STRUCTURAL and BINDING CHARACTERIZATION of the ANTIVIRAL HOST PROTEINS, VIPERIN and VAPC

STRUCTURAL AND BINDING CHARACTERIZATION OF THE ANTIVIRAL HOST PROTEINS, VIPERIN and VAPC

SHAVETA GOYAL ( M.Sc. Biotech )

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOCHEMISTRY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE (2012)

Acknowledgments

The time period between Jan 2008 to Jan 2012 will be one of the most memorable periods of my life. I have learnt a lot during this period. These four years added into me the scientific temperament, which is the foremost requirement for researchers. There are few important people for making this Ph.D. thesis possible and

I take this as an opportunity to thank them.

I would like to offer my most sincere gratitude to my supervisor, Dr. Song

Jianxing who gave me the opportunity to work as a Ph.D. student in his laboratory. He gave me the freedom to explore my project on my own, yet he was always there for discussions and valuable comments. His door was always open for the consultation.

His guidance, enthusiasm for science, support and giving me full independence for my project is highly appreciated.

I am grateful to my cosupervisor Dr. Vincent T.K. Chow for his expert advice, comments and suggestions. I thank him for his support throughout my Ph.D. candidature. I appreciate and thank Dr. Tan Yee Joo for being a collaborator in HCV project and helping me with the constructs and her guidance for the project. I am thankful to Dr. Jingsong Fan for NMR experiment training and his help in collecting

NMR spectra on the 800 MHz and 500MHz spectrometer.

I would like to thank Dr. Shi Jiahai, an exPh.D. from our lab, who made me feel comfortable in the lab as well as with protein work during my initial days in

NUS. I want to say thanks to my lab mates for maintaining healthy work space. I extend my gratitude to Dr. Qin Haina for being there whenever I needed help in NMR experiments and data processing work. I thank Huan Xuelu, Garvita, Wang Wei and

Miao Linlin for their help and support.

I also want to thank Mr. Lim Ek Wang (Microbiology) for allowing me to use anaerobic chamber, which was of great help for my first project. I thank Janarthan for helping me with the chemicals.

I thank all the structure biology labs supervisors and lab members for helping me with the chemicals or experiment related materials. I am thankful to NUS for providing me the scholarship during my Ph.D. candidature, which was a great support during all these years.

This thesis work would not have been possible without the support and encouragement of my family. Their trust and my stubbornness, always kept me keep going with my work. They are my life line and a pillar of support and have always encouraged me to do good work.

This acknowledgment will be incomplete if I do not mention about my friends, who have always been there for my help. I thank Chhavi, Suma, Hari, Atul, Karthik,

Priya and Mukesh. I also thank my housemates Madhu, Asfa and Anusha for being so much accommodating and keeping the environment healthy and lively, which have always helped me to regain energy after the daylong work.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS I

TABLE OF CONTENTS III

SUMMARY VII

LIST OF FIGURES VIII

LIST OF TABLES X

LIST OF SYMBOLS XI

CHAPTER 1 INTRODUCTION 1

1.1 Protein structure studies 2

1.2 Features of NMR spectroscopy 3

1.2.1 NMR for proteins 3

1.3 Principle of NMR 4

1.3.1 Larmor frequency 5

1.3.2 Chemical shift 6

1.3.3 Coupling 6

1.3.4 Free induction decay 7

1.3.5 Relaxation 8

1.3.5.1 Spinspin relaxation time (T1) 8

1.3.5.2 Spinspin relaxation time (T2) 8

1.3.5.3 NOE (Nuclear Overhauser Enhancement) 9

1.4 Structure details by NMR

1.4.1 1Dimension NMR 9

1.4.2 The 1H15 N coupling for the heteronuclear NMR analysis 11

1.4.3 Sequential assignment

1.4.3.1 Homonuclear 1HNMR spectroscopy 12

1.4.3.2 Heteronuclear sequential assignment 13

III

1.4.4 Chemical shift analysis 14

1.5 Structure Determination by NMR 15

1.6 Outline of NMR experiment 1617

1.7 Proteinligand interaction by NMR 17

1.7.1 Mapping of Chemical Shifts 1819

1.8 Circular Dichroism 1921

CHAPTER 2 BIOLOGICAL SIGNIFICANCE OF VIPERIN

2.1 Introduction

2.1.1 Viperin sequence details 2325

2.1.2 Viperin in Immune response 2627

2.1.3 Viperin Induction and Action 2728

2.1.4 Influenza virus inhibition by viperin 2930

2.1.5 Radical SAM domain proteins 3032

2.1.6 AIMS 33

2.2 MATERIALS AND METHODS

2.2.1 Vector Construction 3536

2.2.2 Protein Expression and Purification

2.2.2.1 Expression and purification of insoluble proteins 37

2.2.2.2 Expression, Purification and Reconstitution of the [Fe 4–S4] cluster in Viperin (45–361) 3839

2.2.3 Media prepration for NMR sample 39

2.2.4 UV–visible Spectroscopy 39

2.2.5 Circular dichroism (CD) 3940

2.2.6 NMR sample prepration 40

2.3 RESULTS AND DISCUSSION

2.3 Structure details of Viperin

2.3.1 Structural characterization of the human viperin fragments 4245

2.3.2 Reconstitution of the [4Fe–4S] cluster in Viperin (45–361) 4549 and viperin (45361) mutant 2.3.3 Structural characterization of the bufferinsoluble Viperin 4951 (214–361)

2.3.4 Conclusion 5253

2.3.5 Future work 53

CHAPTER 3 BIOLOGICAL SIGNIFICANCE OF VAPC

3.1 Hepatitis 56

3.1.1 Acute and Chronic Hepatitis 5657

3.1.2 HCV genotype 5758

3.1.3 Life cycle of HCV 5859

3.1.4 Genome organization 6061

3.1.5 NS5B (RNA dependent RNA polymerase, RdRp) 6163

3.1.6 VAP Proteins 6365

3.1.7 Interaction of VAP proteins with HCV proteins 6567

3.1.8 Therapeutics for HCV 6769

3.1.10 AIMS 70

3.2 MATERIALS AND METHODS

3.2.1 Vector Construction 72

3.2.2 Codon optimization 7273

3.2.3 Preparation of Competent E.coli Cells 73

3.2.4 Transformation of E. coli Cells 73

3.2.5 Protein Expression and Purification

3.2.5.1 Expression and purification of full length VAPC and

truncated constructs 7374

3.2.5.2 Expression and purification of NS5B 7475

3.2.6 Preparation of Isotope Labeled Proteins 76

3.2.7 Determination of Protein Concentration by Spectroscopy 76

3.2.8 Circular Dichroism Spectroscopy 76

3.2.9 NMR experiments 77

3.3 RESULTS AND DISCUSSION

3.3.1 Protein purification 7980

3.3.2 Structure characterization of full length VAPC protein 8185

3.3.3 Interaction of VAPC to HCV NS5B 8688

3.3.4 VAPC Cterminal constructs 8889

3.3.4.1 Structural characterization of VAPC43, VAPC31 and VAPC14 9093

3.3.4.2 Interaction of VAPC43 with HCV NS5B 9498

3.3.3.3 Interaction of VAPC14 with HCV NS5B 9899

3.3.4 Determination of dissociation constant (K d) through HSQC titration 100 102

3.3.5 Discussion 103105

3.3.6 Future work 105

CHAPTER 4. PERSPECTIVE 107

4.1 Association of Viperin and VAP proteins 107108

4.2 Other Cellular proteins as target for antiviral drugs 108109

REFERENCES 110119

PUBLICATION 120

SUMMARY

Cellular proteins with antiviral properties have always been the priority area for researchers. Recent advances in the structural characterization of the proteins have provided a strong foundation towards these efforts. Human immune system act strongly against viral infection by upregulation of certain proteins which are active against such infections. The present work is about two such cellular proteins, Viperin and VAPC, which show antiviral properties. Viperin (Virus inhibitory protein, endoplasmic reticulum associated, interferoninducible), which is an evolutionary conserved gene and the research work done in past decade proves its antiviral activities against whole range of viruses ranging from DNA virus to RNA virus. But these studies lack structural and biochemical details about viperin. My Ph.D. thesis work showed for the first time that viperin is a radical SAM domain protein and it was done by systematic removal of Nterminal domain and reconstitution of purified protein under anaerobic conditions.

Another cellular protein, VAPC (vesicleassociated membrane protein associated protein (VAP subtype C) inhibits HCV virus by interaction with HCV unstructured protein NS5B. Our results indicate that VAPC is a member of intrinsically unstructured protein (IUP) with no secondary and tertiary structures.

Extensive NMR characterization reveals that the Cterminal half of VAPC is involved in binding with NS5B and the isolated Cterminal 43 residues shows even tighter binding affinity with NS5B than the full length protein. The results demonstrate that the intrinsically unstructured VAPC form a “ fuzz ” complex with NS5B and also for the first time we designed a shorter VAPCpeptide which specifically bind NS5B with a Kd of 49.13 µM. In the future, functional characterization needs to be done to evaluate its potential as peptide mimic in treatment against HCV infection.

VII

LIST OF FIGURES

Figure 1.1 Dependence of secondary structure elements on Φ/Ψ angles 4

Figure 1.2 The spinning nucleus with a charge precessing in a magnetic field 5

Figure 1.3 Dihedral angle (φ) 7

Figure 1.4 The free induction decay (FID) 8

Figure 1.5 . NOE patterns associated with secondary structure 10

Figure 1.6 Comparison of NMR spectra of folded and unfolded protein 11

Figure 1.7 Protein backbone highlighting the amide hydrogen/nitrogen pair correlated in the 2D 12

Figure 1.8 HNCACB and CBCACONH connectivity 14

Figure 1.9 Chemical shift analysis of the peptide backbone NMR signals 15

Figure 1.10 Strategy of structure determination by NMR 17

Figure 1.11 CD spectra of various secondary structure 20

Figure 2.1.1 Sequence comparison of human viperin 25

Figure 2.1.2 Schematic representation of immune response pathway that leads to the disruption of viral release from the plasma membrane 28 Figure 2.1.3 Model diagram showing the Influenza A virus release upon viperin expression and interaction with FPPS 30

Figure 2.1.4 SAM domain conserved sequence and reaction 3132

Figure 2.2.1 Secondary structure prediction and truncation representation 36

Figure 2.3.1 FPLC and DLS profiles of viperin 4344

Figure 2.3.2 Far UV and Near UV CD and 2D NMR spectra of unreconstituted viperin 4546

Figure 2.3.3 CD, UV and 1D characterization of viperin (45361) 4849

Figure 2.3.4 CD and NMR characterization of viperin Cterminal 5051

Figure 2.3.5 2D HSQC for Viperin (SAM+Cterminal) domain 55

Figure 3.1.1 HCV global prevalence 2010 57

VIII

Figure 3.1.2 Schematic diagram of life cycle of HCV 59

Figure 3.1.3 HCV genome organization, polyprotein processing and topology 62

Figure 3.1.4 Crystal Structure of NS5B 63

Figure 3.1.5 General domain organization of VAP protein 64

Figure 3.1.6 Alignment of the amino acid sequences of hVAPC with

hVAPB and hVAPC. 64

Figure 3.1.7 Model for the mechanism of formation of HCV replication complex on lipid raft 6869 Figure 3.3.1 Purification profiles of VAPC and NS5B 7980 Figure 3.3.2 VAPC sequence, CD and 1D spectra representation 82

Figure 3.3.3 2D1H 15 N HSQC 83

Figure 3.3.4 VAPC full length Cα (observedrandom) 85

Figure 3.3.5 Far UV CD spectra,1D NMR and CSD calculation of VAPC 8688

Figure 3.3.6 Representation of VAPC cterminal constructs 89

Figure 3.3.7 CD and 1D NMR of VAPC constructs 90

Figure 3.3.8 HSQC and sequential assignments of VAPC43 and VAPC14 91

Figure 3.3.9 Hα Chemical shift deviation plot of VAPC 43 93

Figure 3.3.10 NOE pattern representation for VAPC43 93

Figure 3.3.11 VAPC43 structure 93

Figure 3.3.12 VAPC43 titration experiment to HCV NS5B 9598

Figure 3.3.13 VAPC14 on titration to HCV NS5B 99

Figure 3.3.14 Fitting curves for VAPC43 and VAPC14 102

LIST OF TABLES

Table 2.3.1 Brief description of truncated fragments of Viperin with their expression patterns and the techniques used to study the purified protein 44

