the whole system is entropy (Perozzo,

Folkers, & Scapozza, 2004).

The mechanism of protein-ligand binding is modelled based on three different models that are “lock-and-key”, induced fit and conformational selection (Csermely,

Palotai, & Nussinov, 2010; Du et al., 2016). In lock and key model, there is a perfect match between both protein and ligand due to their rigid characteristics. The unexplainable experimental evidence that protein binds its ligands when their initial shapes do not match well lead to the assumption of induced fit model. This model assumes that there is flexibility in the binding site of protein while there is induction of conformational change by the interacting ligand at the binding site (Du et al., 2016).

Both models treated protein as a single and stable conformation under the given experimental conditions. In contrary, due to inherently dynamic of most protein as well as conformational selection based on this inherent, it is postulated that native proteins exist as vast ensemble of conformational state and not as a single and stable conformation. This conformational selection model is based on free energy landscape

(FEL) theory where population distribution and redistribution of protein conformational states/substrates is taken into consideration (Henzler-Wildman &

Kern, 2007; Miller & Dill, 1997). In term of energy, the lock and key binding model is based on entropy-dominated process, induced fit is dominated by enthalpy while a sequential manner of process involving entropy and enthalpy take place in conformational selection model (Bronowska, 2011; Du et al., 2016; Li, Xie, Liu, &

Liu, 2014). However, according to Kastritis and Bonvin (2013), all the three mechanisms may happen in a simultaneous or sequential manner upon covering a broad spectrum of binding events.

There are two methods used to investigate the protein ligand binding affinity that are experimental and theoretical or computational methods. Among the experimental methods that are commonly use are X-ray crystallography, small angle

X-ray scattering, nuclear magnetic resonance (NMR), single molecule fluorescence spectroscopy and cryo-electron microscopy (Du et al., 2016; Weiss, 2000). These methods can be used to study the changes in structure and/or dynamic between free and bound forms together with relevant binding events. Results are based on their atomic resolution or near atomic resolution structures of the unbound proteins and the protein-ligand complexes provided through the experimental methods. Another method that is carry out experimentally to study protein-ligand binding affinity are

Isothermal Titration Calorimetry (ITC) (Chaires, 2008; Ladbury & Chowdhry, 1996;

Perozzo et al., 2004), Surface Plasmon Resonance (SPR) (Torreri, Ceccarini, Macioce,

& Petrucci, 2005; Willander & Al-Hilli, 2009), and Florescence Polarization (FP)

(Rossi & Taylor, 2011; Weber, Pantoliano, & Thompson, 1992). However, time consuming, laborious and expensive factors has contributed towards experimental design for determining the binding affinity of protein and ligand. This event has broadened the techniques of investigating the association of protein and ligand using theoretical/computational methods (Du et al., 2016).

Two class of theoretical methods that have gain an attention are protein ligand docking and binding free energy calculation. Currently, molecular docking is widely used since it is fast and computational based tools that is economical in order to predict in silico in term of binding modes and affinities of molecular recognition events (Sousa et al., 2013). There are various softwares available such as AutoDock (Morris et al.,

2009), DOCK (Ewing, Makino, Skillman, & Kuntz, 2001; Mukherjee, Balius, &

Rizzo, 2010), CABS-dock (Ciemny, Kurcinski, Kozak, Kolinski, & Kmiecik, 2017) and FlexX (Rarey, Kramer, Lengauer, & Klebe, 1996) which use different algorithm to deal with docking problems. Protein-ligand docking is a method that predicts the orientation (poses) of a stable complex (bounding of one molecule to another).

Through docking, the best fit orientation of the ligand which goes and binds to a protein will be described by estimating the binding affinities of the generated poses, ranking them and consequently determining the most favourable binding model(s) of ligand to target (Du et al., 2016).

Docking function is based on two separate platforms that are ; search algorithm and scoring function. Common search algorithms are Monte Carlo, Genetic algorithms, Fragment-based methods and Molecular Dynamics (vlab.amrita.edu.,

2013). This search algorithms provide an optimum number of configurations that are evaluated using scoring functions in order to compare the difference between binding modes which are determined through experiment. The scoring function is a mathematical method that is used to access the binding affinity between two molecules that are docked (Du et al., 2016; Ewing et al., 2001). Another class of theoretical methods is free energy calculations that are divided into three main types. They are alchemical calculation, path sampling and the endpoint methods. Low efficiency and potential high accuracy of free energy calculation enable them to be applied in more detail to study the protein ligand interactions. The challenge that is commonly encounter through free energy calculation is in term of speed of the methods and improvement of the accuracy and reliability of the calculated results (Du et al., 2016).

CHAPTER 3

METHODOLOGY

3.1 List of Reagents/Chemical

All materials used in this study are listed in tables below : Table 3.1 Bacterial strains and plasmids No Strain/plasmid Description Brand, Origin 1. E. coli DH5α This strain was developed for New England Biolabs, laboratory cloning; multiple USA mutations enable high-efficiency transformations

2. E. coli BL21 (DE3) B strain of E. coli that contained Merck, USA λDE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter

3. E. coli ER2738 Strain of E. coli that contain F pilus New England Biolabs, which function in infection of USA bacteriophage; tetracycline resistance

4. Plasmid MNV-1 Plasmid harboring full length MNV- Obtain as a gift from cDNA clone 1 genome based on CW1 strain: Prof Ian Goodfellow, (pT7MNV3’Rz) ampicillin resistance Cambridge University, UK 5. Expression plasmid Expression plasmid that carry Obtain as a gift from (pET26Ub) histidine at the C-terminal; contain Prof Ian Goodfellow, T7 polymerase promoter and the Cambridge University, ubiquitin gene UK

6. Cloning plasmid Cloning vector that has high copy New England Biolabs, (pUC19) number USA

7. pCG1 Plasmid that encodes for a ubiquitin- Obtain as a gift from Dr speciﬁc, carboxy-terminal protease Craig E. Cameron, (Ubp1) Pennsylvania State University, USA

Table 3.2 Antibodies No Antibodies Description Brand, Origin (dilution factor) 1 Rabbit anti-His Act as primary antibody to detect 6X Cell Signaling polyclonal antibody Histidine-tagged recombinant proteins Technology, USA (1:1000 ) in western blotting; raised in rabbit

2 Goat anti ProPol Antibody that target Protease and Received from anti serum Polymerase of MNV1 in western Dr.Stephanie Karst (1:1000) blotting; raised in goat Lab, University of Florida

3 Goat Anti rabbit Antibody specific to the host species Life Technologies, IgG (H+L) HRP (rabbit) of the primary antibody; USA conjugate complexed with HRP. ( 1:10000)

4 Rabbit Anti goat Antibody specific to the host species Invitrogen, USA HRP conjugate (goat) of the primary antibody; (1:20000) complexed with HRP.

5 Mouse anti-M13 Detection of M13 phage in ELISA GE Healthcare Life monoclonal Science, USA antibody, HRP conjugate (1:5000)

6 Biotin HRP Detection of streptavidin in ELISA Invitrogen, USA (1:5000)

Table 3.3 Commercial kits No Kits Brand, origin 1. GoTaq® Flexi DNA Polymerase Promega, USA 2. Q5 HotStart High Fidelity DNA polymerase New England Biolabs, USA 3. PureYield™ Plasmid Midiprep System Promega, USA 4. HisTALON™ Gravity Column Purification Kit Clontech, USA 5. IllustraTM GFX PCR DNA and Gel Band purification kit GE Healthcare, USA 6. PierceTM BCA protein assay Thermo scientific, USA 7. WesternBrightTM ECL Advansta, USA 8. Ph.D.TM Phage Display Peptide Library New England Biolabs, USA

Table 3.4 Bacterial culture media and antibiotics No Reagents Brand, Origin 1. Luria Bertani Broth (LB broth), Miller R & M Chemical, UK 2. Peptone from casein (Tryptone) Merck, USA 3. Yeast extract Merck, USA 4. Sodium chloride Fisher Scientific, USA 5. D (+) Glucose, anhydrous Merck, USA 6. Bacteriological agar Oxoid, USA 7. SOC outgrown medium New England Biolabs, USA 8. Kanamycin sulfate GIBCO, USA 9. Chloramphenicol Amresco, USA 10. Carbenicillin disodium salt Invitrogen, USA 11. Tetracycline hydrochloride Fisher scientific, USA

Table 3.5 Reagents No Reagents Brand, Origin 1 Bovine Serum Albumin R & M Chemical, UK 2 Calf alkaline phosphatase (CIP) New England Biolabs, USA 3 CutSmart buffer New England Biolabs, USA 4 DNA marker: 100bp DNA ladder New England Biolabs, USA 5 DNA marker: 1kb DNA ladder New England Biolabs, USA 6 DNTPs mix Promega, USA 7 Dithiothreitol (DTT) Thermo Scientific, USA 8 Phenylmethylsulphonyl fluoride (PMSF) Thermo Scientific, USA 9 MagicMarkTM XP Western Standard Invitrogen, USA 10 SeeBlue ® Plus2 Prestained Standard Invitrogen, USA 11 Restriction enzyme: BamH1 New England Biolabs, USA 12 Restriction enzyme: Sac II New England Biolabs, USA 13 RNase A Invitrogen, USA 14 T4 DNA ligase New England Biolabs, USA 15 TMB substrate Thermo scientific, USA 16 Ultrapure nuclease free water Ambion, USA

Table 3.6 Chemicals No Chemical Brand, Origin 1 Acetic acid glacial, Grade AR R & M Chemical, UK 2 Acrylamide Amresco, USA 3 Agarose Promega, USA 4 Ammonium persulphate (APS) Calbiochem, USA 5 Bis-acrylamide Amresco, USA 6 Bromophenol blue QRec, New Zealand 7 Calcium chloride (CaCl2) Fisher, USA 8 Coomassie Brilliant blue G250 Merck, USA 9 Dimethyl Formamide, DMF Amresco, USA 10 di-sodium hydrogen phosphate Merck, USA 11 EDTA Merck, USA 12 Ethanol QRec, New Zealand 13 Ethidium bromide (EtBr) Merck, USA 14 Glycerol Emsure (Merck), Germany 15 Glycine Fisher, USA 16 Hydrochloric acid R & M Chemical, UK 17 Imidazole R & M Chemical, UK 18 Isopropanol, Grade AR QRec, New Zealand 19 Isopropyl-β-D-thiogalactoside, IPTG 1st BASE, Singapore 20 Magnesium chloride (MgCl2) QRec, New Zealand 21 Methanol QRec, New Zealand 22 Orange G Sigma Aldrich, USA 23 Polyethylene glycol-8000 Promega Incoporation, USA 24 Ponceau S Sigma, USA 25 Potassium acetate Fisher, USA 26 Potassium chloride Merck, USA 27 Potassium dihydrogen phosphate Merck, USA 28 Sodium Azide, NaN3 Systerm, Malaysia 29 Sodium Bicarbonate, NaHCO3 Amresco, USA 30 Sodium chloride (NaCl) Fisher, USA 31 Sodium dihydrogen phosphate R & M Chemical, UK 32 Sodium dodecyl sulphate, SDS Systerm, Malaysia 33 Sodium hydroxide R & M Chemical, UK 34 Sodium Iodide, NaI Sigma Aldrich, UK 35 Sulphuric acid 95-97 % Qrec, New Zealand 36 TEMED Thermo scientific, USA 37 Tris base Fisher, USA 38 Tween 20 Detergent Calbiochem, USA 39 XGal 1st BASE, Singapore

Table 3.7 Consumables No Consumable Brand, Origin 1 1.5 ml microcentrifuge tube Greiner Bio-one, Austria 2 15 ml and 50 ml Centrifuge tube LabServ, China 3 96 well ELISA plate, immunoplate SPL life science, Korea 4 96 well plate, Flat-bottom Thermo Fisher Scientific, USA 5 PCR tube Labcon, USA 6 Petri Dish Thermo Fisher Scientific, USA 7 Pipette tips Greiner Bio-one, Austria 8 PlateMax axyseal sealing film Axygen, USA 9 PVDF western blotting membrane Merck Millipore, Germany 10 Dialysis tubing cellulose membrane Sigma Aldrich, Germany 11 Serological pipette Greiner Bio-one, Austria 12 Spreader SPL life science, Korea 13 Sterile filter Jet biofil, China 14 Syringe Terumo Corporation, Philipine

Table 3.8 List of Equipments No Equipment Brand, Origin 1 Autoclave Hirayama, Japan 2 Belly dancer shaker Stovall Life Science, USA 3 Centrifuge 5424 Eppendorf, USA 4 Centrifuge 5810R Eppendorf, USA 5 Centrifuge Pro-Research Centurion Scientific Ltd., UK 6 Electrophoresis system BioRad, Califonia 7 Gel dryer Model 583 BioRad, Califonia 8 Heating block Thermo, USA 9 Incubator Incucell, Germany 10 Incubator shaker Thermo Scientific, USA 11 Incubator shaker (Refrigerated) New Brunswick Scientific, USA 12 Laminar Air Flow Esco, USA 13 Mastercycler nexus GX2 Eppendorf, USA 14 Microplate reader Thermo Fisher Scientific, USA 15 Microwave Samsung, Korea 16 Mini Spin Eppendorf, USA 17 Mini-PROTEAN® Tetra System BioRad, Califonia 18 Molecular Imager ® VersaDoc TM MP Imaging BioRad, Califonia 19 Nanodrop 2000c Thermo Scientific, USA 20 pH meter Mettler Toledo, USA 21 PowerEase 90W Life Technologies, USA 22 Spectrophotometer Thermo Electron Corp, USA 23 Trans-Blot D Semi-Dry Transfer Cell BioRad, Califonia 24 Transluminator Sigma Aldrich, Germany 25 Vortex mixer Ika ® MS 3 digital, USA 26 Water Bath Memmert, UK 27 Weighing Balance Mettler Toledo, USA

Table 3.9 Recipe for preparation of reagents No Chemical reagents Formulation 1 10X Ligation mix 2.5 mM ATP, 20 mM DTT, 2 mM NaEDTA (pH 8), 10 mM spermidine, 1 mg/mL BSA 2 Ligation salt 150 mM NaCl, 150 mM Tris (pH 7.5), 38 mM MgCl2 3 Resuspension buffer 50 mM Tris/HCl (pH 7.5), 10 mM EDTA, 100 µg/ml RNase A 4 Lysis buffer 0.2 M NaOH, 1 % SDS 5 Neutralisation buffer 1.32 M potassium acetate (pH 4.8) 6 Destaining solution 40 % Methanol, 10 % Acetic acid, H20 6 Blocking buffer for protein-based 0.1 M NaHCO3 (pH 8.6), 5 mg/ml BSA, ELISA and phage display selection 0.02 % NaN3. Filter sterile 7 Tris Buffer Saline 50 mM Tris-HCI (pH 7.5), 150 mM NaCI. Autoclave 8 Tris Buffer Saline + Tween 20 50 mM Tris-HCI (pH 7.5), 150 mM NaCI + Tween 20. Autoclave 9 PEG/NaCI 20 % (w/v) polyethylene glycol-8000, 2.5 M NaCI. Autoclave 10 Iodide Buffer 10 mM Tris-HCI (pH 8.0), 1 mM EDTA, 4 M sodium iodide (NaI) 11 Solution for Streptavidin 10 mM sodium phosphate (pH 7.2), 100 mM NaCI, 0.02 % NaN3 12 TE buffer 1 M Tris-HCI (pH 7.5 or 8.0) and 0.5 M EDTA (pH 8.0)

Table 3.10 List of Primers Primer name Sequence Polarity Tm Description Brand/ Origin Primers for cloning full length MNV-1 protease sequence into E.coli expression plasmid pET26Ub AY7 ATT ATC CGC GGT GGA GCC CCA GTC F 67.5 ˚C MNV-1 N-term NS6 into pET26Ub, SacII TCC ATC site. Addition of extra sequences representing addition of amino acid glycine prior to N-terminus MNV NS6 protease AY8 TTC TAG GAT CCC TGG AAC TCC AGA R 64.5 ˚C MNV-1 C-term NS6 into pET26Ub, BamHI Integrated GCC TCA AG site DNA 51 Technologies

Primers for generating MNV-1 NS6 mutant (C139A NS6) AY17 CTC GGG ACC ATC CCG GGC GAC GCA F 71.4 ˚C Mutation at position 139 (IDT), USA GGC TGT CCC TAT GTT TAT AAG TGT(cysteine) to GCA(alanine) AY18 CTT ATA AAC ATA GGG ACA GCC TGC R 71.4 ˚C Mutation at position 139 GTC GCC CGG GAT GGT CCC GAG TGT(cysteine) to GCA(alanine) Sequencing primer for phage DNA AY25M13F TTA TTC GCA ATT CCT TTA GTG GTA F 57.2 ˚C PCR amplification & sequencing of phage CCT TTC DNA AY26M13R GCC CTC ATA GTT AGC GTA ACG ATC R 58.3 ˚C PCR amplification & sequencing of phage TAA AG DNA

3.2 Plasmid Construction

3.2.1 Bioinformatics Tools

All the primers used as shown in Table 3.10 were designed using Vector NTI software based on the sequences found in GenBank https://www.ncbi.nlm.nih.gov/nuccore/DQ285629.1. Specificity of the designed primer used in this study was cross-checked through Primer-BLAST online server https://www.ncbi.nlm.nih.gov/tools/primer-blast/.

3.2.2 Polymerase Chain Reaction (PCR)

PCR amplification were performed using Q5 Hot Start Hi-fidelity DNA Polymerase according to manufacturer’s protocol. All the amplifications were carried out using

Mastercycler thermal cycler (Eppendorf, USA). For wildtype NS6 protease (WT NS6), the NS6 gene sequence was PCR amplified from the full length MNV-1 cDNA clone

(pT7MNV3’Rz) using primers pair AY7 and AY8 as listed in Table 3.10. The restriction enzyme site SacII and BamHI have been incorporated in the primers sequence of AY7 and AY8 respectively. Extra sequences representing addition of amino acid glycine prior to N-terminus MNV NS6 protease was also included in AY7.

Meanwhile for AY8, a stop codon was not included after the last amino acid residue of C-terminal of NS6 to produce a histidine-tagged recombinant NS6 protease. The

PCR master mix was prepared on ice and the PCR components were shown in Table

3.11. The parameters for PCR cycles were adjusted according to the protocol as shown in Table 3.12.

Table 3.11 PCR components Component 50ul reaction Final concentration 5X Q5 Reaction buffer 10 µl 1X 10mM dNTPs 1 µl 200 µM 10µM forward primer 2.5 µl 0.5 µM 10µM reverse primer 2.5 µl 0.5 µM Template DNA 1 µl ˂ 1000 ng Q5 Hi-fidelity DNA polymerase 0.5 µl 0.02 U/ µl Nuclease free water To 50 µl

Table 3.12 PCR condition for NS6 protease gene amplification Step Temperature Time Cycle Initial denaturation 98 ˚C 30 seconds 1 Denaturation 98 ˚C 10 seconds Annealing 65 ˚C 30 seconds 30 Extension 72 ˚C 30 seconds Final extension 72 ˚C 2 mins 1 Hold 4 ˚C ∞ 1

3.2.3 Site-Directed Mutagenesis

A point mutation has been introduced at amino acid position 139 where the active site of protease is located to deactivate the NS6 protease by overlapping PCR. Based on the WT sequence of NS6 protease, the introduced mutation will cause coding change of sequence from TGT (cysteine) to GCA (alanine). Two stages of PCR amplifications were carried out using pT7MNV3’Rz as template. For the first stage of PCR, primer

AY7 and AY18 were used while primer AY17 and AY8 were used for the second

PCR. The reaction mixture and condition were adjusted according to manufacturer protocol (Table 3.11 and Table 3.12) where the annealing temperature was set to 70

˚C for the first stage and 66 ˚C for the second stage. The PCR amplicons for both stage of amplifications were subjected to agarose gel electrophoresis and the amplicons with expected size 437 bp and 155 bp were purified using IllustraTM GFX PCR DNA and

Gel Band purification kit (GE Healthcare, USA) according to the manufacturer’s protocol (Section 3.2.7). After that, overlapping PCR was carried out by using 30 µg of both purified PCR amplicons. The forward primer (AY7) and the reverse primer

(AY8) were used in the overlapping PCR following the condition in Table 3.12.

3.2.4 Agarose Gel Electrophoresis

PCR amplicons from all of the reactions were visualised and analysed by agarose gel electrophoresis with percentage varying from 0.8 % -2.0 % depending on the estimated product size. Gel was prepared by dissolving agarose in 1X TAE buffer. The gel was allowed to cool down to 60-70 ˚C and approximately 0.5 µg/ml ethidium bromide was added to the mixture. Samples were run alongside DNA ladder markers (NEB, USA).

Gel was then electrophoresed for 30 minutes (mins) at 100 volts followed by visualization under UV (BioRad, Califonia).

3.2.5 Cloning

The pET vector system has been adopted to clone the gene of interest in which pET26Ub was used as the expression plasmid where it carries 6X histidine at the C- terminal of cloning region. The plasmid contains T7 polymerase promoter and the ubiquitin gene from Saccromyces cereviciae upstream of the multiple cloning site

(Gohara et al., 1999). The plasmid that was available in the lab has been cloned with

NS7 sequences. Restriction enzyme reaction was performed to excise out the readily cloned NS7 sequence and obtain the 5.5 Kbp expression vector only (Section 3.2.6).

The plasmid integrity and concentration were verified using gel agarose gel electrophoresis and nanophotometer respectively.

3.2.6 Restriction Enzyme Reaction

In order to obtain the pET expression vector only, double restriction enzyme reaction was set up. The reaction was set up as follows; 1 µg of plasmid was added into 1.5 microcentrifuge tube, 1.5 µl 10X CutSmart buffer and 0.5 µl of each SacII and BamHI restriction enzyme. The reaction mixture was made up to final 15 µl total volume with nuclease free water. The mixture was then incubated at 37 ˚C for 2 hours (h) followed by addition of 1 µl calf alkaline phosphatase (CIP) to the mixture and further incubated 54

at 37 ˚C for 30 minutes. The purified PCR amplicons containing C139A NS6 and

WT NS6 were also subjected to restriction enzyme reaction using the same restriction enzyme as the plasmid vector above but without the treatment with CIP.

Approximately around 12.5 µl of PCR amplicon was used in the reaction. All the digested products were subjected to electrophoresis and purification using GFX PCR

DNA and Gel Band Purification Kit (GE Healthcare, USA) as detailed in section 3.2.7.

3.2.7 Gel and PCR Purification

All the plasmids DNA and PCR amplicons used for cloning were purified using GFX

PCR DNA and Gel Band Purification Kit (GE Healthcare). Plasmid DNA from the restriction enzyme reaction was purified according to the protocol for purification of

DNA from TAE agarose gel. Firstly, DNAse-free 1.5 ml microcentrifuge tube was weighed and it weight was recorded. The DNA band of the amplicon of interest on the agarose gel was cut using scalpel while it was exposed minimally under wavelength

(305 nm) ultraviolet light. The extracted agarose gel with the DNA band was placed into the 1.5 ml microcentrifuge tube and their weight was measured. The weight of agarose slice containing the DNA band was determined. Ten miligrams (mg) of agarose gel slice was mixed with 10 µl of Capture Buffer type 3. The mixture was incubated at 60 ˚C with intermittent agitation for every 3 mins until the agarose gel slice completely dissolved. After that, the mixture was added to the assembled GFX

MicrospinTM Column and collection tube followed by centrifugation at 16000 g for 30 seconds (secs). The flow through collected in the collection tube was discarded and the column was placed inside the same collection tube. Next, 500 µl of Wash Buffer type 1 was added to the column and subjected to centrifugation at 16000 g for 30 secs.

The collection tube was discarded together with the flow through while the column was placed into new DNase-free 1.5 ml microcentrifuge tube. In elution step, 25 µl of

Elution Buffer type 4 or 6 was added to the column and incubated at room temperature for 60 secs. The mixture was then centrifuged at 16000 g for 1 min and the flow through was retained. The purified DNA was stored in -20 ˚C until further used. The purification for PCR amplicon was carried out following the same protocol as the gel purification except that the first step was replaced by mixing 100 µl of PCR product to 500 µl of Capture Buffer type 3 and the mixture was added to the GFX column for further purification as mentioned above.

3.2.8 DNA Quantification

The concentration of DNA was determined by spectrophotometer at the wavelength of 260 nm and 280 nm using nanodrop spectrophotometer (Thermo Scientific, USA).

The ratio of 260/280 indicates sample purity whereby the reading with value of ˃1.8 indicates pure DNA sample (Olson & Morrow, 2012).

3.2.9 Ligation Reaction

The purified plasmid (vector) and the purified PCR amplicon (insert) were subjected for ligation reaction based on the concentration ratio of 1:3 (plasmid:insert). The ligation components for both WT NS6 and C139A NS6 reactions were as follows; concentration 1 vector : 3 insert, 1.5 µl 10X ligation mix, 3 µl 5X ligation salt, 1.5 µl

10X PEG, 1 µl T4 Ligase and nuclease free water was added up to a final volume of

15 µl. The reactions were then mixed and incubated at room temperature for 15 mins.

3.2.10 Transformation

Transformation was carried out according to manufacturer’s protocol with slight modification. Five µl of plasmid DNA from the ligation reaction was added to 25 µl of DH5α competent E. coli cell. The mixture was flicked gently 4-5 times to mix the cell and DNA. After that, the mixture was incubated on ice for 30 mins. The sample

was subjected to heat shock at 42 ˚C for 30 secs followed by incubation on ice for 5 mins. Next, 300 µl of room temperature SOC media was added to the mixture and the sample was placed at 37 ˚C for 60 mins with vigorous shaking. One hundred µl of the sample was then plated onto selection plate (LB) containing 30 µg/ml kanamycin. The plate was incubated for overnight at 37 ˚C. Positive control was included to examine the transformation efficiency using pUC19 plasmid plated on carbenicillin selection plate.

3.2.11 Screening Positive Colonies

Positive colonies were screened by plasmid extraction/mini preparation of the selected colonies from positive plates. Ten single colonies were selected and each of them was cultured in 3 ml LB broth supplemented with 30 µg/ml kanamycin for 3 hours. One ml from each culture was centrifuged at 1500 g for 5 mins while the rest of the culture were kept at 4 ˚C for further use. Following centrifugation, the supernatant was discarded and 150 µl of resuspension buffer was added to the pellet. The mixture was mixed well and briefly vortexed. After that, 150 µl of lysis buffer was added to the sample and mixed in hand rapidly. Prior to centrifugation, 150 µl of neutralization buffer was added to the sample. The sample was mixed well and then centrifuged at

15000 g for 2 mins. Next, 400 µl of the supernatant was transferred into new tube and each tube was labelled properly. A 280 µl of room temperature isopropanol was then added to the sample and mixed by inverting. The mixture was subjected to centrifugation at 15000 g for 5 mins. The supernatant was removed carefully without disrupting the pellet and 300 µl of ice cold 70 % ethanol was added to the pellet. The centrifugation was repeated, and the resulting pellet was left to air dry before subjected to resuspension in 30 µl TE buffer. The extracted plasmid was subjected to confirmation by restriction enzyme reaction as detailed in section 3.2.6 above.

3.2.12 Medium Scale Plasmid Preparation

To prepare a medium scale extraction of plasmid (midiprep), 1 ml of culture from previously stored culture (4 ˚C) was re-culture overnight in 250 ml LB broth supplemented with 30 µg/ml kanamycin. Midiprep was performed using commercial kit PureYield™ Plasmid Midiprep System (Promega, USA) and following the manufacturer protocol with slight modification. The large-scale culture was centrifuged at 5000 g for 10 mins and the supernatant was discarded. The pellet was resuspended in 6 ml resuspension buffer followed by addition of in 6 ml lysis buffer with intermittent mixing. The mixture was incubated at room temperature for 3 mins.

Next, 10 ml of neutralisation buffer was added to the sample and the tube was inverted

5 - 10 times. The sample was then centrifuged at 15000 g for 15 mins. The extracted plasmid was subjected to purification process. Firstly, blue clearing column was assembled on top of white binding column and both columns were placed on top of the vacuum manifold. The supernatant containing the plasmid DNA was poured into the blue clearing column and vacuum was applied until all the solution passed through the white binding column. After all the solution passed through, the clearing column was removed followed by addition of 5 ml endotoxin removal wash into the binding column. The vacuum was applied again until all the solution was eluted from the binding column. Twenty ml of column wash solution was then added and vacuum was applied once again until all the solution has passed through binding column. In the elution step, 1.5 microcentrifuge tube was placed on the base of vacuum eluator device and the device was connected to vacuum manifold while the binding column was assembled on top of the vacuum eluator device. Before elution, 1000 µl of nuclease free water was pre-heated at 60 ˚C prior to elution. Following pre-heating, 400 – 600

µl of nuclease free water was added to the binding column and vacuum was applied.

The pass through was collected as purified plasmid and stored in -20 ˚C for further analysis.

3.2.13 Analysis of Plasmid Preparation

The purified plasmid was verified using PCR and restriction enzyme reaction before proceeding with sequencing analysis. The presence of insert in the purified plasmid

DNA was determined using PCR by employing the GoTaq Flexi DNA polymerase.

The components and condition of the reaction was based on manufacturer’s instruction. The reaction mixture as shown in Table 3.13 was heated to 95 ˚C to denature the double stranded DNA. The reaction mixture was then cooled to 65 ˚C to allow annealing of primer to the target sequence, and the temperature was then increased again to 72 ˚C to allow extension of the DNA sequence from the primers.

This cycle was repeated for 30 times. After the PCR cycles completed, the reaction was subjected to final extension step for 5 mins at 72 ˚C before the PCR reaction was hold at 4 ˚C. Restriction enzyme reaction was carried out following protocol as detailed in Section 3.2.6 above. Both products of restriction enzyme and PCR reactions were analysed on agarose gel electrophoresis. The positive clone was then sent for sequencing analysis. The resulting cloned plasmids were named as pET26Ub:MNV

WT NS6 C-His and pET26Ub:MNV C139A NS6 C-His, for WT MNV-1 NS6 protease and active site mutant MNV-1 NS6 protease, respectively.

Table 3.13 Components for GoTaq DNA polymerase Reagents 1X Reaction mix, µl Final concentration 5X Go taq buffer 10 1X MgCI2, 25mM 5 2.5 mM DNTp mix, 10mM 1 0.2 mM each dNTP Forward primer, 10µM 2 0.4 µM Reverse primer, 10µM 2 0.4 µM Go taq DNA polymerase 0.25 1.25 U Template DNA 1 ˂ 0.5 µg/50 µl Nuclease free water 28.75 Total 50

3.3 Protein Expression and Purification

3.3.1 Preparation of BL21 (DE3) Competent Cell

The competent cell was prepared based on the adapted protocol with slight modification (Sambrook, Fritsch, & Maniatis, 1989). E. coli strain BL21(DE3) that carries pCG1 plasmid was grown in 3 mL LB broth supplemented with 25 µg/ml chloramphenicol for overnight. On the next day, the overnight culture was diluted into ratio 1:100 using LB broth supplemented with the same concentration of antibiotic.

The culture was incubated at 37 ˚C with 250 rpm and its growth was monitored until the density reached 0.5 OD600. After the density reached 0.5, it was quickly immersed and swirled in ice. The culture was centrifuged at 5000 g for 5 mins at 4 ˚C. The supernatant was discarded, and the cell pellet was resuspended in 15 mL ice cold 0.1M

MgCl2. After it was completely resuspended, the mixture was centrifuged at 5000 g and 4 ˚C for 5 mins. The supernatant was discarded, and the cell pellet was resuspended in 15 mL ice cold 0.1 M CaCl2. The mixture was then incubated on ice for 45 mins followed by centrifugation at 5000 g and 4 ˚C. The supernatant was poured off and the cell pellet was resuspended in 5 ml ice cold CaCl2 and 15 % glycerol. Prior to storage at -80 ˚C, the mixture was mixed well and aliquoted into sterile 1.5 ml tubes.

3.3.2 Transformation into BL21 (DE3)

Both the pET26Ub:MNV WT NS6 C-His and the pET26Ub:MNV C139A NS6 C-

His plasmids that have been prepared as in Section 3.2.13 above was further transformed into prepared competent cell in Section 3.3.1. The bacterial strain used for recombinant protein expression was E. Coli BL21(DE3) containing pCG1 plasmid expressing ubiquitin specific protease (Gohara et al., 1999). The expression plasmid used in this study (pET26Ub) was designed to fuse the ubiquitin molecule to the N- terminus of the desired protein in order to increase the expression and the solubility. 60

The presence of pCG1 plasmid will result in the removal of ubiquitin domain thus producing an authentic recombinant protein even without the presence of methionine as start codon. The transformation reaction was carried out according to the protocol in Section 3.2.10 with slight modification. The amount of competent cell E. coli BL21

DE3 (+pCG1) used was 50 µl and the sample was plated on LB plate supplemented with 30 µg/ml kanamycin and 25 µg/ml chloramphenicol.

3.3.3 Growth Curve of Transformed BL21 DE3 (+pCG1)

The above mentioned growth curve was generated by spectrophotometric analysis of cultures at OD600 using spectrophotometer. Initially, single colony of the transformant was inoculated into 10 ml of LB supplemented with 30 µg/ml kanamycin and 25 µg/ml chloramphenicol and cultured for overnight at 37 ˚C with vigorous shaking. On the next day, one ml of overnight starter culture was diluted into 100 ml LB with antibiotics and adjusted until the OD600 reach 0.1. The rest of the overnight culture was kept as glycerol stock in – 80 ˚C freezer. The diluted culture was prepared in 250 ml flask to ensure good aeration and incubated at 37 ˚C with shaking at 250 rpm. Culture’s density was measured for every 30 mins interval for 8 hours and growth curve of OD600 against time was constructed for both cultures of transformant with pET26Ub:MNV

WT NS6 C-His and pET26Ub:MNV C139A NS6 C-His.

3.3.4 IPTG Induction

Ten µl of glycerol stock was inoculated into 50 ml LB broth in the presence of 30

µg/ml kanamycin and 25 µg/ml chloramphenicol. The starter culture was incubated overnight at 37 ˚C with shaking at 250 rpm. Subsequently, twenty ml the starter culture was diluted in 1 litre LB supplemented with specific antibiotic and grown until the

OD600 reach the reading of 0.5 – 0.6 which is the desired log phase. From thereon, the

IPTG with final concentration of 0.1 mM was added to the culture for 4 hours induction 61

at 25 ˚C and 250 rpm shaking. The expression of the target protein was analysed using

Sodium Dedocyl Sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) stained with Coomassie blue.

3.3.5 Sodium Dedocyl Sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)

Expression of protein of interest (induced and non-induced samples) was analysed on

SDS PAGE using 17.5 % resolving gel (Table 3.14) and 5 % stacking gel (Table 3.15).