Table 3.1 Geographical distribution of HCV genotypes 58

Table 3.1.2 Cost of updated HCV treatment 68

Table 3.1.3 HCV DirectActing Antivirals (DAA) in Clinical Trial Phase II or III, or FDAApproved 68

Table 3.3.1 Random Coil Chemical shifts (in ppm) for the 20 common amino acids in acidic 8 M urea 85

Table 3.3.2 Kd values with SD (standard deviation) of shifted residues from VAPC43 and VAPC 14 101

SYMBOLS and ABBREVATIONS

γ Gyromagnetic Ratio

δ Chemical Shift

Cα Cα Conformational Shifts

α Alpha

β Beta

βME Beta mercaptoethanol

µ Micro

φ Phi angle

ψ Psi angle

ω0 Precessional frequency dαN/ dβN / dNN NOE Connectivity Between CαH/ CβH/ NH with NH

Å Angström

I Spin Number

Kd Dissociation constant

B Magnetic field

1D Onedimension

2D Twodimension

3D Three dimensions

CD Circular Dichroism

CSD Chemical Shift Deviation

DTT Dithiothreitol

E.coli Escherichia coli

FID Free induction decay

GST Gluthathione Stransferase

HCV Hepatitis C virus

HSQC Heteronuclear Single Quantum Coherence

IPTG Isopropyl βDthiogalactopyranoside

IUP Intrinsically unstructured proteins

ITC Isothermal calorimetry

LB Luria Bertani

MALDITOF MS MatrixAssisted Laser Desorption/Ionization Timeoff light Mass Spectroscopy

NOE Nuclear Overhauser Enhancement

NOESY Nuclear Overhauser Enhancement Spectroscopy

NMR Nuclear Magnetic Resonance

OD Optical Density

PBS Phosphatebuffered Saline

PCR Polymerase Chain Reaction

PDB Protein Data Bank ppm Part Per Million

RdRp RNA dependent RNA polymerase

RMSD Root Meansquare Deviation

RPHPLC ReversedPhase High Performance Liquid Chromatography

TOCSY Total Correlation Spectroscopy

UV Ultraviolet

XII

Chapter 1 INTRODUCTION

Introduction

1.1 Protein structure studies

The structure based approach to study the biological system has brought advances in understanding the important molecular mechanisms of biological system.

Structural biology has shown explosive growth since late 1980s, with the number of high resolution structures of proteins added to the protein data bank (PDB) currently growing at more than 2000 per year (Kelly S.M. et.al. 2005). This has allowed much more detailed insights into the function of systems. Detailed protein structure and interaction studies constitute the foundation for novel drug discovery and drug design.

Lots of techniques are available to study the proteins like, Xray crystallography,

NMR (Nuclear magnetic resonance spectroscopy), Electron microscopy (EM),

Circular dichroism (CD), ITC (Isothermal calorimetry), Biacore and many more.

NMR spectroscopy and Xray crystallography are currently the powerful techniques capable of determining threedimensional structures of biological macromolecules like proteins and nucleic acids at atomic resolution. This chapter gives description of

NMR technique and usage in protein studies and a brief account about use of CD.

Few years back, Xray crystallography was the main technique for protein structure studies and is superior to NMR in determining structures of much larger macromolecules, in a more automated way but since 1946, when the NMR was first used it has developed into premier organic spectrophotometric technique to study biomolecules. The important role of NMR in structural biology is illustrated by more than 6000 NMR solution structures deposited in the protein data bank. It has some advantages over Xray crystallography technique. With NMR it is possible to study

the time dependent phenomenon. It allows the study of intramolecular dynamics in macromolecules, reaction kinetics, molecular recognition or protein folding.

1.2 Features of NMR spectroscopy

Nuclear magnetic resonance spectroscopy is the technique to study physical, chemical, and biological properties of matter. NMR is a powerful analytical tool to study molecular structure including relative configuration, relative and absolute concentrations and intermolecular interactions. By using NMR spectroscopy proteins can be studied in solution state and conditions like pH, temperature and salt concentrations can be adjusted to mimic the physiological condition. NMR is able to characterize very weak interactions between macromolecules and ligands at atomic resolution by means of chemicalshift changes and makes this technique a major tool in rational drug design and discovery.

1.2.1 NMR for proteins

The signals in NMR spectroscopy are referred to as resonances. Their positions in NMR spectrum depend on the local environment of nucleus producing the signal and referred to as chemical shifts reported in ppm. Protein structure study using NMR starts with assignment of maximum possible resonances of as many hydrogen, carbon and nitrogen on the protein of interest. The resonance of Cα, Cβ, Co and Hα depends on phi (φ)/psi(ψ) angle propensities (Spera S. et.al . 1991; Wang Y. et.al. 2002). The secondary and tertiary structure of protein is decided by its phi(φ)/psi(ψ) angle values.

Figure 1.1 shows the possible φ/ψ values for different conformation of protein. So, it is the basic peptide bond structure which lays the foundation of protein structure studies by NMR. The following section gives the brief overview of NMR in protein structure solution.

(A)

(B)

Figure 1.1 Dependence of secondary structure elements on Φ/Ψ angles. A) Representation of Φ and Ψ angles on peptide bond. B) Ramachandran plot shows the dependence of secondary structure elements on the Φ/Ψ angles. Adapted from Peti W. et al. (2000)

1.3 Principle of NMR

All nucleons (neutrons and protons) composing any atomic nucleus, have the intrinsic quantum property of spin. This means they rotate around the given axis. The overall spin is determined by the spin quantum number I. Nuclei with even number of protons and neutrons (e.g. 12 C, 16 O, 32 S) have I = 0 and has no overall spin as their spins are paired and cancel each other. Isotopes with odd number of protons and/or of neutrons ( 1H, 13 C and 15 N) have an intrinsic magnetic moment and angular momentum, in other words a nonzero spin. Spinning charged particles are associated with magnetic field and behave like small magnets. The magnetic field developed by

the rotating nucleus is described by a nuclear magnetic moment vector or microscopic magnetization vector µ, which is proportional to the spin angular moment vector.

1.3.1 Larmor frequency

A nucleus with magnetic moment (µ), when placed in external magnetic field, orients opposite to the direction of external magnetic field, B (Figure 1.2) and precess around the axis of external magnetic field. This is called Larmor precession. The frequency of this precession is proportional to the strength of the external magnetic field and is a physical property of the nucleus with a spin. The precessional frequency,

ω0 = γ B 0, where γ is the gyromagnetic ratio and is constant for all nuclei of a given isotope.

Figure 1.2 Spinning nucleus with a charge precessing in a magnetic field. Adapted from Van De Ven F. J. (1995) Protons ( 1H) have a high natural abundance (99.9885 %) and gyromagnetic ratio is also high ((γ 1H/γ 13 C is bout 4 and γ 1H/γ 13 N is about 10) (Roberts, 1970), which makes them the most sensitive nuclei for NMR investigations. Introduction of 15 N and 13 C

NMR active stable isotopes has made the structure studies of protein relatively easy.

There are some phenomenon in NMR , which when intelligently used, provides required information about molecular stru cture and dynamics and some of those are described below.

1.3.2 Chemical Shift (δ)

The resonance frequency depends upon the kind of nuclei (γ) and the external magnetic field (B 0). Therefore, similar kind of nuclei would resonate at a same frequency. But i n biological samples we deal with “chemical” protons which are surrounded by different electronic densities. The external magnetic field induces electronic circulations, which in turn create an induced local magnetic field at the nucleus position. The tota l effective magnetic field that acts on the nuclear magnetic moment will therefore be reduced depending on strength of the locally induced magnetic field. Induced magnetic field is the characteristic of chemical nature of group, to which it belongs. This i s called a screening effect or shielding effect, or more commonly known as the chemical shift. It is one of the most basic parameter of

NMR and is usually quoted in parts per million (ppm).

1.3.3 Coupling

Nuclei in molecules are not isolated and so the magnetic moments of nuclei can interact between themselves. The phenomenon of interaction between nuclei through the polarization of the electrons in the orbitals joining the two nuclei is called scalar coupling or J-coupling. The coupling is observed by a splitting of the NMR signal. The value of J is independent of the external magnetic field and its value decays to zero for nuclei separated by more than 4 or 5 bonds . It is an important

phenomenon as it the basis of coherence transfer between nuclei, a crucial step in the development of two and multidimensional NMR spectroscopy. J coupling along with chemical shift can produce characteristic patterns of couplings in many types of the amino acids, which are helpful to identify amino acid types. The size of the coupling depends on structural properties such as, dihedral angles (φ) (Figure1.3) via Karplus equation.

Figure1.3 Dihedral angle (φ)

To determine the torsion angles Ф and χ1, 3JNHHα and 3JHαHβ are used thus providing important information on conformations of peptide backbone and amino acid side chains. Deviation of 3JNHHα values from random coil values provides valuable secondary structural information. For α helices peptide segments where the

ϕ angle is around 60°, coupling constants is around 4 Hz, and it is between 8 and 12

Hz for peptide segments in βstructures, where the ϕ angle is in the 120° range. But for unfolded proteins it is estimated to be around 6~7.5Hz because of the averaging of coupling constant caused by conformational fluctuation (Dyson et al, 2004).

1.3.4 Free Induction Decay (FID)

The nuclear magnetization perpendicular to magnetic field decreases with time and is measured in the receiver coil as fluctuating declining amplitude with time. This measures a frequency decay rate as a function of time (Figure 1.4).

The FID is a function of time; the Fourier transformation converts this to a function of frequency.

Figure 1.4 Free induction decay, FID, is measured as a function of time in the x and ydirections perpendicular to magnetic field. Adapted from Van De Ven F.J. (1995)

1.3.5 Relaxation

The NMR process is an absorption process. Nuclei in the excited state must also relax and return to the ground state and the timescale for this relaxation is crucial to the NMR experiment. The timescale for relaxation gives information about how the

NMR experiment is executed and consequently, how successful is the experiment. T1 and T2 (the inverses of the relaxation rates) are, respectively, the longitudinal (spin lattice) and transverse (spinspin) relaxation times.

1.3.5.1 Spinlattice relaxation time (T1): In T1 relaxation time, longitudinal relaxation energy is transferred to the molecular framework (lattice) and is lost as vibrational or translational energy. The halflife for this process is called the spin lattice relaxation time.

1.3.5.2 Spinspin relaxation time (T2): In this process, energy transfer to the neighboring nucleus. The halflife for this is called spinspin relaxation time. The peak width in an NMR spectrum is inversely proportional to the lifetime and depends on T2. These are influenced by the mobility in the solution, and so the molecular size

of the compound of interest. For large molecules T2 values reduces and the spectra produced is with broader lines.

1.3.5.3 NOE (Nuclear Overhauser Enhancement)

This is also a kind of relaxation phenomenon and was discovered by Alber

Overhauser in 1953. Nuclei close to each other in space transfer energy to each other during relaxation, and extent of transfer depends on the distance between the nuclei. It is normally detected between nuclei separated by a distance of less than 5Å. The NOE between two protons can be used to estimate the distance between them and is helpful in determining the twodimensional and threedimensional structure of the macromolecule (Figure 1.5) (Jeremy N.S Evans, 1995). NOE intensities are classified into three different categories, with distances of 1.82.7Å is classified as strong NOE,

1.83.3Å as medium range NOE and 1.85.0Å weak or long range NOE.

1.4 Structure details by NMR

1.4.1 1Dimension NMR

1dimension NMR gives important information about protein stucture, whether it is folded or is unfolded. The figure (Figure 1.6) below shows the 1D spectrum of folded and unfolded protein. For folded proteins 1D spectra shows well dispersed peaks over the range of 1 to 12ppm and also presence of upfield peaks in 1D is the characteristic feature of folded proteins. While for unfolded proteins peak dispersion is in a narrow range, within just 1ppm range. Due to the large number of protons,

Figure 1.5 . NOE patterns associated with secondary structure. (A) Broken lines indicate some of the NOE interactions that may be observable in polypeptide chains. (B) NOE intensities and NHCaH J couplings in several types of secondary structure. The thickness of thc horizontal lines indicates the intensity of the NOEs. (Adapted from Ad Bax 1989). nitrogens and carbons in a protein, peaks in 1dimensional (1D) spectra of proteins are very overlapped. Peaks are clustered by type of proton, and cluster types are labeled in the spectrum (Figure 1.6). The overlapping in 1dimension spectrum can be separated by adding 2dimension and is done by using specific series of radio frequency (rf) pulses and delay in the transfer of magnetization from first nucleus to second nucleus, and labelling the magnetization with the frequencies of both nuclei.

Figure 1.6 Comparison of NMR spectra of folded (top) and unfolded (bottom) protein (Adapted from Poulsen F.M. 2002).

1.4.2 The 1H15 N coupling for the heteronuclear NMR analysis

The onebond coupling 1H15 N is the most basic experiment important for the heteronuclear NMR analysis of proteins and it arises because the magnetization of the first nucleus is sensitive to the spin orientations of its neighbors. It is considered the fingerprint of the protein. HN bond is present in every amino acid residue except the Nterminal and the proline residues and (Figure 1.7) it correlates the frequency of the amide hydrogen with that of the amide nitrogen to produce a single peak for each residue in the protein with the exception of prolines.

Correlation spectroscopy method used to measure this coupling is called a 1H15 N

HSQC spectrum (heteronuclear single quantum correlation).

(A)

Figure 1.7 A) Protein backbone highlighting the amide hydrogen/nitrogen pair correlated in the 2D [ 1H15 N]HSQC

(B)

(B) 2D [1H15 N]HSQC of protein. Throughbond correlation spectrum between the hydrogen (1H) and the nitrogen (15N) nuclei of amide groups in a protein. The location of the amide groups in the polypeptide backbone are sketched in the formulae on the left. The arrows indicate that each peak in the NMR spectrum corresponds to one NHmoiety. The axes of the NMR spectrum indicate the chemical shift of the hydrogen (1H) and the nitrogen (15N) nuclei. (Adapted from Wilder G. 2000)

1.4.3 Sequential assignment

It is a process by which a particular amino acid spin system identified in the spectrum is assigned to a particular residue in the amino acid sequence.

1.4.3.1 Homonuclear 1HNMR spectroscopy

Sequential assignments for protein sample with 15 N and 1H nuclei can be done by the nuclear overhauser effect (NOE). In the two most common types of secondary structure (helix and beta) the peptide chain accommodates conformation which bring

1H of the peptide backbone and the side chains of neighbouring residues so close together that they can be observable by NOE spectroscopy (Figure1.5). In the αhelix the neighbouring HN are 2.8 (Å) angström apart, and in the βstrand the distance is only 2.2(Å) angström.

1.4.3.2 Heteronuclear sequential assignment

Along with 2D [ 1H, 15 N] HSQC, the sequence specific assignment of the hydrogen, nitrogen and carbon resonances also involves 3D NMR experiments: the (HbHa)CbCa(Co)NH and the HNCaCb. These experiments helps to correlate the amide hydrogen and nitrogen frequencies with those of the Cα and Cβ carbons.

(HbHa)CbCa(Co)NH : It measures the heteronuclear coupling between 1HN and 15 N in one residue and the coupling across 13 C to the 13 Cα and 13 Cβ in the preceding residue.

HNCaCb : This particular 3D experiment measures the one bond coupling between 1HN and 15 N and the one and two bond coupling between 15 N and 13 Cα and 13 Cβ in one residue and also records the coupling across 13 C to the 13 Cα and 13 Cβ in the preceding residue.

The combined analysis of these 2 spectra helps in identification of the 13 Cα and 13 Cβ in the same residue and the preceding residue and thus helps to solve the backbone assignment (Figure 1.8).

Figure 1.8 HNCACB and CBCACONH correlation. Magnetiz ation is transferred from 1Hα and 1Hβ to 13 Cα and 13 Cβ, respectively, and then from 13 Cβ to 13 Cα. From here it is transferred first to 13 CO, then to 15 NH and then to 1HN for detection. (Adapted from Grzesiek S. et.al . 1992 ).

1.4.4 Chemical shift analysis

Chemical shift analysis of 1Hα and 13 CO, 13 Cα, and 13 Cβ of the peptide backbone can be used to determine the secondary structure type of a given peptide segment either by plotting the secondary shifts ( ∆δ = observed shift – random coil shift) or by using the ch emical shift index (CSI) and this gives a consensus value using all of the secondary shifts from the Cα, Cβ and CO nuclei with an output of +1 for betastrand, 0 for random coil and 1 for αhelix (Figure 1.10)

Figure 1.9 Chemical shift analysis of the peptide backbone NMR signals. Each panel represents a chemical shift analysis of the individual residues comparing the observed shift with the shift observed for the same residue type in random coil model peptides. (Adapted from Wishart D.S. et.al . 1994b)

1.5 Structure Determination by NMR

Determination of protein structure by NMR depends in the assignment of

NMR signals. NMR gives information about the structural geometry around the detected nuclei. From the NMR signals the distance to the nearby nuclei in the structure can be read using NOE spectroscopy, coupling constants and chemical shifts gives information about the dihedral angles, the relative angles of a number of bond vectors can be determined by residual dipolar coupling. All these constraints are utilized in the structure calculation.

In the structure calculation process, the computer converts these conformational constraints into a visible structure. The computer program folds the starting structure in a way that the experimentally determined interproton distances are satisfied by the calculated structures. The computer program tries to calculate a structure with a possibly small overall energy.

The calculated structure should be analyzed for its structure quality and this is performed using the following three parameters: constraints violations, the RMSD

(root mean square deviation) of the structure ensemble and the Ramachandran plot

(phi and psiangles of the protein backbone). The good quality structure should have little constraints violation, small RMSD and most phi and psiangles should be within the allowed region of Ramachandran plot. PROCHECK is the most popular program for assessing the quality of a given protein structure (Laskowski et al., 1993).

1.6 Outline of NMR experiment

NMR is a powerful technique to study structure of protein in 3dimension or even in 4dimension. The general outline to carry out NMR (Figure 1.11) experiment for protein is given below:

i) The first step is the preparation of the protein sample in solution. The

recombinant protein is expressed in medium enriched with 15 N and/or 13 C

isotopes. The purified protein is dissolved in around 0.40.5 ml buffer

containing 5%10% D 2O and after adjustment of pH, ionic strength, and

temperature is analyzed by performing different NMR experiments.

ii) The second step is to record high quality NMR spectra. A set of

heteronuclear multidimensional experiments are recorded for chemical

shifts assignments ( 1H, 15 N, 13 C).

iii) Assignment is the process of attributing a resonance in an NMR spectrum

to a particular nucleus in a molecule. The first step is the identification of

certain amino acids with a characteristic pattern of cross signals. The

second stage of assignment is to search the sequential contacts from the

already identified amino acids to the neighboring ones in the NOESY

spectra. Interresidual cross signals can be distinguished from the intra

residual ones by comparing the NOESY with the TOCSY spectrum

(Jeremy N.S Evans, 1995).

iv) Next step is the collection of conformational constraints.

v) Followed by 3D structure calculation.

Figure 1.10 Strategy of structure determination by NMR. Outline of the general strategy used to solve the threedimensional structure of biological macro molecules in solution by NMR (Adapted from Wider G. 2000)

1.7 Proteinligand interaction by NMR

NMR spectroscopy has been widely recognized as an important tool for studying protein interactions (Zuiderweg 2002; Takeuchi and Wagner 2006). A range

of interaction partners can be studied by NMR and it may include macromolecules

(proteins, nucleic acids and carbohydrates), molecular assemblies (cell membranes) and small molecule ligands. NMR spectroscopy gives quality information on binding site and mode, and it is also powerful for detecting interactions even between weakly binding components with dissociation constant Kd>104M (Vaynberg J. and Qin J.

2006).

1.7.1 Mapping of Chemical Shifts

Complex formation leads to selective changes in chemical shift of various nuclei which can be traced to get information of complex structure in the solution.

Chemical shift mapping is commonly based on the 1H15 N HSQC, which is a 2D spectrum containing one signal for each amino acid except proline. The signals of the amides which get perturbed by ligand binding will change position, on the addition of ligand. The perturbed residues can be mapped upon the protein structure to reveal the binding site/s in the protein. This approach has been widely used to study both protein–ligand and protein–protein interactions (Farmer B. T et. al . 1996; Williamson

R. A. et. al . 1997; Lian L. Y et.al 2000). The rate constant for the formation of the complex and rate constant for the dissociation of the complex determines the equilibrium of the interaction when ligand binds to a protein. NMR can determine the reaction rate.

7 If the complex binds tightly, K d<10 M, slow exchange conditions apply and chemical shifts from bound conformation and chemical shifts from free conformation

3 can be observed (Lian L.Y. et.al . 1994). Whereas, for the complex with K d>10 M, the exchange rate is fast on NMR time scale and a single averaged resonance is observed for nuclei at corresponding sites in the free and bound species.

The dissociation constant, K d, is deﬁned as follows:

° − ° − = = =

And, complex concentration is given by,

° ° ° ° ° ° = + + − + + − 4

For slow exchange proteins, the signal of the protein ligand complex will increase with the addition of ligand and at the same time intensity of the free protein signal will decrease but the sum of the intensity of the two peaks will always be constant.

1.8 CIRCULAR DICHROISM

Circular dichroism (CD) is the technique to study the conformations of peptides and proteins in solution (Alder A. J. et.al . 1973). It is a form of light absorption spectroscopy which measures differences in the absorption of lefthanded polarized light versus righthanded polarized light by a substance which arise due to structural asymmetry. Three different wavelength ranges in CD gives great deal of details about a protein. These are;

1. Far UV (190nm250nm): in this range main contribution is by peptide bond.

2. Near UV (250nm300nm): dominated by aromatic amino acid residues.

3. Near UVvisible range (300nm700nm): contributed by extrinsic

chromophore.

Figure 1.11 Far UV CD spectra associated with various types of secondary structure. Solid line, αhelix; long dashed line, antiparallel βsheet; dotted line, type I βturn; short dashed line, irregular structure. Adapted from Brahms & Brahms (1980)

It has been shown (Figure 1.14) that CD spectra between 260 and approximately 180 nm can be analyzed for the di ﬀerent secondary structural types: α helix, parallel and antiparallel βsheet, turn, and other. The application of CD for conformational studies in peptides (like proteins) can be largely grouped into i) monitoring conformational changes (e.g., monomeroligomer, substrate binding, denaturation, etc.) and ii) estimation of secondary structural content i.e. amount and type of secondary structure. It is also an important method to study the stability of macromolecules by chemical denaturants or temperature stability. As addition of 8M urea to protein and studying it in near UV range will give details about the tertiary packing in the protein.

We have used the above mentioned techniques for the structural characterization and the interaction studies of two antiviral proteins, Viperin ((Virus

inhibitory protein, endoplasmic reticulum associated, interferoninducible) and VAPC

(vesicular associated membrane proteinassociated proteinsC). These are cellular proteins and recent studies show their activity against virus propagation. We choose

NMR as a tool as it is the only technique available to study unstructured proteins and also it can help to observe very weak interactions among protein and its binding partner.

PART – I – Viperin a RadicalSAM protein

Chapter –2 BIOLOGICAL SIGNIFICANCE OF VIPERIN

2.1 Introduction

Secretion of interferons (IFNs) from viral infected cells is the hallmark of antiviral immunity. Cells that receive these signals, prepare the cells against infections by increasing expression of interferon stimulated genes (IFGs). Viperin (Virus inhibitory protein, endoplasmic reticulum associated, interferoninducible), is one such gene which is induced by both type1 and type II interferon and is located at endoplasmic reticulum (ER) membrane. Viperin inhibits a number of RNA and DNA viruses.

2.1.1 Viperin sequence details

Zhu et. al . in 1997 identified a gene by differential display analysis during the study of mRNAs which accumulates to enhanced levels in human cytomegalovirus infected cells. It was named as cig-5, cytomegalovirus induced gene5. Further studies

(Boudinot et. al. 1999) show that the rhabdovirus viral homorrhagic septicemia virus

(VHSV) induces a gene in rainbow trout which was named as vig-1 (VHSV induced gene) and (Boudinot et .al . 2000) mouse dendritic cells infected with vesicular stomatitis virus (VSV) and pseudorabies virus (PrV) induces gene named as mvig.