Firstly, 15 µl of culture was mixed with 5 µl of 4X loading buffer and the mixture was heated at 95 ˚C for 5 mins. The sample was separated on SDS-PAGE gel electrophoresis at 150 V for 90 mins using Mini-PROTEAN ® Tetra Cell (BioRad) with 1X SDS-PAGE running buffer. A pre-stained protein marker (Invitrogen, USA) was also loaded on gel for protein size determination. The gel was then stained with

Commassie blue for 5 mins and destained with destaining solution for 1 h followed by overnight destaining with distilled water for better visualisation.

Table 3.14 Resolving gel 17.5 % resolving gel 10ml 30 % acrylamide 5.83 ml 2% Bis-acrylamide 0.37 ml 1M Tris pH 8.8 3.75 ml 10% SDS 0.10 ml Distilled water 0.00 ml TEMED 8.35 µl 10 % Ammonium Persulphate 33.35 µl

Table 3.15 Stacking gel 5 % stacking gel 5ml 30 % acrylamide 0.85 ml 2 % Bis-acrylamide 0.35 ml 1M Tris pH 6.8 0.63 ml 10% SDS 0.05 ml Distilled water 3.15 ml TEMED 12.50 µl 10 % Ammonium Persulphate 25.00 µl

3.3.6 Cell Lysis

After the presence of expression of protein of interest was confirmed, a large-scale culture was prepared from glycerol stock culture. Induction was carried out following protocol as detailed in Section 3.3.4. The induced sample was centrifuged at 8000 g for 20 mins and the cell pellet was subjected to lysis in lysis buffer (20 mM Tris pH

7.9, 500 NaCl, 5 mM Imidazole, 1 mM EDTA, 5 % glycerol) containing protease inhibitor by double freeze-thaw cycle at -80 ˚C. The sample was then centrifuged at

8000 g for 30 mins. The supernatant was collected as soluble fraction and subjected to protein purification.

3.3.7 Protein Purification

Protein purification was performed using HisTALON Gravity Column Purification Kit according to manufacturer’s protocol with slight modification.

3.3.7(a) Equilibration

Initially, 1 ml cobalt resin was transferred into 15 ml sterile tube that can accommodate

10 - 20 times the resin volume. The tube was then centrifuged at 700 g for 2 min to pellet down the resin. After that, the supernatant was discarded and followed by addition of 10 bed volumes of equilibration buffer. The mixture was mixed briefly to pre-equilibrate the resin. The mixture was centrifuged again at 700 g for 2 min and the supernatant was discarded. The pre-equilibration step was repeated with 10 bed volumes of equilibration buffer and subjected to centrifugation at the same condition stated above to remove the supernatant.

3.3.7(b) Sample Application

The crude lysate was added to the equilibrated resin and the mixture was gently agitated at 4 ˚C for 20 min on a platform shaker to allow the histidine-tagged protein

to bind the resin. The mixture was then centrifuged at 700 g for 5 mins and supernatant was removed carefully as much as possible without disturbing the resin pellet.

3.3.7(c) Washing

The resin was subjected to washing using equilibration buffer and washing buffer.

Firstly, 10 – 20 bed volumes of equilibration buffer were added to the resin. The suspension was gently agitated at 4 ˚C for 10 mins on a platform shaker for thorough washing. Next, the mixture was centrifuged at 700 g for 5 mins and the supernatant was discarded. The washing step was repeated with 10 – 20 bed volumes of equilibration buffer. Following removal of supernatant, 1 bed volume of equilibration buffer was added to the resin and the mixture was resuspended by vortexing. After that, the mixture was transferred to a 2 ml gravity-flow column with an end-cap in place. The resin was allowed to settle at the bottom. The end-cap was then removed and buffer was allowed to flow go through colum. The column was then subjected to washing with 5 bed volumes of wash buffer for one time. After all the washing buffer passed through the column, the resin was used for elution.

3.3.7(d) Elution

The histidine-tagged protein was eluted by adding 5 bed volumes of elution buffer to the washed resin in the column. The eluate was collected in 500 µl fractions and subjected for quantification prior to further analysis.

3.3.8 Determination of Protein Concentration

Prior to determination of protein concentration, the eluted protein was dialysed against buffer (25 mM Na2SO4, 50 mM NaCl) for overnight at 4 ˚C. The concentration of purified recombinant MNV-1 C-terminal histidine-tagged WT NS6 and C139A NS6 was determined using Pierce™ BCA Protein Assay Kit with slight modification.

Bovine serum albumin (BSA) (Sigma, USA) standards were prepared by using 10 mg/ml stock (Table 3.16) to establish the standard curve. Elution buffer (from protein purification process) was used as the diluent and blank. Ten µl of each concentration of standard was mixed with 200 µl of BCA reagent. Meanwhile, 10 µl of purified protein was directly mixed with 200 µl of BCA reagent; each sample including the standards and purified proteins were done in triplicate. The samples were mixed thoroughly on plate shaker for 30 seconds and then incubated for 30 mins at 37 ˚C before the readings of absorbance were taken at wavelength 562 nm using plate reader

(Thermo Fisher Scientific, USA). The protein concentration was calculated based on the standard curve that was plotted.

Table 3.16 Standard BCA Tube Concentration (µg/µl) Amount of BSA (µl) Amount of diluent (µl) A 0 0 50 B 0.16 50 from C 50 C 0.31 50 from D 50 D 0.63 50 from E 50 E 1.25 50 from F 50 F 2.50 50 from G 50 G 5.00 50 from H 50 H 10.00 100 0

3.3.9 Western Blot

SDS-PAGE gel were transferred onto polyvinylidene fluoride (PVDF) Immobilon-P transfer membrane with 0.45 µm pore size (Merck Milipore,Germany) using Trans-

Blot D Semi-Dry Transfer Cell (BioRad, Califonia) for 40 mins at 20 V. A 0.1 % (w/v)

Ponceau Red (Sigma) solution in 5 % acetic acid was used to check for successful protein transfer. Membrane was immersed in the solution for 5 mins and then deionised water was used to wash the membrane in order to visualise band. Upon successful transfer of protein onto the membrane, the membrane was blocked with 5% skim milk diluted in PBST (0.1 % Tween 20 in PBS) for 1 h at room temperature. After blocking,

the membrane was probed overnight at 4 ˚C with primary antibody in 5 % (w/v) skim milk dissolved in PBST (0.1 % Tween 20 in PBS). The membrane was then washed 3 times with PBST, 5 % (w/v) milk. Subsequently, the secondary antibody diluted in

PBST, 5 % milk was applied for 1 h and the membrane was washed 3 times and 2 times with PBST and PBS respectively. Finally, the antibody-bound protein was visualised using WesternBright ECL detection substrate (Advansta, USA) accoding to manufacturer’s instruction. The processes membrane was visualised using Molecular

Imager ® VersaDoc TM MP Imaging (Biorad. Califonia).

In addition, SDS gel with C139A NS6 proteins were also transferred onto PVDF following all the steps as mentioned above except the primary goat anti ProPol anti serum and secondary anti-goat HRP (Invitrogen) were used for detection.

3.4 Preparation and Optimisation for Peptide Phage Selection

3.4.1 Quantification of Phage’s Host Bacterial Cell

3.4.1(a) E. coli ER2738 Culture

E. coli ER2738 (New England Biolabs, USA) was cultured from the original stock by adding 10 µl of the stock into 20 ml of LB broth (R & M Chemical, UK) supplemented with 20 µg/ml Tetracycline (Fisher Scientific, USA), (LB + Tet). The culture was subjected to overnight shaking at 200 rpm, 37 ˚C. Meanwhile, some of the stock was streaked on LB plate with 20 µg/ml tetracycline and the plate was incubated in incubator (Memmert, UK) at 37 ˚C for overnight.

3.4.1(b) E. coli ER2738 Growth Curve and Quantification

Growth curve of E. coli ER2738 was constructed following protocol as detailed in

Section 3.3.3 but using tetracycline as antibiotic for selection. Graph for ER2738

growth was plotted as time against absorbance taken at OD600. Growth of E. coli

ER2738 from two methods of starting culture was monitored based on the graph constructed. The first culture was prepared by inoculating a single colony of E. coli

ER2738 from a plate into 250 ml conical flask filled with 20 ml of LB broth supplemented with Tetracycline (LB + Tet). Another method was carried out by diluting overnight culture of ER2738 in LB + Tet broth in 250 ml conical flask according to 1:100 ratios. Both cultures were incubated at 37 °C with shaking at 200 rpm. Initial OD600 for both cultures were recorded and the growth was monitored for 4

- 8 hours (mid-log phase, OD600 ̴ 0.5) at 30 mins interval. Two ml of each culture was kept as glycerol stock for subsequent analysis.

3.4.2 Phage Titration

Overnight culture of ER2738 was prepared by inoculating the bacterial cell from the glycerol stock into the 10 ml (LB + Tet) broth. On the next day, the overnight culture was diluted in a ratio of 1:100 and incubation was continued at 37 ˚C with vigorous shaking until it reached mid-log phase. Serial dilutions of the phage were prepared while the cells are growing by diluting the phage stock with LB broth. Initially, the phage stock which is included in the kit was subjected to re-titration and dilution scale of 108 to 1011 was used. For subsequent titration in biopanning experiment, the unamplified phage was diluted from scale 101 to 104 while amplified phage was diluted from scale 108 to 1011. After the bacterial ER2738 culture had reached mid-log phase,

200 µl of culture was dispensed into sterile 1.5 ml tubes (each phage dilution was done in triplicate). The aliquoted E. coli cell of ER2738 was then subjected to infection with 10 µL of each phage dilutions, 101 - 104 for unamplified phage and 108 - 1011 for amplified phage. Then, the infected cultures were vortexed quickly and incubated at room temperature for 1-5 mins. After that, the infected cultures were transferred to 15

ml tube containing 4 ml pre-warmed top agar, briefly vortexed and immediately poured onto pre-warmed LB/IPTG (1st BASE, Singapore)/XGAL (1st BASE,

Singapore) plate. The plates were gently tilted and swirled to spread the top agar evenly. The plates were allowed to cool down for 5 mins. Ater the top agar had solidified, the plates were inverted and incubated at 37 ˚C incubator (Incucell,

Germany) for overnight. The plaques formed on plates were counted and plaque forming unit per ml (pfu/ml) were calculated.

3.4.3 Determination of Optimum Concentration of Protein to be Coated on Plate

Different concentrations (10, 25, 50, 75, 100 µg/ml) of Streptavidin-dissolved in 0.1

M NaHCO3 (pH 8.6) was coated overnight on 96-well ELISA plate and the plate was incubated at 4 ˚C with gentle agitation in humidified condition. On the next day, the solution was discarded and the well was completely blocked with blocking buffer for

1 h at 4 ˚C. After that, the wells were washed for six times with TBST 0.25 % (v/v).

Following washing steps, the well was incubated with Biotin-HRP (Invitrogen, USA) diluted in PBST 0.05 % (v/v) pH 7.4 (1:5000) and the plate was then incubated for 30 mins at room temperature with gentle agitation. Then, the solution was discarded and followed by ten times washing with TBST 0.25 % (v/v). Next, 100 µl of TMB substrate

(Cell Signalling Technology, USA) was added and the plate was incubated until different blue colour intensity formed. Stop solution (2 M sulphuric acid) was added to stop the reaction once the blue colour appeared at the required intensity. The absorbance value was recorded at 450 nm using a plate reader (Thermo Fisher

Scientific, USA).

The same protocol was applied to the purified C139A NS6, except the primary rabbit anti-His antibody (Cell Signalling Technology, USA) and secondary anti-rabbit HRP

(Life Technology, USA) were used for detection. 68

3.5 Subtractive Screening of Peptide Phage Display Library against Target

Protein

3.5.1 Panning against Streptavidin

Validation of phage library was carried out by panning against streptavidin. The experiments were performed according to the kit manufacturer’s instruction (Ph.D.-7 Phage Display Peptide Library, New England BioLabs, USA) with a slight modification. Briefly, 150 µl of 100 µg/mL streptavidin in 0.1 M NaHCO3

(pH 8.6) as well as BSA were immobilised on separated wells of 96 well ELISA plate and incubated overnight at 4 ˚C with gentle agitation. Next, blocking solution was added to the BSA-coated well and the plate was incubated at 4 ˚C for 1 h. Meanwhile, double negative selections were carried out by adding 100 µl of 2 x 1011 plaque forming units per ml (pfu/ml) of phage from the library to an empty well of ELISA plate for 1 h incubation at room temperature. The unbound phage from this well was then transferred to the BSA-coated well and incubated for another 1 h for the second- negative selection. Concurrently, the streptavidin-coated well was blocked with blocking buffer at 4 ˚C for 1 h and then washed for 6 times with TBST (TBS + 0.1 %

[v/v] Tween 20). The unbound phage from BSA coated well were added to the streptavidin-coated well and incubated at room temperature for 1 h to allow phage- protein binding. Then, the well was washed 10 times with TBST 0.1 % (v/v). The bound phage was eluted with 100 µl of 0.1 mM Biotin (New England Biolabs, USA) in TBS by incubating the plate with gentle agitation for 30 mins at room temperature.

The concentration of Tween 20 in TBS used during washing steps was increased in the range of 0.25 % (v/v) to 0.5 % (v/v) respectively for subsequent round of panning in order to increase the stringency of panning. The eluted phage was titrated, amplified and purified for further used in the successive round of panning. In total, four rounds

of panning were carried out. After 4th round of panning, 20 positive clones (represented as blue plaques on selection plate) were randomly selected. The selected plaques were amplified and the DNA from phage were then extracted. The phage DNA was subjected for PCR to amplify the region with insert and the purified PCR product with insert was then sent for sequencing to verify whether the sequence contain specific motif for Streptavidin. The summary of phage display selection steps is described in

Figure 3.1.

Step 1 Peptide Step 2 Step 3

library (M13

The bound phage was amplified

Target protein was coated on plate and The unbound phage was removed exposed to peptide after extensive washing steps; phage library the bound phage was eluted Step 4

The eluted phage 1011 of amplified pool of phage is used from each round is

for the successive round of selection. titrated.

Step 5 Step 6 Individual phage The phage clones were plaque is isolated analysed by sequencing and amplified.

Figure 3.1 Diagrammatic representation of the phage display method used to select specific peptides that bind to the target protein. This figure was adapted from the manual book provided by New England Biolabs.

3.5.2 Panning against C139A NS6 Protease

The validated peptide phage library was further used for selection against target protein, the purified C-terminal histidine-tagged C139A NS6 following methodology as described in Section 3.5.1. The bound phage was eluted using 100 µl general elution buffer (0.2M Glycine-HCl [pH 2.2], 1 mg/ml BSA) and subsequently neutralised using

1 M Tris-HCl (pH 9.1). The eluted phage was then amplified, purified, re-titrated and used for successive round of panning. Six rounds of panning were conducted and 98 single clones in total were isolated for further analysis.

3.5.3 Phage Titration

Ten µl of the eluted phage from each rounds of panning were subjected to titration/quantification as described previously in Section 3.4.2 while the rest of the eluted phage was stored in 4 ˚C for amplification.

3.5.4 Phage Amplification, Precipitation and Purification

Fourty µL of the eluted phage was used to infect the E. coli ER2738 that previously been diluted in (LB + Tet) broth from the overnight culture and incubated at 37 ˚C with vigorous shaking for 4 to 4.5 hours. Then, the infected bacterial culture was centrifuged at 12000 g for 10 mins at 4 ˚C. After that, 80 % of the supernatant was transferred to a new tube and subjected to precipitation by addition of 1/6 volume of

20 % PEG/2.5 M NaCI. The mixture was incubated overnight with gentle agitation at

4 ˚C to precipitate the phage. After that, the PEG precipitates were centrifuged at 12000 g for 15 mins at 4 ˚C. The supernatant was discarded and the remaining pellet was resuspended in 1 mL TBS. Then, the suspension was centrifuged again at 14000 g for

5 mins at 4 ˚C. The supernatant was transferred into a new tube and re-precipitated by adding 1/6 volume of 20 % PEG/2.5 M NaCI. Next, the sample was incubated on ice for 15 to 60 mins and before centrifuged again. The pellet was re-suspended in 200 µL 71

of TBS and centrifuged for 1 min to pellet any remaining insoluble material. The supernatant which is the purified and amplified phage was transferred into a fresh tube and 10 µl was subjected to titration (Section 3.4.2). The rest of the amplified phage was stored in 4 ˚C for next round of panning.

3.6 Isolation of Phage DNA

3.6.1 Plaque Isolation and Amplification

Ten to 20 single plaques (representing single clone phage) were selected from the 4th round of panning elution. Each single blue plaque from the LB/IPTG/XGAL plate was picked and transferred using a sterile pipette tip into tube containing 1 ml of diluted

ER2738 culture (1:100 ratio) and incubated at 37 ˚C with shaking for 4 to 4.5 hours.

The infected culture was then centrifuged at 14000 g for 30 secs at 4 ˚C. The supernatant was transferred to fresh tube and subjected to brief centrifugation. The upper 80 % of the supernatant containing the amplified phage stock was then transferred to a fresh tube. Five hundred µl of the amplified phage was used for phage

DNA extraction while another 300 µl was diluted in ratio 1:1 with glycerol and stored at -20 ˚C for further use.

3.6.2 Phage DNA Extraction

Phage DNA was extracted from the amplified plaque. A 200 µL of 20 % PEG/2.5 M

NaCI was added into tube containing 500 µl of amplified phage. After that, the tube was inverted, mixed and incubated for 10 – 20 mins at room temperature. Then, the tube was centrifuged at 14000 g, 4 ˚C for 10 mins. The supernatant was discarded and any remaining supernatant was discarded using pipette. The pellet was re-suspended in 100 µL of Iodide buffer with vigorous tapping followed by addition of 250 µL of ethanol. The tube was then incubated for 10 to 20 mins at room temperature. After 20

mins, the tube was centrifuged at 14000 g for 10 mins at 4 ˚C. The supernatant was discarded, and the pellet was subjected to washing with 0.5 ml of cold 70 % ethanol.