Similar gene was found in rat which was named best5 (Grewal et. al . 2000). All these genes have similar sequences and are induced by viral particles suggesting their functional importance. Later in 2001 Chin and Cresswell named it Viperin (virus inhibitory protein, endoplamic reticulumassociated, interferoninducible). Homology study of human viperin (Figure 2.1.1) with mouse, rat (Best5) and bony fish (vig1) shows that the gene is evolutionary conserved among species as diverse as fish, rodents and primates, thus implying the conserved functionality of the gene. Sequence analysis of viperin suggests that it contain an Nterminal amphipathic domain which shows the least homology among the sequences from different species. Nterminal

contains leucine zipper which facilitates in proteinprotein interaction or binding of protein to cell membrane. Rest of the sequence shows more than 95% similarity with a middle domain containing CXXXCXXC motif which is conserved domain of the radical Sadenosylmethionine (SAM) superfamily protein and highly conserved C terminal domain.

Study done by Hinson and Cresswell (2009) show that viperin localize to the cytosolic phase of the ER through Nterminal amphiphatic αhelix. Upon over expression viperin alters ER structure by forming dimers and induces a tightly ordered array of ER membranes, which are called as crystalloid ER which reduces the secretion of soluble, but not membranebound proteins. Nterminal αhelix is necessary for ER localization of viperin and to inhibit protein secretion and this N terminal is important for its antiviral activity.

Later, it was found that this αhelix is required to localize viperin to lipid droplets and thus in HCV inhibition. But it has also been studied (Jiang D. et. al .

2008) that although Nterminal is important for membrane association and for the antiviral activity, removal of Nterminal region does not completely abolishes the antiviral activity. The Nterminal truncated protein still can reduce HCV replication observed by cell colony formation. Trp361, which is the last residue is very well conserved in all the species and mutating this residue to Ala, Asp and Pro completely abolish the antiviral activity of viperin but on mutating it to residues with aromatic ring like Phe and Tyr, it still retain the antiviral activity. This implies that all the conserved motifs in viperin along with Nterminus are essential for its antiviral activity.

Figure 2.1.1 Sequence comparison of human viperin, with sequence from mouse, rat (Best5) and bonyfish (vig1). The asterisks indicate amino acid residues that are identical in all four sequences. Residues in boxes represent highly conserved and important for viperin functionality.

2.1.2 Viperin in Immune response

Viperin was initially identified as a human cytomegalovirus (HCMV) – inducible gene in primary skin cultures (Zhu et. al 1997). Recent investigations show that it interferes with the propagation of a broad spectrum of viruses, for example,

Human cytomegalovirus (HCMV) (Chin & Cresswell 2001), Human immunodeficiency virus (HIV) (Rivieccio M. A et. al 2006 ), Hepatitis C virus (HCV)

(Jiang. D. et. al . 2008), Alphavirus (Yugen Z et.al 2007), Influenza virus (Wang X et. al 2007), Sendai virus (Severa M. et. al . 2006), Vesicular stomatitis virus (VSV)

(Boudinot et. al . 2000), Yellow fever virus (Svetlana F. K. et. al 2005) and also West

Nile virus and Dengue virus (Jiang D. et. al . 2010) and thus suggesting its important role in innate immune response. Studies (Severa M. et. al . 2006) show that it is also induced by microbial products like lipopolysaccharide (LPS) and doublestranded

RNA. Work done by (Helbig et.al 2005) shows that viperin has antiHCV activity invitro also. In HCMV infected cells, the expression of viperin downregulates HCMV structural proteins (gB, pp28 and pp65), required for viral assembly and maturation

(Chin & Cresswell 2001). In influenza A virusinfected cells, viperin prevents virus budding from the plasma membrane by disrupting the lipid rafts (10) and it does this by binding to and inhibiting Farnesyl pyrophosphate synthase (FPPS) which is a key enzyme in isoprenoid biosynthesis (Figure 2.1.2).

Viperin is active not only against viruses but also against bacteria. Work done on fish red drum Sciaenops ocellatus viperin gene (Sovip) indicate that Sovip is involved in host immune response during bacterial infection & by different bacterial pathogens it is differentially regulated at transcription level (Dang W. et. al . 2010).

Viperin expression has also been noticed in inflammatory disease, atherosclerosis

(Olofsson P.S et. al . 2005). Most recent study (Hinson E. R et. al . 2010) shows that viperin is highly expressed in-vivo during acute lymphocytic choriomeningitis virus

(LCMV) Armstrong infection in neutophils and macrophages and also in Tcells, B cells and dendritic cells but in chronic infection expression can only be detected in neutrophils and macrophages. Thus, indicating the role of viperin against bacterial and parasitic infections and showing viperin potential more against bacterial infection than viruses. Studies (Qiu L.Q et. al . 2009) on viperin knockdown mice show that

Viperin facilitates TCell receptor mediated GATA3 activation and optimal Thelper

2 (Th2) cytokine production by modulating NFκB (nuclear factor kappalightchain enhancer of activated B cells) and activator protein (AP1) activities, thus is also involved in adaptive immune response.

Although viperin has been recognized for its incredible antiviral activities, study on Japanese encephalitis virus (JEV) (Chan Y.L, et. al . 2008) shows that virus can evade the antiviral effect of viperin. Viperin gene expression is highly induced by

JEV but it is negatively regulated at protein level and it is due to the degradation of protein by proteosome pathway in JEV infected cells.

Over the past few years, significant research on viperin has proved its antiviral activity but the mechanism of its action since its discovery in 1997 against viral infection remains enigmatic

2.1.3 Viperin Induction and Action

Interferon stimulated genes (ISGs) are induced in two ways upon virus entry – IFN dependent induction and IFN independent induction. Viperin can also be induced by any of the two pathways depending upon the infecting virus (Rivieccio

M.A. et al . 2006). Viral infection triggers upregulation of IFN regulatory factor–3

(IRF3) which stimulates IFN production. IFN signaling through JakStat pathway

activates formation of IFN stimulated gene factor3 (ISGF3), which later on translocation to nucleus triggers IFN stimulated responsive element (ISRE). This up regulates several IFNstimulated genes (ISGs) such as viperin (Figure 2.1.2). IRF3 can induce ISRE directly in IFNindependent pathway and its activation is enough to induce viperin gene expression. IRF1 also induces viperin by IFN independent pathway. Viperin interacts with FPPS after migrating to cytosolic face of ER. Viperin

FPPS binding disrupts the lipid rafts and increase membrane fluidity, thereby forming viral buds with daisychain like structures, which cannot be released from the plasma membrane (Wang et. al 2007).

Figure 2.1.2 Schematic representation of immune response pathway that leads to the disruption of viral release from the plasma membrane. Red color represents FPPS , Green color represents Viperin . (Duschene K. S et. al . 2010)

2.1.4 Influenza virus inhibition by viperin

Although viperin has been widely studied for its antiviral activity but the mechanism of action is still ambiguous. Influenza virus infection kicks off with binding of HA (hemagglutinin) to the cell surface receptor, sialic acid. After attachment virus gets the entry inside the cell via clatherin mediated endocytosis. The low endosomal pH triggers the membrane fusion and release of viral genome into the cytosol and eventually to the nucleus, which is hijacked by the virus to synthesize its own mRNA and to replicate its genome. The viral mRNAs are later released into the cytosol for translation of viral transmembrane glycoproteins, HA (hemagglutinin), and NA (neuraminidase). These proteins traffic to plasma membrane lipid raft which is the important site for release of newly formed viral particles. Viperin effect is noticed (Wang X. et. al 2007) (Figure 2.1.3) at the release stage of influenza virus where virions on the surface of viperin expressing cells shows an abnormal stalk or chain like structure in which two or more viral particles are connected but cannot be released. The results suggest that viperin disrupts the lipid raft formation and which in turn lead to inhibition of release of viral particles and it does so by interacting with

FPPS (Farnesyl pyrophosphate synthase) which is an important enzyme for cholesterol and isoprenoid biosynthesis.

Figure 2.1.3 Model diagram showing the Influenza A virus release upon viperin expression and interaction with FPPS. Left panel shows virus budding in the absence of viperin. Right panel shows IFN induced viperin expression followed by its interaction with FPPS and disruption of lipid rafts and finally blockage of viral budding. (Adapted from Wang X. et.al . 2007)

2.1.5 Radical SAM domain proteins

Radical SadenosylLmethionine enzymes constitute a superfamily of enzymes which includes 2800 putative members from all three kingdoms of life. They are involved in numerous biosynthetic pathways. BioB (biotin synthase), LAM (lysine

2,3aminomutase), LipA (lipoate synthase) and MoaA (cofactor for molybdopterin biosynthesis) are few examples from this family. These enzymes catalyze diverse reactions but have one common function that all such enzymes contain i.e., [4Fe–4S] cluster which is coordinated by the cysteins from CXXXCXXC motif, which is the defining characteristic of these proteins. This motif is conserved in all radical SAM enzymes required for the synthesis of molybdopterin (Mao A) (Figure 2.1.4 A), heme

D1 (NIRJ), and PQQ (PQQIII) [Sofia H.J. et.al . (2001) and Hanzelmann P. et. al .

(2004)]. This family of enzymes requires SAM as cofactor to coordinate with the fourth free iron to make complete and functionally active [4Fe–4S] cluster (Figure

2.1.4 B). As such viperin has been hypothesized as a radical SAM domain protein because of the presence of conserved SAM motif (CXXXCXXC) in the sequence.

Below is the representation of typical chemical reaction catalyzed by radical SAM enzymes (Figure 2.1.4 D) (Layer G. et. al . 2004). The main steps of reaction are:

1. First, the [4Fe4S] center reduces to the +1 state by an external electron donor

(usually flavodoxin).

2. The electrons are transferred from reduced [4Fe4S]1+ center to the sulfonium

of SAM which undergoes hemolytic cleavage to methionine and a highly

reactive 5’deoxyadenosyl radical.

3. A hydrogen is abstracted from an appropriately placed substrate (R–H) by 5’

· deoxyadenosyl radical, creating a substratebased radical (R )

This free radical is further involved in catalyzing chain of reactions.

(A) (B)

Figure 2.1.4 A) Conserved CX3CX2C motifs derived from the human viperin (hVip); cofactor for molybdopterin biosynthesis (MoaA); lysine 2,3aminomutase (LAM); anaerobic ribonucleotide reductase (ARR); biotin synthase(BioB); and lipoyl synthase (LipA). (B) The structure of the MoaA [4Fe–4S] cluster incomplex with the ligand SAM.

Figure 2.1.4 C) The crystal structure of MoaA in complex with SAM (1TV8), with the region corresponding to the viperin Cterminus (214–361) colored in greencyan

Figure 2.1.4 D) Schematic representation of SAM domain catalyzed reaction. Reaction steps common to all Radical SAM enzymes. First, an external electron donor (usually flavodoxin) reduces the [4Fe4S] center to the +1state. Second, the [4Fe 4S] 1+ center transfers an electron to the sulfonium of SAM causing the homolytic cleavage of SAM to methionine and a highly reactive 5’deoxyadenosyl radical. Third, the 5’deoxyadenosyl radical abstracts a hydrogen from an appropriately placed substrate (R–H),creating a substratebased radical (R●). (Adapted from Layer G. et.al 2004)

2.1.6 AIMS

As described before Viperin is an important cellular protein which is evolutionary conserved in species from fish to primates. It has widely been accepted as a protein active against whole range of viruses, be it DNA virus or RNA virus. Its activity has also been noticed against bacteria and other pathogens, so is active in innate immune response also. However, so far there is no structure detail available about Viperin. The aims of this project are to:

1. Study the biophysical characteristics of this protein using techniques like CD

and NMR.

2. Study, if this protein is actually a radical SAM domain protein.

The results of this study will be of great contribution to unveil the

mechanism of action of viperin for its antiviral or antibacterial activities.

2.2 MATERIAL AND METHOD

2.2.1 Vector Construction

Total RNA was extracted from human Monocytic cells from HLCZ cell line, using Qiagen RNeasy Mini kit and then the fulllength viperin RNA was reverse transcribed into cDNA by RTPCR using Onestep RTPCR kit (Qiagen).

Subsequently, by using different pairs of primers, the 361residue human viperin was dissected into 12 fragments, which include nine with the Nterminus differentially truncated, spanning over residues 11–361, 22–361, 36–361, 43–361, 45– 361, 54–

361, 60–361, 71–361 and 81–361, respectively, as well as Nterminal fragment (1–

72); putative SAMcontaining region (71–213) and highly conserved Cterminus

(214–361). The schematic representation of truncation sites in sequence is shown in

Figure 2.2.1. Mutant construct of viperin 45361 was constructed by replacing cysteins with alanine from conserved radical SAM domain motif CXXXCXXC by site directed mutagenesis. All the constructs were designed such that it has BamH1 restriction site at Nterminal and Xho1 restriction site at Cterminal. These fragments as well as the entire viperin were subcloned into Histagged expression vector pET32a. The PCR product and vectors were double digested by BamH I and Xho I

(New England Biolab) at 37 ℃ for 4 hours and subsequently purified by QIAquick Gel

Extraction Kit (Qiagen). Ligation was then carried out at 16 ℃ overnight in the presence of T4 DNA Ligase (New England Biolab). The Colony screening was done by using E.coli DH5α for ligation. Plasmids were purified by QIAprep Spin Miniprep

Kit 250 (Qiagen). All of the above experiments followed the protocols provided by the manufacturers. All the procedures were monitored by Agarose DNA electrophoresis (BioRad). All DNA sequences were conﬁrmed by automated DNA sequencing.

Figure 2.2.1 Secondary structure prediction diagram of Viperin (Arrow represents the truncation site)

2.2.2 Protein Expression and Purification

2.2.2.1 Expression and purification of insoluble proteins

The recombinant full length Histag Viperin and also the truncated constructs were expressed in the bacterial strain BL21. A single colony was picked and inoculated to the ampicillin (final concentration of 100µg/ml) containing LB broth and kept in an incubator shaker at 37°C at 200rpm for overnight. This seed culture was used to inoculate 1L LB media (100µg/ml ampicillin) with ratio of 1:100.

Briefly, the cells were cultured at 37 ℃ so as to reach OD of 0.6 at 600nm λ. The culture was then induced by IPTG with a final concentration of 0.4 mM and kept for expression for 16 hours at 20 ℃. Cells were harvested by centrifugation at 6000 rpm, Beckman JA 8.1000 rotor and disrupted in lysis buffer [(50mM TrisHCl buffer

(pH7.5) with 150mM NaCl and 10mM βME)] by using ultra sonicator at 4 ℃. The lysate was discarded and the pellet was washed three times with TBS buffer [(50mM

TrisHCl (pH7.5) 150mM NaCl)] with 0.1% triton x100 and then again washed with same buffer but without triton X100. After every wash the suspension was centrifuged to separate pellet and supernatant. Finally, the pellet was dissolved in 8M urea and the supernatant was separated by ultracentrifugation at 18000rpm for 30 minutes at 4°C. The supernatant of the centrifuged lysate was allowed to bind with

Nickel beads resin. The resin was washed with buffer A [8M urea, 50mM Tris

HCl (pH 8.0), 150mM NaCl, 20mM imidazole]. Protein was eluted from Nickel bead resin by elution buffer (buffer A with 250mM imidiazole). Elute was further purified by HPLC puriﬁcation on a reversephase C4 column (Vydac). The identities of recombinant proteins described above were veriﬁed by MALDITOF mass spectrometry.

2.2.2.2 Expression, Purification and Reconstitution of the [Fe 4–S4] cluster in

Viperin (45–361)

Recombinant containing a putative SAM domain was also expressed the same way as other constructs but with the exception of media content. The LB media for

Viperin (45–361) was supplemented with 0.15mM FeCl 3 to facilitate the reconstitution of [Fe 4–S4]. Brieﬂy, the cells were cultured at 37°C to an OD 600 of 0.6 and then IPTG was added to a ﬁnal concentration of 0.4mM to induce expression of the recombinant proteins for 5h at 30°C. Cells were harvested by centrifugation at

6000 rpm, Beckman JA 8.1000 rotor and disrupted in lysis buffer [(50mM Na 2HPO 4

(pH 6,5), 150mM NaCl, 20mM imidazole, 10mM βME and 10% glycerol] by using ultra sonicator at 4 ℃. The supernatant of the centrifuged lysate was allowed to bind with Nickel beads resin and on elution was subjected to FPLC puriﬁcation using a gel

ﬁltration column (HiLoad 16/60 Superdex 200). Unfortunately initial experiments were performed under aerobic condition. As a result, the protein samples puriﬁed under aerobic condition was colorless, indicating no formation of the [Fe 4–S4] cluster.

Further reconstitution following the previous protocol (Paraskevopoulou C. et. al .

2006) also failed under aerobic condition. Moreover protein tends to precipitate during purification and was highly unstable. To overcome this, viperin (45–361) after binding to nickel resin was purified in anaerobic chamber (DW Scientiﬁc MACSMG

1000 anaerobic chamber). All the buffers used were degassed by purging with nitrogen (N 2) gas. In the anaerobic chamber the resin was washed with buffer A

[50mM Na 2HPO 4 (pH 6,5), 150 mM NaCl, 20 mM imidazole, 10mM βME and

10% glycerol] and after ingel digestion with thrombin for 4hrs at room temperature was eluted with buffer B [50 mM Na 2HPO 4 (pH 6,5), 500 mM NaCl, 10mM βME and 10% glycerol]. The eluate this time was yellow in color, indicating the formation

of the [Fe 4–S4] cluster to some extent. As such, the reconstitution was further performed under anaerobic condition by incubating the puriﬁed Viperin (45–361) protein with 10 mM DTT for 30 min, followed by further incubation for 1 h in the presence of Na 2S and (NH 4)2Fe(SO 4)2 at a 10 M excess (C. Paraskevopoulou et. al .).

The reconstituted protein was desalted by bufferexchange in Amicon centrifuge tubes with a molecular weight cutoff of 10KDa. The identity of recombinant protein was veriﬁed by MALDITOF mass spectrometry and also the protein sequence of Viperin

(45–361) was further conﬁrmed by sequencing 75 overlapped trypsindigested peptide segments with mass spectrometry on a Micromass QTOF 2 machine.

2.2.3 Media prepration for NMR sample

For heteronuclear NMR experiments, 15 Nlabeled proteins were prepared following the similar expression and puriﬁcation procedure except for growing E. coli cells in M9 medium. E.coli cells in the M9 minimal medium (Na 2HPO 4•12H 2O

17.1g/L (sigma); KH 2PO 4 3g/L (sigma); NaCl 0.5g/L; (15 NH 4)2SO 4 1g/L

(Cambridge Isotope Laboratories Inc.); Glucose 4g/L (Sigma); MgSO 4 1mM (Merck);

Thiamine 2mg/L (Sigma); ampicillin 75mg/L). The (15NH 4)2SO 4 salt was used for

15 Nisotope labeling

2.2.4 UV–visible Spectroscopy

UV–visible spectra for viperin (45361) were collected on a Nicolet evolution 300 spectrophotometer with a wavelength ranging from 295 to 585nm.

2.2.5 Circular dichroism (CD)

All CD experiments were performed on a Jasco J810 spectropolarimeter equipped with a thermal controller using 1mm path length cuvettes. Viperin (45–361) was dissolved in 10mM phosphate buffer (pH 6.5). However, the Cterminus Viperin

(214–361) was insoluble in buffers and consequently was solubilized in saltfree

water (pH 4.0). The farUV CD spectra were recorded at protein concentrations of

20M while the nearUV CD spectra were recorded at protein concentrations of

100M at 25°C. The thermal unfolding was conducted from 15°C to 95°C. Final CD spectra were obtained by adding and averaging data from three independent scans.

Estimation of the secondary structure contents by deconvoluting farUV CD spectra is performed with the CDPro software package

(http://lamar.colostate.edu/;sreeram/CDPro ).

2.2.6 NMR sample prepration

NMR samples were prepared by bufferexchanging the proteins into phosphate buffers (pH 6.5) for Viperin (45–361) or by dissolving the powers into

MilliQ water for Viperin (214–361) and Viperin (71–361) at pH 4.0, with an addition of 50 L of D2O into 450 L samples for NMR spinlock. The final protein concentration for 1D and 2D experiments was 100µM. Onedimensional (1D) 1H

NMR and twodimensional (2D) 1H–15 N HSQC experiments were acquired on an

800MHz Bruker Advanced spectrometer equipped with pulse ﬁeld gradient units at

298K. The 1D and 2D NMR data was processed by using NMRDraw and NMRview softwares.

2.3 Results & Discussion

2.3 Structure details of Viperin

2.3.1 Structural characterization of the human viperin fragments

The experiments were done by cloning the fulllength viperin and its 12 dissected fragments into Histagged expression vector. However, the full length viperin was found not to be expressed. For other fragments, only ﬁve were expressible, which include fragments (43–361), (45–361), (71–361), (81–361) and

(214–361). Table 2.3.1 gives a description of constructs made and expression pattern, whether in the soluble fraction or insoluble fraction. Out of them, the fragments (71–

361), (81–361) and (214–361) were only found in inclusion body and could not be refolded by fast dilution or dialysis against various buffers. Viperin (43–361) was found to be partly in supernatant but the purified protein was very unstable, which was prone to aggregation even at very low concentrations (<20M). Interestingly, the majority of the recombinant Viperin (45–361) protein existed in supernatant and consequently it was purified by Ni 2+ affinity column under native condition, followed by FPLC purification after the thrombin cleavage to remove Histag. From 1 liter culture 10mg of purified protein was obtained in approximately 100µl of buffer solution.The FPLC and DLS profiles (Figure2.3.1) show that the protein is homogenous and is monomeric. Moreover, its protein sequence was also confirmed by trypsin digestion followed by sequencing with mass spectrometry.

Table 2.3.1 Brief description of truncated fragments of Viperin with their expression patterns and the techniques used to study the purified protein.

Figure 2.3.1A) FPLC purification profile of Viperin (45361)

Figure 2.3.1B) DLS profile of Viperin (45361)

As presented in Figure 2.3.2 A, although expressed in the medium without

FeCl 3 and puriﬁed under aerobic condition, the recombinant Viperin (45–361) protein has a farUV CD spectrum with one positive signal at 197 nm, and two negative ones at 208 and 222nm, respectively, indicating the presence of a large fraction of αhelix secondary structure. Indeed, further analysis of the CD spectrum indicates that the unconstituted Viperin (45–361) already contains 65% αhelix; 19% βstrand/turn and

16% unordered secondary structures. However, the unconstituted protein was unstable and prone to aggregation. For example, during the thermal unfolding at a protein concentration of 20 M, precipitation started at 30°C and almost completed at 45°C

(Figure 2.3.2 A). On the other hand, Viperin (45–361) appears to have considerable tertiary packing as evident from a dramatic difference between its nearUV spectra in the absence and presence of 8 M urea (Figure 2.3.2 B). Furthermore, the unconstituted

Viperin (45–361) seems undergoing conformational exchanges on µm–ms time scale

or/and dynamic aggregation over some regions. Consequently no very upﬁeld peak is observed located closed to or less than 0ppm in its 1D NMR spectrum and HSQC peaks can only be detected for a small portion of residues (Figure 2.3.2 C). For instance, only one peak is visible for ﬁve Trp side chains in Viperin (45–361).

Figure 2.3.2 Characterization of Viperin (45–361) puriﬁed under aerobic conditions (A) FarUV CD spectra of Viperin (45–361) at a concentration of 20µM in 10mM phosphate buffer (pH 6.5) at different temperatures. The spectrum at 15°C is colored in blue and that at 95°C is in red. Inlet: thermal unfolding curve as monitored at 222 nm. (B) NearUV CD spectra of Viperin (45–361) at a concentration of 100µM in 10mM phosphate buffer (pH6.5) at 25°C in the absence (blue) and presence of 8M urea (red).

2.3.2 Reconstitution of the [4Fe–4S] cluster in Viperin (45–361) and viperin (45

361) mutant

Since viperin has been hypothesized to be a radical SAM enzyme, we thus attempted to reconstitute the [Fe 4–S4] cluster in Viperin (45–361). Initially experiments were conducted under aerobic condition and consequently the reconstitution failed. However, when experiments were performed under anaerobic condition, the Viperin (45–361) sample puriﬁed from E. coli cells cultured in the medium containing FeCl 3 is yellowish (Figure 2.3.3A). This implies that the sample may already gain a small amount of the [Fe 4–S4] clusters. Therefore, we further

conducted the reconstitution under anaerobic condition by incubating Viperin (45–

361) with 10mM DTT for 30 min, followed by further incubation for 1 h in the

Figure 2.3.2(C) 1H–15 N heteronuclear single quantum correlation (HSQC) spectrum of Viperin (45–361) at a concentration of 100µM in 10mM phosphate buffer (pH6.5) at 25°C. The green arrow is used to indicate the peak resulting from the side chain of Trp residue presence of Na 2S and (NH 4)2Fe(SO 4)2 at a 10M excess (C. Paraskevopoulou et. al .