Next, the tube was centrifuged, and the pellet was dried briefly. The dried pellet was re-suspended in 30 µl of TE buffer and stored at -20 ˚C until further use.

3.6.3 PCR Amplification of DNA Insert from Phage Genome

Phage DNA for each clone was used as template in PCR to amplify the DNA insert.

The specific set of primer was designed as shown in Table 3.10. The PCR master mix was prepared on ice and the PCR components were shown in Table 3.13. The PCR programme used is shown in Table 3.17 and the amplification was carried out using the Mastercycler thermal cycler (Eppendorf, USA). The PCR product was visualised and analysed by agarose gel electrophoresis (Section 3.2.4) with percentage of 2 %

(w/v) agarose gel prepared in TAE buffer. A 100 bp DNA marker was used to analyse the size of the PCR product. The gel was electrophoresed for 45 mins at 100 V and then visualized under UV light.

Table 3.17 PCR condition for amplification of DNA insert from phage genome Steps Temperature (oC) Time (min) Cycle Initial denaturation 95 2 1 Denaturation 95 0.5 Annealing 57.8 0.5 30 Extension 72 0.5 Final extension 72 5 1

3.6.4 Purification of PCR Amplicon

All the PCR products from Section 3.6.3 were purified using GFX PCR DNA and Gel

Band Purification Kit (GE Healthcare) as described in Section 3.2.7. The purified

DNA was electrophoresed as described in Section 3.2.4 to ensure the success of purification steps. All PCR amplicons from corresponding phage clones which carried the DNA sequences that encode for randomised 7-mer peptide was sent for

sequencing. Specific sequencing primer (5’ GCC CTC ATA GTT AGC GTA ACG ATC

TAA AG 3’) was used for single pass DNA sequencing base (1st Base Laboratories Sdn

Bhd).

3.6.5 DNA Sequencing Analysis

From the sequencing data, the complete DNA sequences were reverse complemented

(https://www.bioinformatics.org/sms/rev_comp.html). Then, sequences spacer and conserve leader sequences which are (5’-GGT GGA GGT-3’) and (5’-GTG GTA CCT

TTC TAT TCT CAC TCT-3’) were identified respectively. The start position of the first nucleotide of the randomised DNA sequences were identified whereby they are located exactly before the spacer sequences. Then, the third nucleotide of each codon in the randomized region was examined. The third nucleotide of each codon in randomized region should be G or T. After that, the sequences were translated into peptide sequences using the ExPASy (https://web.expasy.org/translate/) for further analyis.

3.7 Binding Affinity Assessment of Phage

The ability of the selected phage to bind the target protein was determined using enzyme-linked immunosorbent assays (ELISA). In this assay, each row of 96-well

ELISA plate was coated overnight with 100 µl of 100 µg/ml of selected target protein

in 0.1 M NaHCO3, pH 8.6 and incubated at 4 ˚C with humidified condition.

Subsequently, the coated wells were blocked completely with blocking buffer for 1 h and washed with 0.5 % TBST. Ten-fold serially diluted of the selected phage clones were prepared. Then, 100 µl of each phage dilution were transferred into target protein- coated well and incubated at room temperature for 1 h. After 1 h, the unbound phage was washed with TBST 0.5 % (v/v) for 6 times. After that, 100 µl of anti-M13 antibody

conjugate with HRP (GE Healthcare Life Science) in blocking buffer was added into each well and the mixtures were incubated for 1 h at room temperature with agitation.

Then, the plate was subjected to washing again with TBST 0.5 % (v/v) for 10 times.

After the washes completed, 100 µl of TMB substrate was added into each well and the reactions were left until blue color appeared at the required intensity. Finally, the reactions were stopped by the addition of 100 µl of stop solution (2 M sulphuric acid) and the absorbance was measured at 450 nm using a plate reader (Thermo Fisher

Scientific, USA).

3.8 Bioinformatics Study

3.8.1 Prediction of Protein Structure

The C139A NS6 protease’s structure was predicted using online server for protein structure prediction software, Iterative Threading ASSEmbly Refinement

(I-TASSER) at https://zhanglab.ccmb.med.umich.edu/I-TASSER/. Full sequence of

NS6 protease was obtained from GenBank NCBI database with accession no

(DQ285629.1). Single nucleotide of NS6 sequence was changed in order to introduce mutation at the active site 139 which changes cysteine to alanine. The FASTA sequence was inserted through I-TASSER and protein structure prediction was acquired. The structure protein was viewed using Visual Molecular Dynamic (VMD) software while Ramachandran plot was identified using RAMPAGE

(www.mordred.bioc.cam.ac.uk) to check the quality of the predicted structure.

3.8.2 Prediction of Selected Peptide Phage Binding Sites on C139A NS6 Protease

The PDB file for predicted structure of C139A NS6 protease was used as an input file and full sequence of each identified peptides from phage clones were keyed-in into the CABS-dock (www.biocomp.chem.uw.edu.pl). The binding simulation of each 75

peptides with the targeted protein known as docking was conducted using (CAB-dock) server for protein-protein interaction. The result provided by CABS-dock includes 3D- structure, clustering details, contact maps between peptides and receptor were used to identify the binding sites of peptides towards C139A NS6 protease.

CHAPTER 4

RESULTS

4.1 Recombinant MNV-1 C139A NS6 Production and Purification

The first part of this study was aimed at producing a purified recombinant MNV-1

NS6 protease with the active-site mutant (C139A NS6) to be used in the subsequent phage display bio-panning selection assay. To achieve this objective, series of PCR amplifications, site-directed mutagenesis, cloning, protein expression and purification were carried out successfully.

4.1.1 Cloning of C139A NS6 Gene into Expression Plasmid

Expression plasmid of active-site mutant NS6 protease (C139A NS6), was generated by cloning the NS6 protease C139A coding sequence into the expression vector pET26Ub with C-terminal histidine (Figure 4.1A). Site directed mutagenesis was carried out to introduce a point mutation at amino acid position 139 where the active site of MNV-1 protease is located. Two set of primers were designed to obtain the amplicon with single mutation through polymerase chain reaction. Polymerase chain reaction was carried out and the PCR product was verified through gel electrophoresis.

The series of overlapping PCR is summarised in Figure 4.2. The first reaction involved

DNA template of full length MNV-1 cDNA clone pT7: MNV 3’Rz (Figure 4.1B) and first set of primer (AY7 and AY18). The resulting amplicon size was 437 bp (Figure

4.3). Second set of primer (AY17 and AY8) was used for PCR with the same DNA template resulted in production of amplicon with the size of 155 bp (Figure 4.4). Both

PCR amplicons were purified and further used as templates for overlapping PCR using

AY7 as forward and AY8 as reverse primer. The final amplicon of overlapping PCR with the size of 549 bp was successfully generated (Figure 4.5).

The resulting final amplicon of overlapping PCR (Figure 4.5) was purified and subjected to restriction enzyme reaction using SacII and BamHI in order to be cloned into pET26Ub plasmid (Figure 4.6) that was also digested with the same restriction enzymes. Both digested and purified PCR product (insert) and plasmid (Figure 4.7) were ligated and cloned successfully, producing the plasmid namely pET26Ub: MNV

C139A NS6-His which was then transformed into E. coli DH5α competent cell.

Several colonies of transformant were screened through restriction enzyme reaction and PCR reaction. Positive clone was further confirmed by Sanger DNA sequencing analysis using both AY7 and AY8 (Refer Appendix A).

3C/NS6

Figure 4.1 Schematic diagrams illustrating the plasmid map used in this study. (A) The expression plasmid was originated from the pET26Ub: MNV 3D-His which contains expression cassette for MNV-1 RNA dependent RNA polymerase (RdRp). For this study, the RdRp gene was removed from the plasmid and replaced with a PCR amplified NS6 protease sequence. (B) The pT7:MNV3’Rz plasmid that contains the full genomic sequence of MNV-1. This plasmid was used as template to PCR amplified the NS6 protease sequence. The 3D/NS7/RdRp gene (red circle) that has been incorporated in pET26Ub plasmid was removed and replaced with amplified 3C/NS6 (blue square).

Figure 4.2 This schematic diagram shows an illustration of polymerase chain reaction series to construct recombinant MNV-1 C139A NS6. A point mutation is introduced at the amino acid 139 where TGT (cysteine) had been changed to GCA (Alanine). First PCR involves forward primer of AY7, AY18 as reverse primer and MNV-1 cDNA clone (pT7: MNV 3’Rz) as template. The same template as 1st PCR is used for the second PCR while AY17 and AY8 were use as forward reverse primer respectively. The first overlapping PCR involved the product of first and second PCR as templates while AY7 is forward primer and AY8 is reverse primer.

1 2 3 4

500bp 437 bp 400bp

Figure 4.3 The PCR amplicons from 1st pair of primer. Lane 1, 2 and 3 are the identical products from the first PCR using primer set AY7 and AY18 while the 4th lane is the negative control. The amplicon size is 437 bp. A 100 bp DNA standard was used as ladder.

1 2 3 4

155 bp 200bp 100bp

Figure 4.4 The PCR amplicons from 2nd pair of primer. Lane 1 is the negative control while lane 2, 3 and 4 are the identical products from the second PCR using primer set AY17 and AY8. The amplicon size is 155 bp. A 100 bp DNA standard was used as ladder.

1 2

600bp 549 bp 500bp

Figure 4.5 The final PCR amplicon from 3rd pair of primer. Lane 1 and 2 are the identical amplicons from PCR using primer set AY7 and AY8. The final amplicon is the product of overlapping PCR using product of 1st and 2nd pairs of primer as template. The amplicon size is 549 bp. A 100 bp DNA standard was used as ladder.

1 2 3 4 5

7.0 kb

6.0 5.0 5.5 kb

2.0

1.5 1.5 kb

1.0

0.5

Figure 4.6 Product of restriction enzyme (SacII and BamHI) reaction for expression plasmid pET26Ub: MNV 3D-His. Lane 1 is the 1 kb DNA ladder, lane 2 is the undigested plasmid while lane 3, 4 and 5 are the identical product of the digested plasmid. The expected size of pET26Ub-His is 5.5 kb while MNV 3D is 1.5 kb.

1 2 3

10.0

8.0

6.0 5.5 kb 5.0

4.0

3.0

2.0

1.5

1.0

0.5 549bp

Figure 4.7 Purified DNA products of restriction enzyme reaction. Lane 1 is the 1kb DNA ladder. Lane 2 is the digested and purified plasmid pET26Ub-His (5.5 kb) while lane 3 is the digested and purified MNV C139A NS6 (549 bp) DNA fragment (insert).

4.1.2 Overexpression and Purification of MNV-1 C139A NS6

The resulting expression plasmid (pET26Ub: MNV C139A NS6-His) from Section

4.1.1 was then subjected to transformation into E. coli BL21(DE3) competent cell in the presence of pCG1 plasmid (Gohara et al, 1999) for inducible overexpression. The recombinant NS6 in this study was designed to be expressed as Ubiquitin fused to its

N-terminus. The pCG1 plasmid carrying sequence that encode for ubiquitin-specific, carboxy-terminal protease (Ubp1) that will co- and/or post-translationally cleaves the fusion protein and releasing the recombinant NS6 with the precise N-terminus as seen in the virus.

Growth curve of the selected transformant carrying the pET26Ub: MNV

C139A NS6-His was generated by measuring the absorbance (turbidity of the culture) at several time points through the growth course (Figure 4.8). The resulting graph showed that the transformant culture took approximately around 1 h 30 mins to reach log phase (OD600 ~ 0.5 - 0.6). For overexpression of recombinant NS6, the transformant culture was subjected to induction when the growth reached the log phase using 0.1 mM IPTG for 4 hours at 25 °C. The overexpression was analysed using SDS-

PAGE whereby the expected overexpressed protein band size of 19 kDa was compared to the uninduced sample (Figure 4.9). The overexpressed protein was further confirmed via western blot analysis with the presence of band at expected size 19 kDa using anti histidine antibody and anti MNV ProPol antisera (Figure 4.10). Following the western blot analysis, the culture was further lysed and purified using immobilised metal affinity chromatography (IMAC). The purified protein was again verified using

SDS-PAGE (Figure 4.11) and all fractions was pooled together for quantification. The concentration of protein obtained was 2.6 mg/mL.

Growth curve of C139A NS6-His 2

1.5

0.5

0 Absorbance reading at 600 nm 600 at reading Absorbance 0 200 400 600 800 Time (min)

Figure 4.8 Growth curve of C139A NS6-His. The growth curve was constructed prior to induction with IPTG. Initially the selected transformant was grown overnight in 10 mL of LB culture with aeration by shaking at 250 rpm, 37 °C. After that, 1 mL of the overnight culture was diluted in 100 ml LB supplemented with antibiotics until OD600 reach 0.1. The culture was grown at the same condition and sampled out for every 30 mins to measure the turbidity. The growth curve is plotted as absorbance vs time.

A) 1 2 3 4 B) kDa 1 2 kDa

245 135 190 100 135 80 100

80 58

58 46

46 32

32 25

22 25 C139A 22 17 NS6 C139A 17

11 NS6 11

Figure 4.9 Expression of C139A NS6 protein. The sample of total protein was denatured and loaded onto 17.5 % SDS PAGE together with SeeBlue ® Plus2 Prestained Standard protein marker. The gel was stained with 0.1 % coomassie blue for visualization. The expected size of protein is 19 kDa. (A) Lane 1 is the non-induced sample while lane 2, 3 and 4 are the identical induced protein. (B) Lane 1 and 2 are the expression of C139A NS6 protein after subjected for lysis.

A) B) 1 2 C139A NS6

Figure 4.10 Western blot analysis of purified C139A NS6 protein. The sample of total protein was denatured and loaded onto 17.5 % SDS PAGE together with MagicMarkTM XP Western Standard. The gel was transferred onto PVDF membrane and the membrane was probed against antibody for detection. The expected size of protein is 19 kDa. (A) Detection of histidine-tagged C139A NS6 using rabbit polyclonal anti-His tag. The lane 1 is the non-induced protein sample while the lane 2 is the induced protein sample. (B) Detection of C139A NS6 using goat anti ProPol anti serum. All protein samples were total protein samples from the lysed bacterial cells.

1 2 3 4 5

C139A NS6 (19 kDa)

Figure 4.11 Purified C139A NS6 protein. The total protein from lysed bacterial cell was subjected to IMAC using His-Talon column. After a few washes, the bound his- tagged protein was eluted with elution buffer into 500 µl fractions each. The eluted fractions were subjected to SDS PAGE analysis before been quantified using the BCA assay. Lanes 1, 2, 3 and 4 are the purified C139A NS6 from different fractions while lane 5 is the non-purified protein/total protein.

4.2 Identification of Peptide Phage with binding capacity towards the Target

Proteins

The resulting purified C139A NS6 protein as described in Section 4.1 was employed as target in the biopanning phage display selection to fulfill the second objective of this study. This specific part of the study was aimed to identify peptide phage with binding activity towards the purified C139A NS6. To accomplish this objective, a Ph.D-7 Phage Display Peptide Library kit (NEB) was used in the biopanning selection. Several optimisations on bio-panning parameters and efficiency verifications of the kit were carried out before the selection towards target protein

(C139A NS6) was successfully done.

4.2.1 Parameters Optimisation for Peptide Phage Selection

4.2.1(a) Optimum Growth Time for E. coli ER2738 Culture

Growth curve of E. coli ER2738 (host for M13 bacteriophage) was constructed by measuring the absorbance at the OD600 for every half an hour. This step was carried out to determine the optimum time for the cultures to achieve mid-log phase (OD600 = 0.5); the rate at which E. coli growth is doubled. Two types of ER2738 starter cultures were prepared. One culture was started by inoculating a single colony of ER2738 grown overnight on LB + Tet plate into 20 mL of medium supplemented with tetracycline while another culture was prepared by diluting the overnight culture of ER2738 in LB broth according to 1:100 ratio. Both cultures were incubated at 37

°C with shaking at 200 rpm. From the graph constructed in Figure 4.12, it shows that the diluted overnight culture took only two hours to achieve mid-log phase as compared to the single colony culture where it took around five hours. The result obtained from the growth curve indicated that the diluted overnight culture was the

most preferable method to initiate the growth of E. coli ER2738 for subsequent experiment.

Growth Curve of E. Coli ER2738 in LB Broth 1.8 1.6 (nm) 1.4 600 1.2 1 0.8 0.6 0.4 0.2

Absorbance at OD at Absorbance 0

1 hr

2hrs 6 hrs 7 hrs 8 hrs 9 hrs

3 hrs 4 hrs 5 hrs

0 min

10 10 hrs 11 hrs 12 hrs 13 hrs 14 hrs

30 30 mins

1 30 minshr

2 30mins hrs 3 30mins hrs 4 30mins hrs 5 30mins hrs 6 30mins hrs 7 30mins hrs 8 30mins hrs 9 30mins hrs

10 10 hrs 30 mins 11 hrs 30 mins 12 hrs 30 mins 13 hrs 30 mins Time (mins)

OD600 (dilute overnight culture) OD600 (single clone)

Figure 4.12 Growth curve of E. coli ER2738 from a diluted overnight culture and a single colony culture in LB + Tet broth supplemented with Tetracycline. Both cultures were incubated at 37 °C with shaking at 200 rpm. The absorbance reading was taken at OD600 for every half an hour interval.

4.2.1(b) Peptide Phage Concentration in the Ph.D-7 Phage Display Peptide

Library Kit

The concentration of the phage library supplied by the manufacturer was determined through phage titration as described in detail in Section 3.4.2 in the previous chapter. The blue plaques formed on the plate after overnight incubation were counted to get the amount of plaque forming unit per ml (pfu/ml). The number of phage calculated from the titration is 3.675 X 1013 pfu/ml.

4.2.1(c) Determination of Optimum Concentration of Streptavidin to be Coated

on Plate

Before biopanning against streptavidin was performed, ELISA was carried out to determine the optimum concentration of streptavidin required to be coated on plate. The graph of binding signal of streptavidin and BSA protein at different concentration was constructed.

From the graph in Figure 4.13, it showed that 100 µg/ml of streptavidin has the highest binding as compared to other concentrations while for BSA, the absorbance does not show any major changes even the concentration of BSA used were increased.

Therefore, 100 µg/ml of streptavidin has been determined as the optimum concentration to be used in coating the ELISA plate.

Binding of Strepatvidin and BSA on ELISA plate 0.800 0.700 0.600 0.500 0.400 0.300 0.200

Absorbance 450 nm 450 Absorbance 0.100 0.000 0 20 40 60 80 100 120 Concentration (µg/mL)

bsa streptavidin

Figure 4.13 Optimum concentration of streptavidin and BSA. Different concentration of Streptavidin and BSA were coated on the plate, washed with TBST 0.25 % (v/v) and detected with Biotin-HRP enzyme substrate reaction to determine the binding ability of Streptavidin towards biotin and the maximum concentration of protein required to be coated on plate. Absorbance reading was taken at wavelength OD450. The reactions were carried out in triplicate and the error bars represent standard deviation.

4.3 Peptide-phage with Binding Activity towards Streptavidin

4.3.1 Panning of Peptide Phage Library against Streptavidin

The efficiency and ability of peptide phage library to select the specific peptide was validated through in vitro selection of known target protein (streptavidin) and its interaction with biotin. The known consensus motif for streptavidin-binding peptide includes histidine-proline-glutamine (HPQ) and histidine-proline-methionine (HPM).

Subtractive panning of a Ph.D-7 Phage Display Peptide Library (NEB) against

Streptavidin was performed to validate the phage library as described in Section 3.5.1.

The number of input phage panned against Streptavidin was kept constant for each round of panning at 2 X 1011 pfu/ml. Phage titration were performed after each round of screenings and blue plaque formed on the plate was counted (Figure 4.14). The input, output and amplified phage for 1st, 2nd ,3rd and 4th round of panning was shown

in Table 4.1. Results showed that there was 1.1-fold and 315-fold enrichment from 1st round to 2nd round and after 3rd round of panning, respectively. Twenty single phage clones were randomly selected from the fourth round of panning and propagated independently for further screening as described in Section 3.5.4.

blue plaque

Figure 4.14 Blue plaques formed by individual phage appeared on LB/IPTG/XGAL plate after titration of recovered phage from 4th round streptavidin panning

Table 4.1 Number of recovered phages for every round of panning. Round of Input phage Output phage Phage Phage panning (pfu/mL) (pfu/mL) enrichment/fold amplification 1st 2 X 1011 5.10 X 105 - 5.37 X 1012 2nd 2 X 1011 5.60 X 105 1.1 2.03 X 1012 3rd 2 X 1011 4.19 X 105 - 2.80 X 1012 4th 2 X 1011 1.32 X 108 315.0 *phage enrichment: Recovered phage after successive round of selection divided by recovered phage before selection

4.3.2 Identity of Peptide Sequence from Streptavidin-Binding Peptide Phage

Phage genomic DNA from 20 individual phage clones were successfully isolated and were subjected to PCR as described in Section 3.6. PCR was performed to confirm whether the clones selected contain the DNA insert that encodes the 7-mer randomized peptide sequences using specific forward and reverse primers. Based on Figure 4.15, all 20 clones yielded PCR amplicons with the expected size of 180 bp. This outcome indicated that the DNA extracts contained the sequence that encodes the 7-mer randomised peptide display on the surface of the phage. All the PCR amplicons containing DNA sequence that encodes the randomised peptide sequences were then sent for DNA sequencing.

The DNA insert sequence for the randomised 7-mer peptide is located between pIII leader sequence and spacer sequence. There is also restriction enzyme site in between the insert sequence as shown in Figure 4.16. The DNA sequence (triplet codon) of the single clone phages were translated into peptide sequence and the corresponding displayed 7-mer peptides sequences were shown in Table 4.2. Ten clones out of 20 clones that have been sent for sequencing, shared the same HPQ motif.

200bp 100bp

200bp

100bp

Figure 4.15 PCR products from amplification using purified phage genomic DNA of single clones from Streptavidin panning as template. All 20 clones contained randomised peptide sequences as indicated by the presence of amplicons with 180 bp in size. A 100 bp DNA standard was used as ladder.

Figure 4.16 Library insert sequences fused to the pIII leader sequence and located in between 2 restriction sites (Kpn1/Acc65I and EagI). The insert sequences containing 7 mer amino acids sequences followed by spacer GGT GGA GGT (Gly-Gly-Gly)

Table 4.2 The identified sequences from 20 single clone phages. 10 clones sharing HPQ motif No Clone 5’ … 3’ Protein sequencesb motif 1 C5, C9, C14, C19 HFEGHPQ 2 C11, C12, C13, C18 SLIAHPQ HPQ 3 C17 GLLAHPQ 4 C20 LVPNHPQ 5 C1, C6, C15 ISTTLFP 6 C3, C4, C8, SILPVTR 7 C2 TLLEYNW 8 C7 NGATYPS 9 C10 NGATHPR 10 C16 TLLEYNW bSingle letter abbreviations of amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr

4.3.3 Binding Kinetics of Selected Peptide Phage towards Streptavidin

Phage ELISA was performed to establish the binding affinity of the selected peptide-bearing phage towards streptavidin as described in detail in Section 3.7. The peptide phage namely C9 clone with HPQ motif, was selected as a representative of

Streptavidin clones while the C7 clone, which does not contain HPQ motif was selected as a control phage. BSA was used as an irrelevant protein control (negative control). The selected phage pool was subjected to serial dilution and were exposed to the target coated wells. The bound phage was then detected with HRP-conjugated M13 antibody. Figure 4.17 shows that the ELISA signal for the C9 clone on Streptavidin coated plate increase at phage concentration 2 X 107 pfu/ml of phage with Kd value of

5.772 X 109 pfu/ml while for the C7 clone, the signal started to increase at phage concentration 2 X 109 pfu/ml with Kd value 2.672 X 1011 pfu/ml. ELISA signal of both clones on BSA coated plate does not show any increase until 2 X 1010 pfu/ml of phage concentration added.

Based on the result obtained from the peptide-phage selection towards biopanning against Streptavidin, we have successfully validated the efficiency of the

Ph.D-7 Phage Display Peptide Library (NEB) that was purchased and readily available in the laboratory.

Binding Kinetics of Selected Peptide-bearing Phage Clones towards Streptavidin 2.500

2.000

1.500

1.000

0.500

0.000 Absorbance at 450 nm 450 at Absorbance 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage Concentration (pfu/ml)

C9-Streptavidin C7-Streptavidin C9-BSA C7-BSA

Figure 4.17 Binding kinetics illustrating the specificity and affinity of the peptide phage clone towards Streptavidin and BSA protein. Different concentration of purified clones of phages were subjected to the binding reaction with Streptavidin and BSA in the ELISA plate. After extensive washes with TBST, the binding affinity was detected using HRP- conjugated anti M-13 antibody. The TMB solution was used as the substrate, the reaction was stopped by adding HCl and the absorbance was taken at 450 nm. Phage clone C7 was used as control phage. Graph showed that clone C9 binds specifically towards Streptavidin and with greater affinity compared to clone C7. The reactions were carried out in triplicate and the error bars represent standard deviation.

4.4 Peptide-phage with Binding Activity towards MNV-1 C139A NS6

4.4.1 Determination of Optimum Concentration of C139A NS6 to be Coated

on Plate

Prior to selection against C139A NS6 protein was carried out, ELISA was performed to identify the optimum concentration of C139A NS6 required to coat the well of ELISA plate. Different concentration of C139A NS6 and BSA protein were coated on a 96-well plate whereby BSA was used as negative protein control in this case. Rabbit primary anti-His and secondary anti-rabbit HRP were used to detect the histidine-tagged C139A NS6 on the well and TBST 0.25 % was used during washing 95

steps. The absorbance was measured at OD450 by using a microplate reader. Detailed protocol was as described in Section 3.4.3. As shown in Figure 4.18, ELISA signal of

C139A NS6 coated wells increases as the concentration of the protein increases. In contrast to signal from C139A NS6 coated wells, the BSA coated wells does not show any changes in signal even the concentration was increased. From this assay, we have identified that 100 µg/ml of C139A NS6 was the optimum concentration to be used in subsequent biopanning assay.

Binding Ability of C139A NS6 towards anti-His Antibody 0.700 0.600 0.500 0.400 0.300 0.200

0.100 Absorbance 450 nm 450 Absorbance 0.000 0 20 40 60 80 100 120 Concentration (µg/mL)

BSA ∆C139A NS6

Figure 4.18 Optimum Concentration of C139A NS6 to be Coated on Plate. Different concentration of C139A NS6 and BSA protein were coated on the plate, washed with TBST 0.25 % (v/v) and detected with primary rabbit anti-His antibody and anti-rabbit HRP enzyme substrate reaction to determine the optimum concentration of protein coated on plate. Absorbance reading was taken at wavelength OD450. The reactions were carried out in triplicate and the error bars represent standard deviation.

4.4.2 Panning of Peptide Phage Library against C139A NS6

One hundred microgram of C139A NS6 was coated onto the wells of 96-well ELISA plate and the library of peptide expressing phage (Ph.D-7 Phage Display Peptide Library) was exposed to the protein in the wells as described in Section 3.5.2. Negative selections against plastic (empty well) and BSA (well coated with BSA) were carried out. Unbound phage from this selection, which have eliminated most plastic and BSA binders, were panned against C139A NS6 coated on the well of ELISA

plate. The unbound phage was then removed by washing while the bound phage was eluted using general elution buffer. The eluted phage was then amplified through passage in E. Coli ER2738, purified, re-titrated and used for successive round of panning. In total, six rounds of panning were performed. Number of input phage used for panning, recovered phage (unamplified), phage enrichment and phage amplified after every round of panning were summarised in Table 4.3. Result showed that there was phage enrichment after third round of panning.

Table 4.3 Total number input phage, recovered phage (output phage), amplified phage and phage enrichment for each round of C139A NS6 panning. Round of Input phage Output phage Phage Amplified panning (pfu/mL) (pfu/mL) enrichment/fold phage (pfu/ml) 1st panning 2 X 1011 4.65 X 106 - 2.10 X 1012 2nd panning 2 X 1011 3.34 X 106 - 5.88 X 1012 3rd panning 2 X 1011 7.97 X 106 2.4 4.29 X 1012 4th panning 2 X 1011 1.43 X 107 1.8 4.65 X 1011 5th panning 2 X 1011 8.70 X 107 6.1 2.32 X 1013 6th panning 2 X 1011 3.35 X 108 3.9 *phage enrichment: Recovered phage after successive round of selection divided by recovered phage before selection

4.4.3 Identity of Peptide Sequence from C139A NS6-Binding Peptide Phage

Peptide-phage genomic DNA of 98 individual clones from 3rd until 6th round of panning was isolated and purified. PCR was carried out using a specific pair of forward and reverse primers (Table 3.10) to amplify the region of interest in the phage genome and confirm the presence of DNA insert that encodes for the 7-mer randomised peptide in each phage clones. All 98 single clone phage contain the DNA insert for the 7-mer randomised peptide sequences as indicated by PCR amplicons of 180 bp in size (Figure

4.19). All these PCR amplicons were purified and subjected to DNA sequencing.

The DNA insert sequences of the clones were flanking by conserve leader sequence and spacer sequence as shown in Section 4.3.2 (Figure 4.16). From the sequences analysis, the identified displayed 7-mer peptides sequences were shown in Table 4.4 and the bar chart of percentage of hits of the identified peptides were plotted as in

Figure 4.20. All the identified peptide sequences were subjected to further analysis using website-based software, PepBank (http://pepbank.mgh.harvard.edu/) and

SAROTUP (immunet.cn/sarotup/) to analyse any similarities of selected peptide with the published sequences and towards unintended materials respectively from databases. Result from the analysis showed that from the 98 peptide sequences, several sequences are repetitive resulting in 38 unique peptide sequences. Among all the selected peptide sequences, there were several conserved peptide sequences with core motif Glu-Lys-Asn (EKN) for 28 %, 41 % for Asp-Ala-Arg (DAR) motif, 41% for

Tyr-Lys-Ser (YKS) and 26 % for Lys-Asn-Pro (KNP). The peptide sequence varies according to their respective position from 1st position to 7th position. It shows that the dominant amino acid at position one is Ser/Ala/Gln, 2nd position is Asp (50 %) or Thr

(20 %), 3rd position is Glu or Ala, 4th position is Arg or Lys, 5th position is Asn (33 %) or Tyr (44 %), 6th position is Lys (44 %) or Pro (31 %) and at 7th position is Leu or Ser

(Table 4.4). ADARYKS was the most dominant peptide sequence identified after total of 6 rounds panning carried out with 38% hits and followed by QTEKNPL and

NSLKVLG with 16 % and 8 % hits respectively. Both peptide sequences QTEKNPI and IV*RMLG showed 2 % percentage of hits while the rest are 1% hits.

Figure 4.19 PCR products from amplification using purified phage genomic DNA of single clones isolated from panning against C139A NS6. All the 98 clones containing randomized peptide sequences which is 180 bp in size. A 100 bp DNA standard was used as ladder.

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Table 4.4 Amino acid sequences analysis of 98 samples of C139A NS6 phage clones No Clones Sequences 1 C7, C14, C23, C26, C32, C34, C44, C51, C55, A D A R Y K S C57, C60, C62, C63, C64, C68, C71, C72, C73, C74, C75, C76, C77, C78, C79, C80, C82, C83, C85, C88, C89, C90, C92, C95, C96, C98, C99, C100 2 C22, C29, C30, C31, C33, C39, C40, C41, C42, Q T E K N P L C45, C49, C50, C61, C65, C66, C67 3 C15, C20, C81, C86, C87, C91, C93, C94 N S L K V L G 4 C46, C59 Q T E K N P I 5 C16, C97 I V * R M L G 6 C21 R S E K N P L 7 C25 L S E K N P L 8 C35 I P E K N P F 9 C36 S A E K N Q S 10 C69 S A E K N T S 11 C43 S D E K N Q S 12 C47 S D E N Y P S 13 C48 S D E K N P S 14 C54 R D E K N P L 15 C56 A D E K N P S 16 C84 A D A Q N K S 17 C27 T D E N Y P S 18 C52 T D E N Y K S 19 C37 T D A R Y K S 20 C38 T D A R N K S 21 C53 S D A R Y K S 22 C58 R D E N N P I 23 C24 R L R L G W T 24 C1 M M S Y P K H 25 C5 M P R L P P A 26 C9 M R L S V P N 27 C17 M T T T H M H 28 C2 A W P Y V T L 29 C8 A G K S W T I 30 C19 N H D I T L T 31 C3 S I Q A N L E 32 C4 L L A P P Y W 33 C18 Q T Q K N P H 34 C6 Q S G T Y V P 35 C10 V F Q T T Y K 36 C11 H W N T V V S 37 C12 H W H T A V Q 38 C13 H Y Y S S Q V Single letter abbreviations of amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr

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Percentage of hits for peptide sequences from six rounds (98 clones) of panning 40% 38%

35%

30%

25%

20% 16% 15%

102 Percentage of hits (%) hits of Percentage 10% 8%

5% 2% 2% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% 1%

I E P K N F P

Q Q T E K N I P I S Q A N L E

N N H D L IT T

R DRE N N I P

L E S K N L P

I R V L* G M SRKN PE L D S E N Y S P D S E K N S P

S A A E S N K T S T D E N Y S P

Q Q T E K N L P

M M R P PP L A

A A G K W S I T

S A A S E K N Q S D S E K N Q S DRE K N L P A D E K N S P Q G S T Y V P

N S L K V L G T D E N Y K S D S A R Y K S V F Q T T Y K

T D A Y R K S T D A N R K S M L R V S N P L L A Y P P W H Y Y S S QV

Q Q T Q K N H P

A A D A YRKS L RRGW L T

A A D A Q N K S A W L Y P V T

H H W V V N S T

M M M Y S K P H

H H W A V H Q T M T T T T M H T M H T Sequence of peptides

Figure 4.20 Percentage of hits of the identified peptides. 98 clones from six rounds of panning were send for sequencing. Among 98 clones, there are 38 unique peptide sequences that show repetitive sequences. Peptide sequence ADARYKS is the most dominant peptide sequence identified after total of 6 rounds panning carried out with 38% hits.