2006) and was spinned down at 3000 rpm for 10min. to remove any precipitation.

Strikingly, the reconstituted sample becomes brownish (Figure 2.3.3A), indicating that the [Fe 4–S4] cluster has been successfully reconstituted in Viperin (45–361). The similar studies when done with Viperin mutant it didn’t show any brown color formation, indicating the importance of CXXXCXXC motif in the chemistry of radical SAM domain protein.

Further evidence for [Fe 4–S4] cluster formation in the reconstituted viperin

(45361) was confirmed by the UV–visible spectroscopic characterization (Figure

2.3.3 B). The reconstituted sample has the absorption property characteristic of [Fe 4–

S4] clusters: a shoulder at 325nm and a broad peak at 415nm. By contrast, the sample puriﬁed under aerobic condition has no absorption at the two wavelengths.

Interestingly, the unconstituted sample but puriﬁed under anaerobic condition also has the two peaks but their intensities are much smaller, indicating the presence of a small amount of residual [Fe 4–S4] clusters only. Lack of infrastructure for Electron

Paramagnetic resonance (EPR) spectroscopy hindered our future experiments for the more convincing results regarding the Radical SAM chemistry of viperin but UV visible spectroscopy, Circular dichroism together with NMR help us to give convincing results to prove the radical SAM behavior as well as for the structure characterization of viperin. Notably, as revealed by deconvoluting the farUV CD spectrum (Figure 2.3.3C), the reconstituted Viperin (45–361) has a higher content of ordered secondary structures than unconstituted one, with 70% αhelix; 25% β strand/turn. This suggests that the reconstitution of the [Fe 4–S4] cluster can induce further formation of the ordered secondary structures from unordered regions in

Viperin (45–361). Furthermore, upon reconstitution, the thermal stability of Viperin

(45– 361) increased. The reconstituted sample started to precipitate at 45°C (Figure

2.3.3 C) while the unconstituted one began at 30°C (Figure 2.3.2 A). In particular, the very upﬁeld NMR peaks are observed for samples 2 and 3 (Figure 2.3.3 E), but not 1, indicating that the reconstitution also triggered the formation of the tight tertiary packing. As such, the very broad NH peaks may mostly result from the paramagnetic effect of Fe ion.

Viperin (45–361) was characterized by CD spectroscopy to have largely formed secondary structures and considerable tertiary packing even before the reconstitution of the [4Fe–4S] cluster. This implies that the unconstituted Viperin

(45–361) may already adopt the overall fold common to radical SAM enzymes, which contains a large content of ahelix, as shown by the MoaA structure (Figure 2.1.4 C).

However, the unconstituted Viperin (45–361) appears less stable and is more prone to aggregation than the reconstituted one.

Figure 2.3.3 Characterization of Viperin (45–361) after reconstitution (A) NMR samples of Viperin (45–361) puriﬁed under aerobic (1); anaerobic condition (2); and after reconstitution (3) (B) UV–visible spectra (295–585nm) of different Viperin (45– 361) samples at protein concentrations of 30 µM, puriﬁed under aerobic (1, black); anaerobic conditions (2, blue) and after reconstitution (3, red)

Figure 2.3.3 C) FarUV CD spectra of Viperin (45–361) after reconstitution at a concentration of 20 µM in 10mM phosphate buffer (pH 6.5) at different temperatures. The spectrum at 15°C is colored in blue and that at 95°C is in red. Inlet: thermal unfolding curve as monitored at 222nm. (D) NearUV CD spectra of the reconstituted Viperin (45–361) at a concentration of 100µM in 10mM phosphate buffer (pH 6.5) in the absence (blue) and presence of 8M urea (red) at 25°C.

Figure 2.3.3 E) Onedimensional 1H NMR spectra of Viperin (45–361) at 25°C and a concentration of 250µM in 50mM phosphate buffer (pH 6.5) containing 500mM NaCl, 10%Glycerol, 10mM βME, 3mM DTT; puriﬁed under anaerobic conditions (upper panel) and after reconstitution (lower panel). The red arrows indicate the very upﬁeld NMR resonance peaks.

2.3.3 Structural characterization of the bufferinsoluble Viperin (214–361)

Since the Cterminal region is highly conserved within all viperin proteins but has no signiﬁcant homology with other proteins, here we present the detailed characterization of the human Viperin (214–361) by CD and NMR spectroscopy.

Viperin (214–361) was highly expressed in E. coli cells but all existed in inclusion body. As such, we purified the fragment first by Ni 2+ affinity column under denaturing condition, followed by HPLC purification on a C4 column. Intriguingly the lyophilized powder of Viperin (214–361) was insoluble in buffers but again could be dissolved in saltfree water (pH 4.0) at a protein concentration even up to 1mM. As seen in Figure 2.3.4A, Viperin (214–361) in saltfree water has a farUV CD spectrum with a positive signal at 192nm and two negative signals at 207 and 220 nm, respectively. Deconvolution of the CD spectrum indicates that it contains 24% α helix; 16% βstrand/turn and 60% unordered secondary structures. Furthermore,

Viperin (214–361) showed no precipitation during thermal unfolding even up to

95°C. Noticeably, as judged by the farUV CD spectrum (Figure 2.3.4A), even at

95°C its structure is not completely random coil. On the other hand, the non cooperative unfolding curve implies that Viperin (214–361) has no tight tertiary packing. Also it is worthwhile to note that introduction of salt signiﬁcantly induces the aggregation of Viperin (214–361) even at a protein concentration of only 20mM.

At 12mM NaCl, almost all protein precipitated (Figure 2.3.4B). Consistent with CD results, the small HSQC spectral dispersions (Figure 2.3.4C) again indicate that

Viperin (214–361) has no tight tertiary packing. Interestingly, for Viperin (214–361)

HSQC peaks can be detected for all three Trp side chains (Figure 2.3.4C). Similar results were obtained for viperin 71361 construct which constitutes SAM domain and

Cterminal domain (Figure 2.3.5). HSQC spectrum for this construct shows very small dispersion and only 1 Trp residue peak can be seen.

Figure 2.3.4 Characterization of Viperin (214–361) solubilized in saltfree water. (A) FarUV CD spectra of Viperin (214–361) at a protein concentration of 20µM in salt free water (pH 4.0) at different temperatures. The spectrum at 15°C is colored in blue and that at 95°C is in red. Inlet: thermal unfolding curve as monitored at 222nm. (C) FarUV CD spectra of Viperin (214–361) at a protein concentration of 20µM at different concentrations of NaCl (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10 and 12 mM) at 25°C. The spectrum in saltfree water (pH 4.0) without addition of NaCl is colored in blue while that with a ﬁnal NaCl concentration of 12mM is in red. The Viperin (214– 361) forms visible aggregates at 12mM NaCl.

Figure 2.3.4 (B) 1H15 N HSQC spectrum of Viperin (214–361) at a protein concentration of 1mM in saltfree water (pH 4.0) at 25°C

Figure 2.3.5 1H15 N HSQC spectrum of Viperin (71–361) at a protein concentration of 100µM in saltfree water (pH 4.0) at 25°C.

2.3.4 Discussion

Interferons (IFNs) initiate the first line of defense against viral infection and they do so by stimulating hundreds of interferon stimulated genes (IFG’s) present in the system. Viperin is an example of one such gene which is highly inducible by both type I and type II IFNs. The key role of viperin in innate immunity is strongly implicated by its evolutionary conservation across both mammals and lower vertebrates and its antiviral activity has constantly been proved against whole range of viruses. This extra ordinary activity of viperin can be of great interest to target for drug discovery. It can be a wonder drug like penicillin, which is a potent antibiotic against almost all bacteria.

Since the discovery of Viperin in 1999, a lot of research work has been done on it to access its antiviral or antimicrobial activities against different infectious agents but there is no work reported for the structure or biophysical studies of this protein. Therefore, we initiated the present structural study on the viperin protein in an original attempt to determine its structure. Although we have not solved the 3D structure of viperin but our attempt to study the structure by CD and NMR shows that viperin is a well folded protein. Based on the presence of a characteristic CX3CX2C motif, it was extensively hypothesized that viperin might be a radical SAM enzyme.

However, previous attempts to reconstitute the [4Fe–4S] cluster in viperin all failed.

In the present study, by a systematic dissection of viperin into 12 fragments, the

Viperin (45–361) fragment only with the Nterminal transmembrane helix deleted has been successfully identified to be soluble and structured in buffers. The successful attempt to reconstituting the [4Fe–4S] cluster in Viperin (45–361) under anaerobic condition provides the first experimental evidence confirming that viperin is indeed a radical SAM enzyme. This finding is novel and quite important to study the

mechanism of viperin activity. Our findings show that the unreconstituted protein is unstable and tend to aggregate soon while reconstituted protein is more stable.

In different radical SAM enzymes, the region corresponding to Viperin

(214–361) is highly variable and recognized to be responsible for binding to specific substrates. The experiments here demonstrate that despite having some helix secondary structure, the Viperin Cterminus over residues 214–361 is only soluble in salt free water but not buffers. Furthermore, as revealed by CD analysis, a large portion of Viperin (214–361) appears to become unfolded upon being isolated from

Viperin (45–361). The high insolubility of Viperin (214–361) in buffers is caused by a significant exposure of the hydrophobic side chains to bulk solvent, which likely results from the inappropriate dissection. If Viperin (45–361) adopts the SAM enzyme fold, the Cterminal residues over 214–361 should have extensive packing interactions with residues over 45–361. Consequently, the dissection of Viperin (214–

361) will result in a dramatic loss of these packing interactions, thus leading to the unfolding of some regions, as well as a significant exposure of hydrophobic side chains. Taken together, the results from the present study imply that most residues of

Viperin (45–361) may be required to form the SAM enzyme fold, which is not further dissectible. This speculation is further supported by detailed CD and NMR characterization of another fragment, Viperin (71–361). Viperin (71–361) was also insoluble in buffers but again could be dissolved in saltfree water at high protein concentrations. Similarly, Viperin (71–361) is only partially folded in salt free water, with no tight tertiary packing.

2.3.5 Future Work

Studies show that viperin inhibits several viruses in vitro but still viruses can evade immune response caused by it. Future work should be done to get a better

perspective about it. Viperin is very active against viruses but its mechanism of action is still not clear. Our work shows that it is a radical SAM enzyme. To uncover the molecular mechanism by which viperin functions, it is important to identify the chemical reactions catalyzed by viperin. It may be the chemical molecules catalyzed by Viperin radical SAM enzyme, which are responsible for its antiviral activity or some pathway controlled by viperin carrying out the antiviral function. Moreover, the chemical molecules formed in the catalytic reaction by viperin may bear key information for further design of antiviral drugs of significant therapeutic applications. Also, structure studies of viperin along with binding partner, FPPS can help to solve the mystery behind viperin activity.

PART –II VAPC interraction with NS5B

CHAPTER – 3 Biological significance of

VAPC

3 Introduction

3.1 Hepatitis

What is Hepatits C ? Hepatitis C is an infectious disease caused by Hepatitis C virus, causing liver inflammation. It was first discovered in mid 1970s by Alter H.J. and group. He and his research team found that most posttransfusion hepatitis cases were not due to hepatits A or B and named as “nonA, nonB hepatitis” (NANBH).

For a decade there was no progress in identification of virus but then in 1989 Choo Q.

L. and team isolated HCV virus genome for the first time using random polymerase chain reaction (PCR) assays in plasma of Chimpanzee chronically infected with non

A, nonB hepatitis and hence the virus was renamed as Hepatitis C virus.

Hepatitis C virus infection is a global problem infecting millions of people worldwide. According to World Health Organization (WHO Fact sheet N°164) it infects 170180 million people globally which accounts to about 3% of world’s population (Figure 3.1.1). Every year 34 million people get infected with HCV and

350,000 dies of infection. It is the major reason for liver transplants. As per WHO

2010 report about 1% of Singapores population which estimates to 46090 individuals are infected with HCV.