4.4.4 Binding Kinetics of Selected Peptide Phage towards C139A NS6

Phage ELISA was performed to establish the binding affinity of five selected phage- clones towards C139A NS6 protein. Briefly, C139A NS6 protein was coated on 96- well ELISA plate and subsequently blocked with BSA for 1 hour followed by washing with 0.5 % TBST. Phage clones carrying peptide sequence ADARYKS, QTEKNPL and NSLKVLG with highest percentage of hits, clone bearing the VFQTTYK with almost similar sequence as the previously mentioned three sequence and clone

MPRLPPA with 1 % hit were selected for ELISA while C9 with HPQ motif was selected as a control phage. Streptavidin, BSA and other recombinant protein (NS7) were used as an irrelevant protein control. The selected phage pool was subjected to serial dilution and were exposed to the target coated wells. The bound phage was then detected with HRP-conjugated M13 antibody. The result obtained indicated that clone

QTEKNPL has the highest binding ability towards C139A NS6 protein as compared to other five clones (Figure 4.21). Figure 4.22 shows that the ELISA signal for clone

QTEKNPL, ADARYKS, NSLKVLG and VFQTTYK increase slowly as the phage concentration increases and then they started to increase exponentially at phage concentration 2 X 108 pfu/ml. All the six selected clones were further analysed statistically using Graphpad software to identify the Kd value that is the ligand concentration that binds to half the receptor sites at equilibrium and Bmax that is the maximum number of binding sites. Table 4.5 shows that clone QTEKNPL has the lowest Kd value and higher maximum binding value (Bmax) which is 3.493 X 109 pfu/ml and 2.871 nm respectively as compared to other selected clones.

ELISA signal of all four clones on BSA, streptavidin and NS7 coated plate does not show any increase until 2 X 1010 pfu/ml of phage concentration added except for clone

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MPRLPPA which started to increase once 2 X 108 pfu/ml of phage concentration was added (refer Appendix B).

Binding Ability of Selected Peptide-bearing Phage Clones against Different Targets 3 2.5 2 1.5 1

0.5 Absorbance at 450 nm 450 at Absorbance 0 QTEKNPL ADARYKS NSLKVLG VFQTTYK MPRLPPA Single clone phage

C139A BSA STREP NS7

Figure 4.21 Binding ability of selected peptide-bearing phage clones at phage concentration 2 X 011 pfu/ml towards different target protein. ELISA was carried out by immobilising 100 µg of MNV 1 C139A NS6-His (C139A), BSA, Streptavidin (strep) and MNV 1 NS7-His (NS7) across the ELISA plate. 2 x 1011 pfu/ml of purified single clone phages were subjected to the binding reaction with respective targets in the ELISA plate. After extensive washes with TBST, the binding affinity was detected using HRP-conjugated anti M-13 antibody. The TMB solution was used as the substrate, the reaction was stopped by adding HCl and the absorbance was taken at 450 nm. The streptavidin-binding peptide phage clone was used as the positive control. The reactions were carried out in triplicate and the error bars represent standard deviation.

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Binding kinetics of single clones towards target protein C139A NS6 3.000

2.500

2.000

1.500

1.000

Absorbance at 450 (nm) 450 at Absorbance 0.500

0.000 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage concentration (pfu/ml)

QTEKNPL ADARYKS NSLKVLG VFQTTYK MPRLPPA HPQ

Figure 4.22 Binding kinetics of selected phage clones towards target protein C139A NS6. ELISA was carried out by immobilising 100 µg of MNV 1 C139A NS6-His (C139A) onto the ELISA plate. Different concentrations of purified single clone phages were subjected to the binding reaction with respective target in the ELISA plate. After extensive washes with TBST, the binding affinity was detected using HRP-conjugated anti M-13 antibody. The TMB solution was used as the substrate, the reaction was stopped by adding HCl and the absorbance was taken at 450 nm. The reactions were carried out in triplicate and the error bars represent standard deviation.

Table 4.5 Maximum binding value, Bmax and dissociation constant, Kd of selected clones. Clone Bmax (nm) Kd (pfu/mL) QTEKNPL 2.871 3.493 X 109 ADARYKS 2.754 5.710 X 109 NSLKVLG 2.038 6.340 X 109 VFQTTYK 1.100 7.040 X 109 MPRLPPA 0.456 4.117 X 1010 HPQ 0.493 1.979 X 1010

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4.5 Structure Prediction of C139A NS6 Protein

The structure of C139A NS6 protein was predicted using web server I-TASSER as described in detail in Section 3.8.1. The outcome of the analysis from the software is shown in Figure 4.23. The percentage sequence identity of the templates in the threading aligned region with the newly deposited sequence is 1.00 identical to the crystallised structure of protein database ID 4ASH (Leen et al., 2012) with structure consisted of helix, coil and strand while the C-score is 1.51.

The quality of predicted tertiary structure C139A NS6 protein were identified using Ramachandran plot. The result indicated that the number of residue of the predicted structure in favourable region, allowed region and outlier region is 89.5 %,

8.8 % and 1.7 % respectively (refer Appendix C). The “core” and “allowed” regions are the most favoured regions where they correspond to 10° x 10° pixels having more than 100 and 8 residues in them respectively while there are "generous" regions that is defined as an extending out by 20° (two pixels) all round the "allowed" regions.

Despite this, there is some residues that is located in the space left after these regions and it had been defined as disallowed (Morris, MacArthur, Hutchinson, & Thornton,

1992)

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Figure 4.23 Schematic representation of protein structure. (A) Crystal structure of the active site mutant NS6 protease from murine norovirus 1 with addition of non-native GS sequence at the N-terminus (PDB ID 4ASH) (Refer Appendix D). (B) The predicted structure of C139A NS6 protein from I-TASSER. The 3D structure is viewed as tertiary structure and represented with different colors (yellow: beta strand, purple: α and 3/10 helix, cyan: beta turn, white: coil, red: active site 139, blue: 1st amino acid residue)

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4.6 Binding Simulation (Docking) of Selected Peptide Sequences towards C139A

NS6

Docking analysis was performed using three selected peptide sequences with the highest binding affinity in order to study the protein-protein interaction between those peptides and C139A NS6 protein. The simulation was analysed using CABS-dock web server as described in detail in Section 3.8.2. The docking analysis from the three peptides showed that there were similarities in a few amino acid residues on C139A

NS6 receptor that were lysine, leucine, glutamic acid, serine, valine, glycine, histidine and methionine.

4.6.1 Binding Simulation of 7-mer Peptide ADARYKS on C139A NS6

Peptide ADARYKS bound to C139A NS6 protein receptor at one site, indicated as magenta as shown in Figure 4.24. The value of ligand RMSD (root-mean-square deviation) for the interaction between C139A NS6 receptor and peptide ADARYKS is 3.444. According to contact maps (refer Appendix E), there are a few residues that interact with the peptide on the protein receptor. Amino acids residues on C139A

NS6 protein such as valine and histidine bound mostly to the residue of the ADARYKS peptide. In addition, on ADARYKS peptide, amino acid residue tyrosine made the highest contact with the residue on the protein receptor as compared to other peptide residues. The summary of the peptide-receptor interaction was shown in Table 4.6.

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Figure 4.24 Predicted structure of ADARYKS peptide C139A NS6 receptor complex as predicted by CABS-dock software. Structure highlighted in magenta indicated the peptide ADARYKS bound to C139A NS6 protein. The structure of the proteins C139A NS6 are indicated by the following description: α helix structure (purple); 3/10 helix structure (blue); extended structure (yellow); turn structure (cyan); coil structure (white). Red and green color indicate first and 139th amino acid on C139A NS6 protein respectively.

Table 4.6 Peptide-receptor interactions between ADARYKS peptide and C139A NS6 receptor. ADARYKS Peptide residue C139A NS6 receptor residue ala 1 gly 173, ser 52, thr 50, his 172, ser 51 asp 2 his 172, thr 50 ala 3 his 172, lys 57, val 24 arg 4 leu 73, gly 173, asn 75, glu 174, his 172 tyr 5 val 72, ser 21, leu 73, glu 74, met 71, val 24 lys 6 val 72 ser 7 glu 74, val 72

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4.6.2 Binding Simulation of 7-mer Peptide QTEKNPL on C139A NS6

Peptide QTEKNPL bound to C139A NS6 protein receptor at one site, indicated as magenta colour as shown in Figure 4.25. The value of ligand RMSD (root-mean- square deviation) for the interaction between C139A NS6 receptor and peptide

QTEKNPL is 1.796. According to contact maps (refer Appendix F), there are a few residues that interact with the peptide on the protein receptor. Amino acids residues on

C139A NS6 protein such as serine, histidine and threonine bound mostly to the residue of the QTEKNPL peptide. Other than that, lysine, leucine and valine also show a few interactions with the receptor. The summary of the peptide-receptor interaction was tabulated in Table 4.7.

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Figure 4.25 Predicted structure of QTEKNPL peptide C139A NS6 receptor complex as predicted by CABS-dock software. Structure highlighted in magenta indicated the peptide QTEKNPL bound to C139A NS6 protein. The structure of the proteins C139A NS6 are indicated by the following description: α helix structure (purple); extended structure (yellow); turn structure (cyan); coil structure (white). Red and green color indicate first and 139th amino acid on C139A NS6 protein respectively.

Table 4.7 Peptide-receptor interactions between QTEKNPL peptide and C139A NS6 receptor QTEKNPL Peptide residue C139A NS6 receptor residue gln 1 ser 51, ser 52 thr 2 ser 52, thr 50, his 172, ser 51 glu 3 glu 174, thr 50, gly 173, his 172 lys 4 his 172, lys 57, thr 50, thr 48 asn 5 his 172, leu 73, lys 57 pro 6 lys 57, tyr 59, val 24, leu 73 leu 7 leu 73, val 72, val 20, met 71

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4.6.3 Binding Simulation of 7-mer Peptide NSLKVLG on C139A NS6

Peptide NSLKVLG bound to C139A NS6 protein receptor at one site, indicated as magenta colour as shown in Figure 4.26. The value of ligand RMSD (root-mean- square deviation) for the interaction between C139A NS6 receptor and peptide

NSLKVLG is 3.739. According to contact maps (refer Appendix G), there are a few residues that interact with the peptide on the protein receptor. Amino acids residues on

C139A NS6 protein such as valine, lysine and leucine bound mostly to the residue of the NSLKVLG peptide. Among all peptide residue, leucine at third and sixth positions bind to the highest amino acid residue C139A NS6 protein receptor as compared to other peptide residues. The peptide-receptor interaction was summarized in Table 4.8.

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Figure 4.26 Predicted structure of NSLKVLG peptide C139A NS6 receptor complex as predicted by CABS-dock software. Structure highlighted in magenta indicated the peptide NSLKVLG bound to C139A NS6 protein. The structure of the proteins C139A NS6 are indicated by the following description: α helix structure (purple); extended structure (yellow); turn structure (cyan); coil structure (white). Red and green color indicate first and 139th amino acid on C139A NS6 protein respectively.

Table 4.8 Peptide-receptor interactions between NSLKVLG peptide and C139A NS6 receptor NSLKVLG Peptide residue C139A NS6 receptor residue asn 1 gly 173, his 172 ser 2 lys 57, his 172 leu 3 leu 73, ile 26, val 24, lys 57 lys 4 lys 57, tyr 59 val 5 ser 21, tyr 59, val 24 leu 6 ala 70, leu 73, met 71, val 72 gly 7 glu 74, leu 73

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CHAPTER 5

DISCUSSION

5.1 Production of Recombinant C139A NS6

The expression cassette for MNV active-site mutant NS6 protease (C139A NS6) was constructed by cloning the NS6 protease C139A coding sequence into the expression vector pET26Ub with C-terminal histidine residues. Site-directed mutagenesis was carried out by employing an overlapping PCR in order to introduce a point mutation at amino acid position 139 where the active site of MNV-1 protease is located. The inactive MNV NS6 plasmid construct that incorporates a change from Cysteine to

Alanine coding capacity which knock out the active site nucleophile was performed due to inability to express the wildtype protease. The inactivated version of MNV protease helps to prevent autolysis that causes production of truncated enzyme with reduction in their function (Leen et al., 2012). The production of recombinant proteins that has been expressed using standard pET system through employment of E. coli machinery system as expression host typically yield proteins that consist of formylmethionine that is removed co- and/or post-translationally. Plus, there is also a presence of around 40 % formylmethionine and 50 % methionine as the first amino acid residue of the resulting purified recombinant protein that has been overexpress in

E. coli. However, according to Tan and Board (1996), production of recombinant protein without the necessity to introduce initiation codon at the N-terminus of the protein is possible through expression of the ubiquitin fusion construct in the presence of ubiquitin-specific carboxyl-terminal protease (Ubp1). The presence of pCG1 plasmid that encode for a Ubp1 enhances the production of the authentic protein whereby it capable of recognizing the specific cleavage site upstream of the first amino acid residue of the target protein and co- and/or post-translationally cleaves the fusion

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protein. Production of authentic form of recombinant viral protein is important because study conducted using RNA-dependent RNA polymerase of poliovirus (PV3D) shows that the presence of any substitution or deletion in the residue of the first few amino terminus might lead to loss of the activity as well as production of enzyme with a heterogeneous amino terminus upon high-level expression in E. coli. The authentic form of PV3D with the first amino acid residue of the protein glycine has been successfully produced using this system (Gohara et al., 1999). Thus, the utility of this expression system for production of authentic recombinant protein could also be employed for production of norovirus protease given that the first amino acid residue is alanine instead of methionine. The schematic representation in Figure 5.1 illustrates the cloning strategy used to construct the pET26Ub:MNV C139A NS6 His plasmid.

Figure 5.1 Schematic representation of the expression plasmid pET26Ub:MNV C139A NS6 His. A forward primer which contains the SacII site and part of the N-terminus of the MNV NS6 coding region and a reverse primer which contains the BamHI site. Six times polyhistidine tag was included after the last amino acid residue of NS6. Both primer was used to amplify the full length of C139A NS6 gene upon introduction of a point mutation at the position 139 of protease and the amplicon was subsequently cloned into the pET26Ub plasmid. The cleavage site for the ubiquitin-specific carboxyl-terminal protease is indicated by a vertical arrow.

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The successfully cloned plasmid which carries the C139A NS6 expression cassette was confirmed through sequencing analysis. The obtained results indicated the presence of coding mutation at the position 139 where cysteine has been substituted with alanine. The cloned plasmid was further transformed into E. coli BL21 DE3

(+pCG1) for induction purpose to obtain protein overexpression. The overexpression of the plasmid was performed using IPTG as inducer. Results from SDS PAGE analysis showed that the recombinant protein was successfully overexpressed by distinguishing the intensity of the band produced by expressed protein between induced and non-induced sample. The induced E. coli BL21 carrying the C139A

NS6 expression cassette was further lysed and subjected to protein purification by which the fusion protein is separated according to their affinity towards chelated metal ions that are in the form of insoluble matrix (Winzerling, Berna, & Porath, 1992). The proteins can be readily eluted with buffer containing imidazole or low pH buffer. This purification concept that has been introduced by Porath in 1975 is known as immobilized metal affinity chromatography (IMAC) and is widely used to purify proteins tagged with amino acids such as histidine, tryptophan, and cysteine that are able to form complexes with the chelated metal ions at neutral pH. The recombinant

C139A NS6 protease produced in this study was designed to be cloned into the expression vector pET26Ub with C-terminal histidine. The availability of histidine residues at the C-terminal of the overexpressed C139A NS6 protease will aid in the purification process using IMAC technique since the strongest interaction with metals is showed by the amino acid histidine. This took place upon coordination bonding between the electron donor groups on the imidazole ring in histidine and the immobilized transition metal (Giacometti & Josić, 2013). Furthermore, the versatile purification technique using polyhistidine tag has been established successfully using

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E. coli expression system (Van Dyke, Sirito, & Sawadogo, 1992). In this study, western blot analysis was performed to further identify and confirm the C-terminally his-tagged C139A NS6 protease from complex mixture of proteins extracted from E. coli cells. Simply, the technique that initially separated the protein based on molecular weight using SDS PAGE was then transferred to a solid support and subjected for marking using proper antibody to visualise the target proteins (Mahmood & Yang,

2012). The use of animal derived antibody rabbit polyclonal anti-His tag antibody and primary goat anti ProPol anti serum confirmed the presence of recombinant C139A

NS6 protease that has been tagged with histidine. However, the results indicated multiple bands of few different products in western blot analysis that has been probed with anti ProPol anti serum. This observation might probably due to the nature of polyclonal antiserum that are not purified.

5.2 Optimisation Parameters for Peptide Phage Selection

Several optimisations were carried out prior to panning against the target proteins. The optimised condition such as growth time to achieve mid-log phase of E. coli ER2738, peptide phage concentration in the Ph.D-7 Phage Display Peptide

Library kit and the concentration of proteins required to be coated on ELISA plate were determined and used in the subsequent biopanning against target proteins.

5.2.1 Optimum Growth Time for E. coli ER2738 Culture

In order to determine the optimum growth time for M13 phage bacterial host, the growth curves of ER2738 were prepared based on absorbance reading at OD600 from two cultures. The first ones were from dilution of ER2738 overnight culture in

LB broth according to 1:100 ratio and the second ones was from inoculation of a single colony of ER2738 grown overnight on LB + Tet plate. Result obtained shows that the diluted overnight culture took only two hours to achieve mid-log phase as compared 117

to the single colony culture where it took around five hours. It is important to obtain optimum growth curve at which the bacterial reach mid-log phase (OD600 ~ 0.5) because at this stage, phage infection is maximize due to high number of bacterial cells. During this stage, bacterial grow rapidly and the number is doubled as compared to stationary-phase where bacterial cultures tend to lose F' episomes that is important for this strain of bacteria to be infected by the phage (Sambrook et al., 1989). Study conducted indicated that the exponential phase of E. coli growing in Luria-Bertani broth ends when the OD600 is between 0.6 and 1.0 (Sezonov, Joseleau-Petit, & D'Ari,

2007). The mid-log phase of bacterial cell growth is selected for phage infection in this study as it is an established condition that has been used in a few studies for selection of peptide phage that is specific towards target protein. To date, there were several studies of phage display and selection that used OD600 ~ 0.5 (Kramer et al.,

2003; Kushwaha, Schäfermeyer, & Downie, 2014; Larsen, Meldgaard, Lykkemark,

Mandrup, & Kristensen, 2015; Wang et al., 2016). Therefore, the diluted overnight culture of ER2738 was used throughout the selection for each round of panning to shorten the duration of the experiment.

5.2.2 Peptide Phage Concentration in the Ph.D-7 Phage Display Peptide

Library Kit

The quantification of the number of phage in the library is important prior to panning due to certain requirement of phage number for biopanning. The amount of phage was determined through phage titration and the amount is calculated based on plaque forming unit per ml (pfu/ml). Phage titration is carried out using plaque assay that is the most commonly and widely used method for titration of M13 phage

(Hertveldt et al., 2009). The result showed that the concentration of phages is 3.675 X

1013 pfu/ml which is similar to the original concentration given by the manufacturer.

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The number of phage is sufficient to be used as the input phage because according to the recommended protocol by NEB and others (’t Hoen et al., 2012), 2 X 1011 pfu/ml is needed for input phage for each round of panning.

5.2.3 Optimum Concentration of Target Proteins on ELISA Plate

Prior to biopanning against target proteins, ELISA was carried out to determine the optimum concentration of both streptavidin and C139A NS6 proteins required to be coated on plate. The optimization procedure is performed to ensure that the sufficient proteins are used and they should coat most of the surface of the ELISA.

During coating, hydrophobic interaction is formed between protein and the surface of plates which will then lead to adsorbtion of protein forming a monomolecular film.

The amount of protein bound to the surface at low concentration of protein is proportional to its concentration in the solution while constant amount of protein is bound regardless of its concentration upon reaching saturation level (Kenny &

Dunsmoor, 1983). The fully coated plate surface will increase the successfulness and confidence in isolating specific peptide phage for target proteins throughout the panning process.

5.3 Peptide Phage Library Panning against Streptavidin

Initially, panning against streptavidin was carried out before proceeded to the selection against C139A NS6 protease in order to validate the Ph.D-7 Phage Display

Peptide Library kit. Validation of the library is important to ensure that the phage display library is reliable in isolating specific peptide for the target protein. In this study, streptavidin was used as a target protein to validate the library due to its tetrameric structure with four potential binding sites that have been well characterized in previous studies. Other than that, the interaction of biotin-streptavidin has made them as one of the known strongest noncovalent biological interaction (Weber, 119

Ohlendorf, Wendoloski, & Salemme, 1989). Due to their strength and specificity in term of interaction, they are most widely used affinity pairs in the field of molecular, immunological, and cellular assays (Holmberg et al., 2005).

The availability of four biotin binding sites on the surface of streptavidin with high affinity enable the capturing approach to be widely used in biopanning to select all specific streptavidin phages (Vodnik, Zager, Strukelj, & Lunder, 2011; Weber et al., 1992). The most recognized and established tripeptide motif that are specific for streptavidin is histidine-proline-glutamine (HPQ) and also there are various studies shows the present of HPQ motif at different locations within the amino acid sequence of the isolated peptides from various selection against streptavidin (Giebel et al., 1995;

Katz, 1995; Weber et al., 1992). There are also established studies which identified other streptavidin binding peptides without HPQ motif such as peptide with consensus sequence HPM (Gissel et al., 1995), EPDW(F/Y) (Caparon, De Ciechi, Devine, Olins,

& Lee, 1996), GD(F/W)XF and PWXWL (Roberts, Guegler, & Winter, 1993).

The panning procedure was carried out according to manufacturer’s protocol with a few modifications. Firstly, a negative selection was performed to eliminate binder phage with no actual affinity toward the targets such as subtractive panning against plastic binders and BSA phage binders respectively (Aina, Marik, Liu, Lau, &

Lam, 2005; Tang, Li, Huang, Zheng, & Li, 2013). This is due to possibility of phage binding towards other components of the screening system that may predominate during earlier rounds of biopanning. There are phage clones that displayed certain peptides with greater affinity for plastic surfaces by which that clone able to adhere to the plastic surface due to their ability of withstanding washing steps and are subsequently amplified (Menendez & Scott, 2005; Vodnik et al., 2011). However, it does not necessarily to exclude the ability of phage binders to bind certain targets even

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though it binds to plastic (Rambert et al., 2009). Secondly, the use of BSA during panning could potentially give rise to false positive result due to the nature of albumin that display various binding sites on its surface (Vodnik et al., 2011). Therefore, in this study we did carry out subtractive panning against plastic and BSA at the initial round of biopanning before exposing the selected phage towards the C139A NS6 protease.

In each round of panning, Tween-20 was included in washing buffer with increasing concentration. This detergent helps to reduce non-specific hydrophobic interactions between binding ligands and panning target (Wakayama, Sekiguchi,

Akanuma, Ohtani, & Sugiyama, 2008). The specific binding is usually resistant to

Tween-20 as compared to non-specifically bound proteins that can be prevented from binding or easily washed out. The stringency of washing was increased gradually from low concentration to higher concentration in order to prevent loss of peptide phages that maybe specific to target protein being washed away and to ensure that all the non- specific target peptide phages were eliminated respectively. It is necessary to increase the stringency of washing step to select for phage clones that signify higher affinity or specificity towards the target (Frei & Lai, 2016).

Multiple round of selection was performed to ensure the selected phage is enriched throughout panning as well as to reduce the background of non-specific binders (’t Hoen et al., 2012). It is important to monitor phage enrichment throughout rounds of panning so that phage that was isolated after each round of panning is actually specific towards streptavidin and as indicator that selection process is working succesfully. The enrichment was observed based on the input and output of the titration of phage of the successive round of panning (Frei & Lai, 2016). From the selection against streptavidin, 315-fold of enrichment was seen after third round of panning. The phage enrichment with ratio of 10-fold or more is considered as ideal during later

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rounds of panning whereas little enrichment is observed in early round where only small population of phage that was specific is present (Frei & Lai, 2016). This event is due to elimination of high portion of non-specific phage during washing steps throughout the early rounds of panning since phage that display proteins that specific to target will remain. The repetition of panning process will ensure that phage that is specific to target protein is enriched as the same clones is amplified for multiple times while the undesired materials are removed.

In this study, twenty single phage clones were isolated, amplified and purified from the fourth round of panning. The single clones in the form of blue plaque that were selected from LB/IPTG/XGAL plate must be distinct and far apart from each other to ensure a homogenous DNA is present. The purified DNA that were extracted from the clones using PEG precipitation was then subjected to PCR using AY25M13F and AY25M13R. PCR analysis was carried out to amplify the region of genomic DNA that contained randomised peptide sequences and it was observed that all the clones carry the randomised peptide sequence with a product size of 180 bp. There is possibility of identifying clones with lower than 180 bp in size as this condition indicated the absence of randomised peptide sequences due to wild type M13 phage contamination from the surrounding.

As round of selection/panning progressing, there are higher probability of emerging of the library members. However, upon several rounds of selection, there is convergence of sequence to a consensus (Frei & Lai, 2016). Out of 20 DNA clones that were sequenced, 10 clones showed streptavidin motif HPQ. There are four different sequence carrying HPQ motif at the same position from the positive 10 clones showing streptavidin-binder motif. The obtained peptide sequences of streptavidin clones from the analysis shows no similarities with the existing sequences available in

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databases when they were scanned through PepBank, SAROTUP (Scanner and

Reporter of Target-Unrelated Peptides) and MimoDB (Mimotope Database and

Beyond). These are specific databases and computational tools that are important for phage display work and is widely used for detection of peptide similarities (Huang,

Ru, & Dai, 2011). The screening against database is important to aid the possibility of identifying a false positive result during the phage display selection (’t Hoen et al.,

2012).

5.4 Binding Kinetics of Selected Peptide Phage towards Streptavidin

Binding specificity of streptavidin phage was accessed using ELISA technique that is a reliable and rapid way to confirm the binding specificity of selected population of phage to the target of interest. This technique that was introduced by Engvall and

Perlmann is able to determine quantitative or semiquantitative data on the concentration of a certain antigen (Engvall & Perlmann, 1971). For validation purpose in our study, clone 9 that carries HPQ motif was selected to be used in ELISA while clone 7 that does not carry HPQ motif was selected as irrelevant phage control. On the other hand, BSA was also used as a protein control. The OD450 reading for clone 9

7 started to increase at the phage concentration of 10 pfu/ml with Kd value of 5.772 X

109 pfu/ml. However, for clone 7, the signal started to increase at phage concentration

9 11 of 2 X 10 pfu/ml with Kd value of 2.672 X 10 pfu/ml. This observation suggests that clone 9 are specific binder to streptavidin as they can fill in the binding sites on streptavidin even at low amount. Clone 9 also show low Kd value, which means it has a high affinity interaction since it takes less ligand to fill half the proteins (Lambert,

2004).

Increasing in the ELISA signal at OD450 was observed when clone 7 was incubated with streptavidin. Indeed, such increment was also seen in clone 9 and clone

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7 when they were incubated with BSA. This observation might be due to the non- specific binding of phage towards streptavidin especially when they are used in abundance. Published studies showed that non-specific phage binding during ELISA was encountered when using non-ionic detergent such as Tween-20 that able to interact with the PEG-precipitate phage (Hakami, Ball, & Tarr, 2015; Kenna, Major, &

Williams, 1985). Therefore, we conclude that this ELISA analysis indicated that the

HPQ phage clones were specific for streptavidin. This finding also proves the ability of phage display peptide library to select specific peptides for any target protein; thus, validating the library. The validated library was then subjected for selection against target protein, C139A NS6 to identify specific peptide for the protein.

5.5 Peptide Phage Library Panning against C139A NS6 Protein

Development of small molecule inhibitors that efficiently able to inhibit norovirus infection could act as a good strategy to control the outbreak. There have been few studies that work on small molecule that has potential in inhibiting norovirus replication. These includes in vitro anti-norovirus activity of the protease inhibitor rupintrivir (Rocha-Pereira, Nascimento, et al., 2014), the use of commercially available protease inhibitors chymostatin ( Chang, Takahashi, Prakash, & Kim, 2012), a novel set of triazole-based macrocyclic inhibitors ( Weerawarna et al., 2016), a series of optimized dipeptidyl inhibitors for norovirus protease (Arias, Emmott, Vashist, &

Goodfellow, 2013; Galasiti Kankanamalage et al., 2015; Kim et al., 2012) and designation of peptide-mimetics that effectively inhibit protease activity (Deng et al.,

2013; Muhaxhiri et al., 2013).

Instead of chemical compound based inhibitors, viral protease inhibitor could also be develop based on peptides that possess a high affinity and specific interaction

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for this viral enzyme (Hyde-DeRuyscher et al., 2000). This method has been used against HCV NS5B and successfully identified peptides with inhibitory effects (Amin et al., 2003; Kim, Park, Lee, & Myung, 2008). In this project, phage display technology was use as a selection technique where libraries of filamentous phage clones which carry a short-randomised peptide sequence was subjected for screening against immobilized target protein. Once the library was successfully validated, the library was used in panning against C139A NS6 protein.

Phage display technique was selected as compared to other selection technique such as aptamers due to its advantage. The nature of target protein C139A NS6 that is an enzyme; has made the use of phage display more convenient as compared to aptamer. This is because phage display is a protein based technique while aptamer is a DNA/RNA based technique. Both methods that are cyclic repetitive process use randomly created library that primarily aim to reduce the number of combinatorial molecules into manageable amount once they are subjected for screening against target protein (Mascini, Palchetti, & Tombelli, 2012; Ruigrok, Levisson, Eppink, Smidt, &

Van Der Oost, 2011). The application of small peptides and structured nucleic acid have contributed to various purposes including for diagnostic, therapeutic as well as helping in understanding the cell biology. They can be modified in term of their half- life and can also be associated with other molecules in order to increase their potential use (Tonelli, Colli, & Alves, 2013). The advantage of phage display selection is in their ability to identify strong binding candidates within 3-10 cycles and since they use

E. coli for amplification, their selection process is considered more efficient and convenient with the inherent flexibility (Reverdatto, Burz, & Shekhtman, 2015;

Sergeeva, Kolonin, Molldrem, Pasqualini, & Arap, 2006). On the other hand, aptamer needs higher number of selection round between 8 to 20 times to enrich the selected

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DNA. In addition, the amplification of selected DNA in aptamers use PCR or RT-PCR which is quite tedious in term of cost and time as compared to phage display technique

(Blind & Blank, 2015).

Phage display has been successfully used in several applications including autoantibody profiling approach using serological antigen selection (SAS) (Cleutjens et al., 2008; Somers et al., 2002), diagnostic approach to detect metastatic tumour cells in blood (Jia et al., 2007; Rasmussen, Schreiber, Schultz, Mischler, & Schughart,

2002), identification of peptide agonists and antagonists for receptors (Sidhu, 2000), mapping of carbohydrates and protein functional epitopes (Fukuda, 2012; Sidhu,

2000), vaccine development (Lidqvist, Nilsson, Holmgren, Hall, & Fermér, 2008), selection of antibodies recognizing post-translational modifications (Kehoe et al.,

2006), molecular recognition using fluorescently labelled phage (Petrenko, 2008) and detection of biological threat agents (Petrenko & Vodyanoy, 2003).

The commercially available Ph.D.-7 system by NEB consist of a combinatorial library of randomised heptapeptide that was used throughout the selection process in this study. This library consists of randomized linear 7-mer peptides that fused to the minor coat protein pIII of M13 phage via a short flexible linker sequence Gly-Gly-

Gly-Ser. The displayed peptide is expressed at the N-terminus of pIII where the first residue of the fusion protein is the first randomized position of the 7-mer peptide. The library contains 109 independent clones, which is sufficient to encode most, if not, all the 1.28 X 109 possible 7-residue amino acid combinations. According to manufacturer, Ph.D.-7 library may be the most useful for targets requiring binding elements concentrated in a short stretch of amino acids. Shorter peptide libraries can lead to ligands with higher specificity when compared to longer libraries. This is because increased in the length of the randomized segment may allow target to select

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sequences with multiple weak binding contacts, instead of a few strong contacts (New

England Biolabs, 2018).

The purified recombinant C139A NS6 protein, prepared earlier in this study was used as target throughout the biopanning experiment instead of using cells expressing higher levels of protease. This is because, cells will have a lot of receptors that theoretically capable of pulling a specific ligand out of the library. Thus, these receptors may interrupt the specificity of the panning since they may yield a complex mixture of peptides with no clear consensus thus decreasing the chance of getting the specific peptide phage. Therefore, by targeting immobilised C139A NS6 protein on a plate, the chance of getting potential C139A NS6-specific peptide phage is higher.

Other than that, general elution buffer that is non-specifically disrupt the binding interactions can result in the isolation of all phage in the mixture including the one that specifically bind towards C139A NS6 protein and other components in the well of the ELISA plate (NEB, 2018)

Analysis showed that out of 98 peptide sequences, several sequences are repetitive, resulting in identification of 38 unique peptide sequence. Moreover, there is also presence of TAG stop codon that was considered as glutamine due to suppression of TAG stop codon by glutamine in ER2738 (glnV) strain. A few specific motif were identified such as Glu-Lys-Asn (EKN) motif, Asp-Ala-Arg (DAR) motif,

Tyr-Lys-Ser (YKS) motif and Lys-Asn-Pro (KNP) motif. Furthermore, the peptide sequence also indicated some variation according to their respective position from 1st position to 7th position. Three amino acid residues that appear several times in different sequence contexts is a common observation and it seem that this tripeptide motif provide the minimal framework for structural formation and protein-protein interaction (Vendruscolo, Knowles, & Dobson, 2011). Hence, it is obvious that 127

stronger interaction may be observed in repeated peptides consisting of more residues.

In addition, this is the first study using C139A NS6 protein as a target in peptide phage display selection. Confirmation on the binding affinity of the selected clones on

C139A NS6 protein was further verified by ELISA.

5.6 Binding Kinetics of Selected Peptide Phage towards C139A NS6

ELISA was performed to determine the binding specificity of selected five single clone phages towards C139A NS6 protein. Clones ADARYKS, QTEKNPL,

NSLKVLG, VFQTTYK and MPRLPPA were selected for ELISA while C9 with HPQ motif was selected as a control phage. Streptavidin, BSA and other recombinant protein (NS7) were used as an irrelevant protein control. Different concentrations of each phage clones starting from 2 X 105 pfu/mL to 2 X 1011 pfu/mL were incubated on 96-well plate coated with 100 µg/ml of C139A NS6 protein and control proteins

(C9, streptavidin, BSA and NS7).

Clones QTEKNPL showed highest binding ability towards C139A NS6 protein as compared to other clones while the control phage C9 and clone MPRLPPA have low binding ability towards target protein C139A NS6 suggesting that these clones have a lower binding affinity towards C139A NS6 protein. The increment in the signal of the ELISA for C9 at higher phage concentration might be due to the non- specific binding of phage towards target protein especially when they are in abundance. As mentioned before, the non-specific phage binding during ELISA is facilitated by non-ionic detergent that interacted with the PEG-precipitate phage

(Hakami et al., 2015). ELISA analysis also showed that exposure of all five selected clones towards other proteins (BSA, Streptavidin and NS7) only produced a

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background level of ELISA signals, indicating that the selected clones are only specific towards C139A NS6.

Dissociation constant (Kd) is an equilibrium constant which measures separation of larger objects dissociating into smaller components at equilibrium. It is commonly used to describe the interaction between a ligand and a protein in term of their binding affinity as well as to indicate how tightly the ligand binds to the protein.