3.1.1 Acute and Chronic Hepatitis

Acute hepatitis C infection is asymptomatic, means no visible symptoms of disease can be detected. 1525% of individuals infected with HCV clear the virus from the body during the acute phase while rest develops with chronic hepatitis.

Chronic infection is often asymptomatic until extensive liver damage has occurred. Chronic infection leads to cirrhosis, hepatocellular carcinoma, and hepatic

failure. Within span of few years 10 20% of infected individuals develop cirrhosis and 15% develop liver cancer (Centers for Disease Control and

Prevention, 1998). HCV associated liver damage is the most important cause for liver transplantation among adults.

Figure 3.1.1 HCV global prevalence 2010 (%) (Adapted from D. Lavanchy 2011)

3.1.2 HCV Genotype

HCV is a single stranded positive sense RNA virus which belongs to

Flaviviridae family. This family also includes other Flaviviruses like west nile virus, dengu virus and yellow fever virus. It is the only member of genus Hepacivirus

(Lindenbach et.al, 2007 ). HCV virus consists of RNA genetic material core surrounded by icosahedral protein envelope followed by lipid envelope of cellular origin. The virus has considerable genetic diversity. The re are 6 major genotypes of

HCV, numbered 1 to 6 , with several subtypes like a,b,c. The difference in the 6 major

HCV genotypes at nucleotide level is 31 33% and difference in subtype nucleotide

sequence is between 2025% (Simmonds P et. al . 2005). The geographical distribution of genotypes is different (table 3.1). Genotype 1 and 2 are broadly distributed throughout the globe, and are the dominant genotypes in Europe, North

America and Japan whereas in Asia it is genotype 6 which is prevalent. The geographical distribution of genotypes is important as different genotypes respond differently to the available HCV treatment.

Table 3.1 Geographical distribution of HCV genotypes: Predominant Hepatitis C genotype by region (IBASE, 2008) (DDO limited) HCV exists as a heterogenous population of different viruses, named as quasispecies within each infected individual (Murakawa K et.al . 1992). Lack of proof reading activity in RNA dependent RNA polymerase (RdRp) is the main reason for diversity in HCV genome which replicates the HCV RNA strand with an error rate of

10 2 to 10 3 nucleotide substitutions per site per year (Natyszyn H. J. 2005). The enormous genetic variability is the major hurdle in developing antiviral therapy against HCV.

3.1.3 Life cycle of HCV

The life cycle of HCV is similar to other members of Flaviviridae family.

Figure 3.1.2 depicts the main events in the life cycle of HCV. The life cycle begins with a) circulating infective HCV particles (virions) in the serum which interact with host lipoprotein receptor molecules at the cell surface. These virions are b)

internalized by catherinmediated endocytosis of receptorvirus complex. Low pH of endocytic compartment results in the c) fusion of viral membrane with endosomal vesicle and thus the release of singlestranded , naked viral RNA into the cytoplasm of newly infected cell. The genomic RNA is d) translated by internal ribosomal entry site (IRES) – mediated translation to generate a single large polyprotein that is processed to produce 10 mature HCV proteins in association with virus derived ER like membrane structure termed as the membranous web. Progeny RNA is produced by the e) replication of RNA genome via a minus strand replicative intermediate. Few of the newly synthesized RNA is packaged into nucleocapsid, f) which bud from the

ER after association with the HCV glycoproteins. Viral genomic RNA is g) transferred from membranous web to the nucelocapsid core protein that coats cellular lipid droplets and so the maturation of particles occur. Viral nucleocapsids h) bud into the ER and are released from the cell, thus completing the life cycle.

Figure 3.1.2 Schematic diagram of life cycle of HCV. (Figure adapted from Tellinghuisen T.L et. al . 2007).

3.1.4 Genome organization

The HCV genome is 9.6 kb positive strand uncapped RNA molecule with a single large open reading frame (ORF). It is flanked by highly structured and conserved 3’ and 5’ noncoding regions (Figure 3.1.3) (Tellinghuisen T.L et. al .

2007). Translation of HCV genome depends on internal ribosome entry site (IRES) within 5’noncoding region (NCR). The HCV, IRES binds to 40S ribosomal subunit

(Spahn C. M. et al. 2001) and IRES40S complex then recruits eukaryotic initiation factor (eIF) 3 and MettRNAeIF2GTP complex to form translationally active 80S complex (Ji H. et. al . 2004). The large ORF is translated to yield a polyprotein of

3000 amino acids which is processed during and after translation by combination of host and viral proteases to release 10 proteins, which are two different kinds – structured and nonstructured (NS) (Houghton et al 1991, Van Doorn et al 1994).

Host proteases process the Nterminal part of polyprotein and release HCV structure proteins which are – Core protein, putative envelope proteins E1 and E2 which are glycosylated and a small protein p7. These are involved in the formation of viral particle by the assembly of RNA with core protein, E1 and E2 (Hijikata M. et.al .

1991; Lin C. et.al .1994). The remaining part of polyprotein is processed by NS2 and

NS3 viral proteases. NS2 cleaves at NS2/NS3 junction and release NS2 protein while

NS3 catalyze cleavage at NS3/NS4A, NS4A/NS4B, NS4B/NS5A, NS5A/NS5B to release NS3, NS4A, NS4B, NS5A and NS5B unstructured HCV proteins (Hijikata M et.al . 1993).

HCV unstructured proteins NS3, NS4A, NS4B, NS5A and NS5B form a membranebound replication complex (RC), together with viral RNA and cellular proteins (ElHage N. et.al . 2003). NS3 is multifunctional with protease activity at N terminus and Cterminus displays helicase/NTPase activity (Kolykhalov A. A et.al .

2000). Helicase activity is essential for unwinding of RNA and thus for viral RNA synthesis. NS4A act as a cofactor for NS3 stability, protease activity as well as helicase activity (Failla C. et.al . 1994). NS4B is highly hydrophobic and induces the formation of membranous web structure by inducing specific changes to intracellular membrane (Egger D. et.al . 2002). NS4B, NS4A and NS3 associate with ER via NS4B while NS5A associates with ER through its Nterminal amphiphatic helix and NS5B through its Cterminal hydrophobic region. Synthesis of fulllength negative strand

RNA marks the beginning of HCV genome replication and it is catalyzed by NS5B

(RNA dependent RNA polymerase, RdRp), which is the catalytic subunit for replication within this complex (Behrens S. E. et.al . 1996). Most of the HCV unstructured proteins involved in viral RNA synthesis process are associated with the host cell membrane and replication processes which like most of the positivesense

RNA viruses occurs in close association with host cell membrane (Hijikata et al.,

1993). Recent studies show that some cellular proteins interacts with HCV unstructured proteins NS5A and NS5B and are also involved in the HCV replication process.

3.1.5 NS5B (RNA dependent RNA polymerase, RdRp)

The NS5B is positioned at the Cterminus of virally encoded HCV polyprotein of ~3000 amino acids. NS5B is membrane associated and is distributed in the perinuclear region (Hwang SB et.al .1997). It is 66 KDa protein with composition of 591 amino acids. The GDD motif which is the signature sequence for RdRp is located at Gly317Asp318Asp319 positions respectively. HCV NS5B functions as an

RNAdependent RNA polymerase as well as a RNAbinding protein that is required for viral RNA synthesis (Andino R. et al . 1993). NS5B structure details show that it is a typical ‘right handed’ polymerase similar to most of the RNA dependent RNA

polymerase enzymes (Joyce C et.al . 1995). Its structural organization consists of Palm domain, finger domain and thumb domain, with catalytic site located at the palm domain (Figure 3.1.4). Thumb domain and palm domain encircle the active site located within the palm domain (Lesburg C. A et.al . 1999). Studies show that NS5B

(RNA polymerase) interacts with human VAP both in vitro and in vivo, suggesting that it may serve as a membrane receptor for HCV replication complexes.

Figure 3.1.3 HCV genome organization, polyprotein processing and topology. A) The HCV genome is singlestranded positive sense RNA encoding a single large open reading frame (ORF) of approximately 3000 amino acid residues. The translation of ORF, via IRES elements, generate a large polyprotein, with Nterminus onethird consisting of structured proteins and remaining Cterminus with unstructured proteins. Polyprotein is processes by host and viral proteases to produce 10 individual HCV proteins. B) HCV proteins localization/topology respective to ER membrane. (Adapted from Tellinghuisen T.L et.al . 2007).

Figure 3.1.4 Crystal Structure of NS5B describing the location of finger, palm and thumb domain. (O’Farrell D. et.al . 2003)

3.1.6 VAP proteins

VAMPassociated proteins (vesicular associated membrane proteinassociated proteins also known as synaptobrevinassociated proteins) are expressed in all eukaryotes and are type II integral membrane proteins (Kagiwada S. et.al . 1998;

Nishimura Y. et al . 1999). The human VAP family protein includes VAPA, VAPB,

VAPC, and several newlyidentified spliced variants (Skehel PA et. al. 1995;

Nishimura Y. et. al. 1999; Nachreiner T. et.al. 2010). These proteins have diverse functionality but share same structural organization (Figure 3.1.5). VAPA and VAPB are composed of three conserved domains, with Nterminal immunoglobulinlike β sheet MSP (Major Sperm Protein) domain that has 22% sequence identity to the nematode MSP protein (Sepensowol S. et. al . 1989), a central coiledcoil domain and

Cterminal hydrophobic, membraneanchoring domain. VAPA and VAPB show 60% homology in the sequence. One recently discovered VAP protein, named VAPC, is a splice variant of VAPB. It lacks coiledcoil domain as well as TM (transmembrane)

domain (Nishimura Y. et. al . 1999). VAPC cterminus from 71 to 99 amino acid residues shares no homology to VAPA and VAPB, due to the frameshift

(Figure3.1.6).

Figure 3.1.5 General domain organization of VAP protein, with Nterminal MSP domain, middle amphipathic coiledcoil domain and hydrophobic membrane anchoring Cterminal domain.

Figure 3.1.6 Alignment of the amino acid sequences of hVAPC with hVAPB and hVAPC. The asterisks indicate the amino acid residues that are identical. Sequence in box represents the signature sequence of VAP proteins. Yellow highlighted region represent splice variant region of VAPC.

They are ubiquitously detected in mammalian organs, including the heart, placenta, lung, liver, skeletal muscle, and pancreas (Nishimura Y. et. al . 1999), suggesting their diverse functionality. VAP was first identified in Aplysia californica, a marine mollusc as a VAMP/synaptobrevinassociated protein, by yeast two hybrid screens (Skehel et.al ., 1995). Since then they have been identified in yeast,

Drosophila, mice, rats and humans (Nikawa J. et al . 1995; Weir M.L. et al . 1998;

Nishimura Y. et al ., 1999; Skehel P.A. et al . 2000; Pennetta G. et al . 2002). VAPs are involved in wide range of cellular processes mainly lipid transport and metabolism

(Nikawa, J. et al . 1995; Kawano M. et. al ., 2006), microtubule organization (Skehel

P.A et.al . 2000; Pannetta G et. al . 2002 ), and membrane trafficking (Skehel P.A et. al . 1995) and it does so by interacting with large number of interacellular proteins like

SNARE proteins (Skehel P.A et. al . 1995), tubulin (Pannetta G et. al . 2002), occluding (Lapierre L.A et. al . 1999) and many more. They also interact among themselves through transmembrane domains to form homodimer or heterodimer. VAP proteins also show interaction with viral proteins like Norwalk virus nonstructure proteins (Ettayebi K et.al . 2003), HCV virus proteins (Hamamoto I et.al . 2005; Tu H. et.al . 1999) and cowpea virus proteins (Carette J.E. et. al . 2002).

3.1.7 Interaction of VAP proteins with HCV proteins

VAPA and VAPB form homodimer or heterodimer through cterminal transmembrane domain. Studies show that VAPA and VAPB interacts with NS5B through MSP domain and with NS5A via coiledcoil domain (Tu H. et. al 1999). The first 70 residues of VAPC are same as in VAPB and are 77% homologus to that of

VAPA, suggesting that VAPC also have inclination to bind to NS5B but as it lacks coiledcoil domain it cannot bind to NS5A. The results also prove the same. Kukihara

H. et. al . (2009) by immunoprecipitation shows that VAPC interacts with NS5B but not with NS5A and its expression in the cell interrupts the interaction of VAPA and

VAPB to NS5B and thus impairs the HCV replication and particle formation of

HCV. VAPC is thus the negative regulator of HCV replication.