The smaller the dissociation constant, the tighter the ligand bind; meaning higher affinity of ligand towards the protein. The dissociation constant has a molar unit (M) which indicates the concentration of ligands at which the binding site on C139A NS6 protein is 50 % occupied. Since phage clone is titrated, they are being quantified as pfu/ml unit instead of M. The quantification in M unit is possible by synthesizing the sequence of phage clone. Example of studies that indicated higher affinity of ligand- protein interactions are biotin-avidin and, also ribonuclease inhibitor-ribonuclease

-15 where both showed very small Kd values of 10 M (Johnson, McCoy, Bingman,

Phillips, & Raines, 2007; Livnah, Bayer, Wilchek, & Sussman, 1993). Significant changes of Kd values may be influenced by several factors such as temperature, pH and salt concentration that effectively modify the strength of intermolecular interaction of ligand-protein complex.

Studies on drug binding affinity towards receptor have widely used Kd and Bmax values where Bmax represents the number of binding sites in the assay (Akera & Cheng,

1977). Based on statistical data analysis, interaction between clone QTEKNPL and

9 C139A NS6 protein showed a lower Kd value which is 3.493 X 10 pfu/mL as compared to other clones. This mean that only 3.493 X 109 pfu/mL of clone QTEKNPL phage is required to bind to half of the receptor binding sites on C139A NS6 protein.

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This also suggests that among all the selected clones, clone QTEKNPL binds more tightly and has higher affinity towards C139A NS6 protein.

5.7 Prediction of Protein Interaction between Selected 7-mer Peptide

Sequences and C139A NS6 Protein

5.7.1 Structure Prediction of C139A NS6 using I-TASSER

Protein molecules are composed of a sequence of amino acids from 20 different amino acid residues where each of these residues has their own properties and features.

The unlimited combination of these residues created infinite number of proteins with numerous capability and functions. To understand the protein biological function, it is necessary to determine its three-dimensional (3D) structure that denote the arrangement of atoms in a protein molecule. However, the 3D structure determination is challenging; thus, is being solved by the intermediate step which is the prediction of the secondary structure. Basically, protein structure is divided into four level hierarchy namely primary structure, secondary structure, tertiary structure and quaternary structure (Buxbaum, 2007; Khalatbari & Kangavari, 2015). In this study, the predicted structure is almost identical with the crystallised structure deposited in protein database. The C-score value signifies higher confidence since typically it is in the range of [-5, 2], where higher value signifies a model with a higher confidence and vice-versa. Other than that, the selected model is basically a good model since the indicative of good model will generally have 90 % of its residue in the favoured regions of the Ramachndran plot (Laskowski, MacArthur, Moss, & Thornton, 1993).

5.7.2 Binding Simulation of Selected 7-mer Peptide Sequences on C139A NS6

Nowadays, peptide-based drugs have been constantly in great demand because they are highly selective, effective, easily tolerated and relatively safe compared to

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other class of drugs. Therefore, effective tools and strategies for computer-aided peptide modelling are in the need since they provide knowledge and establish new routes of rational peptide design (Fosgerau & Hoffmann, 2015; Tsomaia, 2015).

Bioinformatics tools, primarily molecular docking, are used to characterize the protein-peptide interactions as it is easy to identify and predict sites of protein interaction based on deposited databases and algorithms. The output can serve as a basic knowledge to validate any protein interaction prior to any experiment in the lab.

CABS-dock is a free web server that provides an interface for modelling protein–peptide interactions to discover new drugs (Ciemny et al., 2017). In this experimental design, binding simulation of all the selected 7-mer peptide sequences to

C139A NS6 was performed using CABS-dock. In CABS-dock single simulation run, the input peptide that is fully flexible will scan through the overall surface of flexible protein receptor without the need to use any information about the shape and location of the binding site on the surface of the protein receptor whereas other docking algorithm such as Rosetta FlexPepDock (London, Raveh, Cohen, Fathi, & Schueler-

Furman, 2011), AutoDock (Morris et al., 2009) and HADDOCK (Trellet, Melquiond,

& Bonvin, 2013) requires pre-defined localisation of the binding site.

The predicted protein structure and each selected peptide sequences were used as the input for CABS-dock. Then, flexible docking was carried out based on 10 different trajectories resulting in 10,000 models. The models are filtered base on interaction energy between peptide and the receptors where zero interaction energy was eliminated. After filtering, up to 100 from each of the 10 trajectories are picked by the lowest interaction energy. After that, 10 clusters were formed from the 1000 filtered models using structural clustering. For each of the cluster, one model representative was selected. The model was ranked 1-10 according to their cluster

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density (Blaszczyk et al., 2016). One representative model in the most density cluster is selected as the final structure of each 7-mer peptide sequences and C139A NS6 receptor complex. The model is selected based on the lowest RMSD value. The quality of docking models were access using ligand RMSD (root-mean-square deviation). The

RMSD is calculated on the peptide only, after superimposition of the receptor structures.The results from the docking of selected peptides and C139A NS6 protein indicated that peptide QTEKNPL has the high quality of prediction as compared to the other two with medium quality of prediction. This indication is based on the range of the RMSD values that has been set as high-quality prediction: RMSD < 3 Å; medium quality prediction: 3 Å ≤ RMSD ≤ 5.5 Å; low-quality prediction: RMSD > 5.5 Å

(Kurcinski, Jamroz, Blaszczyk, Kolinski, & Kmiecik, 2015)

Docking results that were analysed using contact map given by CABS-dock shown that the peptide sequence QTEKNPL give the highest contact number between the receptor and peptide as compared to other two selected 7-mer peptide sequences.

The analysis is in parallel with the binding kinetic shown from the ELISA where it remarks that the binding kinetic of QTEKNPL peptide shows the highest binding ability towards C139A NS6 protein. In addition, even though peptide residue at 4th position of both peptide QTEKNPL and NSLKVLG are the same, they do not show the same number of contact on the protein receptor. This may be due to position of those residues from the binding site which make them indirectly affected and experienced a certain degree of conformational changes upon binding of peptide. In addition, there might be some variation in the protein-peptide interaction due to

CABS-dock procedure itself that employ a Monte Carlo-based algorithm, which may lead to different results in different runs (Chang et al., 2012; Ciemny et al., 2017).

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Also, there is probability of results that are consistent for each run as well as those with qualitatively distinct predictions (Kurcinski et al., 2015)

All the interaction between the peptide and receptor are non-covalent interactions that includes hydrogen bond, van der Waals interaction or hydrophobic

(stabilization of very non-polar ligand) and the electrostatics interaction. Theoretically, when two molecules are docked, the interaction between them are non-covalent instead of covalent. This situation enables transient interaction that are non-permanent or reversible, which will provide equilibrium constant for the formation of the protein- ligand complex. Complex formation is form through non-covalent interaction while covalent interaction lead to chemical reaction (Kumalo, Bhakat, & Soliman, 2015)

Based on the published studies that work on inhibition of norovirus protease, the identified regions on protease that have been determined from this study do not overlap with any of the residue that interact with the inhibitors from the previous studies (Chang et al., 2012; Muhaxhiri et al., 2013; Rocha-Pereira, Nascimento, et al.,

2014; Weerawarna et al., 2016). This suggests that the selected clone identified in this study is a C139A NS6-binding phage clone with unique binding domains that has never been published before. The identification of these domains may potentially be used or manipulated in the development of peptide inhibitor against NS6 protease as well as for the diagnostic purposes, which can eventually be used as an intervention to control norovirus replication.

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CHAPTER 6

CONCLUSION AND FUTURE RECOMMENDATIONS

6.1 Conclusion

In this study, C139A NS6 was successfully cloned, expressed and purified. The peptide-bearing phage Ph.D-7 mer system against recombinant mutant NS6 protease was successfully selected through biopanning assay. The specificity of the binding between selected peptide phages and target proteins were successfully accessed through ELISA. The clones have higher affinity towards C139A NS6 compared to other irrelevant control peptide phages when tested using ELISA. In order to identify the exact location of selected clones on C139A NS6, bioinformatics analysis was performed. Results from this bioinformatics analysis is important as it has given us the basic and fundamental knowledge for future investigation in ligand-receptor interaction involving selected clones and C139A NS6.

6.2 Recommendations for Future Research

More investigations are warranted to verify this ligand-receptor interaction. Although

ELISA and computational method using protein docking have been successfully performed, addition of other specific in vitro analysis using biophysical and theoretical methods such as dynamic light scattering (DLS), X-ray diffraction or fluorescence microscopy techniques may further confirm the binding characteristics. For instance, a synthetic peptide, can be synthesised as a free, soluble peptide. This allows precise control of peptide concentration in binding and inhibition studies. Additionally, without the phage attached, the peptide can be used at much higher concentrations and can be used in-vivo. Furthermore, peptides are easier to be synthesised in larger scale.

The ability of such synthetic peptide to bind C139A NS6 receptor specifically may

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provide a possibility of developing small peptide inhibitor against NS6 protease as well as for the diagnostic purposes, which can eventually be used as an intervention to control norovirus replication.

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APPENDICES

APPENDIX A

RESULT OF DNA SEQUENCING

PET26UbNS6

(sacII)CCGCGGTGGAGCCCCAGTCTCCATCTGGTCCCGTGTTGTGCAGTTCGG CACGGGGTGGGGCTTTTGGGTGAGCGGCCACGTCTTCATCACCGCCAAGCAT GTGGCGCCCCCCAAGGGCACGGAGATCTTTGGGCGCAAGCCCGGGGACTTC ACTGTCACTTCCAGCGGGGACTTCTTGAAGTACTACTTCACCAGCGCCGTCAG GCCTGACATTCCCGCCATGGTCCTGGAGAATGGGTGCCAGGAGGGCGTCGT CGCCTCGGTCCTTGTCAAGAGAGCCTCCGGCGAGATGCTTGCCCTGGCTGTC AGGATGGGTTCACAGGCCGCCATCAAGATTGGTAGTGCCGTTGTGCATGGG CAAACTGGCATGCTCCTGACTGGCTCTAATGCCAAGGCCCAGGACCTCGGGA CCATCCCGGGCGACGCAGGCTGTCCCTATGTTTATAAGAAGGGTAACACCTG GGTTGTGATTGGGGTGCACGTGGCGGCCACTAGGTCTGGTAACACAGTCATT GCCGCCACTCACGGAGAACCCACACTTGAGGCTCTGGAGTTCCAG GGATCC(bamHI)

APPENDIX B

BINDING ABILITY OF PEPTIDE-BEARING PHAGE

Binding Ability of Clone QTEKNPL against NS6 protease C139A, BCA, Streptavidin and NS7 3.000 2.500 2.000 1.500 1.000

0.500 Absorbance at 450(nm) 0.000 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage concentration (pfu/ml)

C139A BCA Streptavidin NS7

Binding Ability of Clone ADARYKS against NS6 protease C139A, BCA, Streptavidin and NS7 3.000 2.500 2.000 1.500 1.000

0.500 Absorbance at 450(nm) 0.000 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage concentration (pfu/ml)

C139A BCA Streptavidin NS7

Binding Ability of Clone NSLKVLG against NS6 protease C139A, BCA, Streptavidin and NS7 2.500

2.000

1.500

1.000

0.500 Absorbance at 450(nm) 0.000 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage concentration (pfu/ml)

C139A BCA Streptavidin NS7

Binding Ability of Clone VFQTTYK against NS6 protease C139A, BCA, Streptavidin and NS7 1.200 1.000 0.800 0.600 0.400

0.200 Absorbance at 450(nm) 0.000 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage concentration (pfu/ml)

C139A BCA Streptavidin NS7

Binding Ability of Clone MPRLPPA against NS6 protease C139A, BCA, Streptavidin and NS7 0.400 0.350 0.300 0.250 0.200 0.150 0.100

0.050 Absorbance at 450(nm) 0.000 2 X 10⁵ 2 X 10⁶ 2 X 10⁷ 2 X 10⁸ 2 X 10⁹ 2 X 10¹⁰ 2 X 10¹¹ Phage concentration (pfu/ml)

C139A BCA Streptavidin NS7

APPENDIX C

RESULT OF RAMACHANDRAN PLOT

Data for Ramachandran plot generated by RAMPAGE (Mordred.bioc.cam.ac.uk, 2017)

APPENDIX D

SEQUENCE OF CRYSTAL STRUCTURE (PDB ID 4ASH)

>4ASH:B|PDBID|CHAIN|SEQUENCE GSAPVSIWSRVVQFGTGWGFWVSGHVFITAKHVAPPKGTEIFGRKPGDFTVTS SGDFLKYYFTSAVRPDIPAMVLENGCQEGVVASVLVKRASGEMLALAVRMGSQ AAIKIGSAVVHGQTGMLLTGSNAKAQDLGTIPGDAGCPYVYKKGNTWVVIGVH VAATRSGNTVIAATHGEPTLEALEFQ

APPENDIX E

CONTACT MAPS OF PEPTIDE ADARYKS ON C139A NS6 PROTEIN

APPENDIX F

CONTACT MAPS OF PEPTIDE QTEKNPL ON C139A NS6 PROTEIN

APPENDIX G

CONTACT MAPS OF PEPTIDE NSLKVLG ON C139A NS6 PROTEIN

LIST OF AWARDS, CONFERENCES AND TRAINING

1) Yayasan Khazanah Scholarship Khazanah Watan 2015-2018

2) Occupational safety and Health Course 10th October 2015 Lecture Hall X, USM

3) 3rd Advanced Medical & Dental Institute International Biohealth Science Conference 2018 19 – 20 January 2018 Riverside Majestic Hotel, Kuching, Sarawak Theme : Emerging Infectious Diseases

4) Viruses 2018 7 – 9 February 2018 Faculty of Biology, University of Barcelona, Barcelona, Spain Theme : Breakthroughs in Viral Replication

5) 4th IPPT Postgraduate Colloquium 7 – 8th August 2018 Seminar Room, animal Research Centre, IPPT, USM Theme : Cultivating Future Young Scientists

1) Yayasan Khazanah Scholarship

2) Occupational safety and Health Course

3) 3rd AMDI, International Biohealth Science Conference 2018

Identification of phages-bearing peptide that bind specifically to murine norovirus 1 (MNV-1) NS6 protease using phage display technique Nur Sakinah Soid, Ida Shazrina Ismail, Kumitaa Theva Das, Muhammad Amir Yunus Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia (USM), Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia Purpose: Norovirus is one of the leading causes of gastroenteritis worldwide. The importance of the enzymatic activity possess by the norovirus NS6 protease has made it as an attractive target for developing antiviral drug. In this study, we identified several peptide phages that bind to the active site mutant (C139A)-recombinant MNV-1 NS6 using phage display technique. Small peptides could subsequently be further developed as a novel inhibitor against norovirus NS6 and their efficiency to act as antiviral therapy could be further assessed. Methods: Initially, C139A NS6 was cloned, expressed and purified. Then, Ph.D-7TM Phage Display Peptide Library (NEB) was used in bio-panning assay for 6 rounds against C139A NS6. In those bio-panning, bound phages were eluted using general elution buffer. A total of 98 phage clones were selected and peptide sequences were identified. The binding interaction of peptides towards C139A NS6 and wildtype NS6 protease were assessed through docking analysis. Results: The C139A NS6 was successfully cloned, expressed and purified. The sequence analysis from 6 rounds of panning showed similar peptide sequences in a few selected phage clones. The identified peptide sequences are ADARYKS, QTEKNPL and NSKLVLG. Docking analysis showed that none of the identified peptide sequences bind to the amino acid residue at position 139 of the NS6 protein. Conclusion: Peptide phages with binding activity towards the C139A NS6 have been identified via bio-panning from the library. Further analysis on specificity, affinity and inhibitory effects are warranted. Keywords: phage display, murine norovirus 1, NS6 protease

4) Viruses 2018

5) 4th IPPT Postgraduate Colloquium

Phage display targeting MNV-1 NS6 Protease Nur Sakinah Soid1, Ida Shazrina Ismail2, Kumitaa Theva Das1, Muhammad Amir Yunus1 1 Infectomics Cluster, Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia (USM), Bertam, Kepala Batas, Penang, 13200, Malaysia; 2 Regenerative Medicine Cluster, AMDI, USM, Bertam, Kepala Batas, Penang, 13200, Malaysia

Norovirus infections are considered the most common causes of epidemic and sporadic cases of acute gastroenteritis worldwide. To date, there are no licensed therapeutic intervention measures either in terms of vaccines or drugs available for these highly contagious human pathogens. Targeting viral non-structural protein such as protease is relevant due to their well conserved regions along with its critical functions in viral replication. In this study, validated Ph. D-7TM Phage Display Peptide Library (NEB) was used in selection against purified C139A NS6. The selection has successfully isolated and identified peptide phage that are specific towards C139A NS6. Upon identification of peptide phage, the peptide sequences were screened using online peptide databases and results indicated that all the clones are unique and do not have common motif with any published peptides. A few clones with highest number of hits were tested in term of their binding ability using ELISA. It has been observed that clone QTEKNPL has the highest binding affinity towards C139A NS6 as compared to other clones and irrelevant phage control. To identify the exact location of selected clones on C139A NS6, binding simulation using molecular docking was performed. Docking analysis showed that all the selected clones interact with C139A NS6 receptor at several putative contacts on the receptor binding sites. The results from this bioinformatics analysis is important as it has given us the basic and fundamental knowledge for future investigation in ligand-receptor interaction involving selected clones and C139A NS6 protein. Thus, the identification of these contact sites may potentially be used or manipulated in the development of small peptide inhibitor against NS6 protease as well as for the diagnostic purposes, which can eventually be used as an intervention to control norovirus replication. Keywords: phage display, murine norovirus 1, NS6 protease, molecular docking.