Gao L. et. al . (2003) (Figure 3.1.7 A) has proposed a model for HCV replication assembly formation and VAP proteins binding on the assembly. They hypothesize that after recruitment of NS4ANS4BNS5A on lipid raft via. NS4B, NS3 also get recruited at lipid raft with the help of its cofactor NS4A. Since hVAP33

(VAPA/VAPB) could be localized on various types of membranes in the cell, NS5B

along with hVAP33 (VAPA/VAPB) is then recruited to join NS4ANS4BNS5A and NS3 on the lipid raft. NS5A binds to the lipid membrane from the Nterminal while Cterminus of NS5B facilitates in its binding to the membrane. hVAP33 act as a linker between NS5A and NS5B, it binds with NS5B by MSP domain which is at the Nterminus and to NS5A by coilcoiled domain. Figure 3.1.7 B is the adapted version of Figure 3.1.7 A and shows the inhibition of replication complex formation after the binding of Nterminus of VAPC to NS5B. NS5B cannot anchor itself into the lipid raft membrane and is therefore dependent on the interaction of hVAP33 with NS5A and as VAPC cannot bind to NS5A, the complete replication assembly cannot be formed because of which HCV RNA replication cannot be executed. This characteristic of VAPC is important and can be studied further for drug discovery against Hepatitis C.

Figure 3.1.7A) Model for the mechanism of formation of HCV replication complex on lipid raft. The uncleaved NS4A4B5A recruits to lipid raft through NS4B. NS3 associates with lipid raft by binding to its cofactor NS4A. NS5B binds to hVAPA/h VAPB and moves to lipid raft and form a complex with NS4A4B5A via the interaction between NS5B and NS5A. The mechanism of entry of RNA into this complex is not known.

Figure 3.1.7 B) hVAPC binding to NS5B inhibits the formation of polymerase assembly complex and so inhibits viral RNA replication.

3.1.8 Therapeutics for HCV

Even after so many years of HCV virus discovery there is no therapeutic vaccine for its treatment. Till now the standard of care (SOC) for HCV consist of combination drug of pegylated interferon–α and antiviral ribavirin. But this interferon

(IFN)based therapy however, has limited efﬁcacy against the major genotype 1 of

HCV. This treatment has a success rate of only <50%, and is plagued with adverse effects (Ghany M.G. et. al. 2009; Schoggins J. et. al. 2011) . New treatment regimens were required that are more efficacious and better tolerated by all patients. On May

2011, FDA approved first direct acting antivirals (DAAs), Merck’s boceprevir

(Victrelis) and Vertex’s telaprevir (Incivek). Although, these target wider HCV genotypes but must be taken with a broadacting antiviral pill, ribavirin as well as with regular injections of pegylated interferon. Interferon stimulates the immune system but also shows side effects ranging from flulike fatigue to severe depression to cardiac problems. Because of adverse reactions all patients cannot take advantages of therapy. Moreover these therapies are quite expensive and cannot reach the people

in under developed nations, were HCV cases are more than other parts. The table below (Table 3.1.2) shows the price range of these new HCV therapies.

Table 3.1.2 Cost of updated HCV treatment with a protease inhibitor in United States (Swan T. 2011)

Table 3.1.3 HCV DirectActing Antivirals (DAA) in Clinical Trial Phase II or III, or FDAApproved (Adapted from Vachon M.L. et. al . 2011)

A lot more direct acting antivirals (DAA), some without inclusion of interferons targeted against HCV are in different phases of development (Table 3.1.3).

But a major concern for drug development is the emergence of drug resistant viruses under the selective pressures exerted by antiviral drugs. The intrinsic low fidelity of the HCV replication machinery and fast turnover rate (up to10 12 virions produced each day) causes every single genome copied to contain at least one mutation (Ogata N et.al 1991). As a result there is coexistence of lots of variant species named quasispecies.

At present the two HCV viral enzymes, NS3/4A serine protease and the

NS5B RdRp are the important targets for drug discovery (Behrens S.E. et.al. 1996), as they are amenable to the development of biochemical assays for inhibitor screening

(Kwo P.Y. et.al. 2011). Targeting enzymatic pathways however, leads to rapid development of drug resistance (Kwo P.Y. et.al . 2011; Halfon P. et. al . 2011).

Therefore, there is promising potential to target nonenzymatic processes such as those required for RNA replication.

A clinically successful HCV drug should be able to suppress all viral variants and inhibit emergence of resistant viruses. One approach to solve the problem is to target cellular proteins involved in viral replication. This can have advantage of higher genetic barrier to resistance and broader genotype coverage. VAPC which was recently identified to block the HCV RNA replication as well as HCV propagation by binding to NS5B could be an interesting and important target in drug discovery against HCV.

3.1.9 AIMS

As stated above, HCV is a worldwide problem and major reason for liver cancer and liver transplants. There is urgent need to find solution for this virus and one approach is to target the cellular proteins interacting with HCV proteins. The previous studies show the negative regulation of HCV replication by cellular protein,

VAPC. This protein could be an important lead in the discovery of effective drug against HCV. In drug discovery, knowledge of the inhibitor site of action is crucial to guiding medicinal chemistry efforts. The aims of the research project are:

1) The purpose of the project is to study the biophysical and structural properties

of VAPC.

2) To investigate the binding pattern of VAPC to HCV unstructured protein

NS5B which helps to gain insight into the interaction details of VAPC and

NS5B using NMR as a tool.

This study is important to discover the new drug lead for hepatitis, which

is a deadly infectious disease.

3.2 MATERIAL AND METHOD

3.2.1 Vector Construction

The gene for VAPC was constructed by using VAPB construct (from Shi

Jiahai), as Nterminal 70 residues of VAPC are homologous to VAPB. The primers were designed to clone Nterminal 70 residues form VAPB and longer primers were designed to synthesize Cterminal 29 residues and then overlapping primers were used to combine the two constructs and get full length VAPC. The full length VAPC was later used to design the truncated VAPC (VAPC43, VAPC31 and VAPC14) constructs. NS5B plasmid was constructed using pXJ40flagNS5B construct (from

Dr. Tan Yee Joe). It is a subtype 1b HCVS1 isolate. The primers were designed so as to truncate the Cterminal 21 residues of full length NS5B. All the constructs were designed such that it has BamH1 restriction site at Nterminal and Xho1 restriction site at Cterminal. The PCR product and vectors (VAPCpET32a, NS5BpET32a and

VAPC43pGEX4T1, VAPC31 pGEX4T1 and VAPC31 pGEX4T1) were double digested by BamH I and Xho I (New England Biolab) at 37 ℃ for 4 hours and subsequently purified by QIAquick Gel Extraction Kit (Qiagen). Ligation was then carried out at 16 ℃ overnight in the presence of T4 DNA Ligase (New England Biolab). The Colony screening was done by using E.coli DH5α for ligation. Plasmids were purified by QIAprep Spin Miniprep Kit 250 (Qiagen). All of the above experiments followed the protocols provided by the manufacturers. All the procedures were monitored by Agarose DNA electrophoresis (BioRad). The DNA sequence was confirmed by automated DNA sequencing.

3.2.2 Codon optimization

The original VAPC constructs when expressed in E.coli cells showed very poor expression. It had lot of amino acid residues, especially at Cterminus, whose

codons relative frequeny is very low in E.coli. So those codons were replaced by the codons, with high frequency E.coli codons by designing suitable primers.

3.2.3 Preparation of Competent E.coli Cells

Overnight culture of DH5α or BL21 cells was diluted into 50 ml fresh LB broth

(Bio Basic Inc.) at a ratio of 1:100. Cells were grown at 37 ℃ with shaking till OD600 reached 0.5~0.6. Then cells were transferred into sterile centrifuge tubes and spun down at 4000×g for 5min. The cell pellet was then gently resuspended with 0.5 volumes (25ml) of ice cold, sterile, fresh 50mM CaCl 2 (Merck) and immediately incubated on ice for at least 20 min. After that, cells were centrifuged down again at

4000×g for 5min. After discarding the supernatant, cells were resuspended with 0.1 volumes (5ml) of cold 50mM CaCl 2 containing 10% glycerol (BDH Laboratory

Supplies). Cell resuspension was then distributed in aliquots into 1.5ml sterile eppendorf tubes. Competent cells were stored at 80℃ after quick frozen by liquid nitrogen.

3.2.4 Transformation of E. coli Cells

1µl plasmid DNA was added to 100 µl of BL21 competent cells and cells were then incubated on ice for 30 min, followed by a heat shock at 42 ℃ for 90 seconds and a subsequent incubation on ice for 2 min. Then 500µl of LB was added to the tube and incubated at 37 ℃ for 1hr. before plating cells onto LB plates containing ampicilin (final concentration of 100µg/ml).

3.2.5 Protein Expression and Purification

3.2.5.1 Expression and purification of full length VAPC and truncated constructs

The recombinant full length histag VAPC and GSTtagged VAPCconstructs were expressed in the bacterial strain BL21. A single colony was picked and

inoculated to ampicillin (final concentration of 100µg/ml) containing LB broth and kept in an incubator shaker at 37°C at 200rpm for overnight. This seed culture was used to inoculate 1L LB media (100µg/ml ampicillin) with ratio of 1:100. Briefly, the cells were cultured at 37 ℃ so as to reach OD of 0.6 at 600nm wavelength. The culture was then induced by IPTG with a final concentration of 0.4mM and kept for expression for 16 hours at 20 ℃. Cells were harvested by centrifugation at 6000rpm, Beckman JA 8.1000 rotor and disrupted in lysis buffer (50mM TrisHCl buffer

(pH7.5) with 150mM NaCl) by using ultra sonicator at 4 ℃. The soluble proteins were separated by ultracentrifugation at 18000rpm for 30 minutes at 4°C. The supernatant of the centrifuged lysate was allowed to bind with Nickel beads resin. The resin was washed with buffer A [50mM TrisHCl (pH 7.5), 150mM NaCl, 20mM imidazole]. The protein elution was performed using buffer B [50mM TrisHCl

(pH 7.5), 150mM NaCl, 250mM imidazole]. Full length histagged VAPC was subsequently purified by HPLC on a reversephase C8 column. The recombinant

GSTVAPC truncated constructs were purified by GlutathioneSepharose 4B resin.

The resin was washed with buffer A [50mM TrisHCl (pH 7.5), 150mM NaCl,

20mM imidazole] and after overnight ingel digestion with thrombin respective protein/peptide was purified by HPLC on a reversephase C18 column. The identities of recombinant proteins described above were veriﬁed by MALDITOF mass spectrometry.

3.2.5.2 Expression and purification of NS5B

For NS5B construct designing Cterminal 21 residues were truncated to design His taggedpET32aNS5B. The construct was expressed in BL21 competent cell. A single colony was picked and inoculated to LB broth containing ampicillin

(final concentration of 100µg/ml) along with 0.5% filter sterilized glucose (NS5B 74

initial expression experiments were failed because of its leaky expression. Addition of

0.5% glucose overcame the problem of leaky expression) and kept in an incubator shaker at 37°C at 200rpm for overnight. This seed culture was used to inoculate 1L

LB media (100µg/ml ampicillin and 0.5% filter sterilized glucose) with ratio of 1:100.

Briefly, the cells were cultured at 37 ℃ so as to reach OD of 0.6 at 600nm wavelength. The culture was then induced by IPTG with a final concentration of 0.5mM and kept for expression for 16 hours at 20 ℃ at 180 rpm. Cells were harvested by centrifugation at 6000 rpm, Beckman JA 8.1000 rotor and disrupted in lysis buffer [(50mM TrisHCl buffer (pH8.0) with 300mM NaCl, 0.1% Triton X100, 10mM MgCl 2 and protease inhibitor tablet (Roche)] by using ultra sonicator at 4 ℃. The supernatant of the centrifuged lysate was allowed to bind with Nickel beads resin. The resin was washed with buffer A [50mM TrisHCl (pH 8.0), 150mM NaCl, 20mM imidazole]. The protein elution was performed using buffer B [50mM TrisHCl

(pH 8.0), 500mM NaCl, 250mM imidazole].

The eluted protein after Ni 2+ affinity chromatography was dialyzed in buffer with 20mM Tris, 200mM NaCl and 10mM βME at pH8.5. It was then purified by affinity purification AKTA FPLC machine (Amersham Biosciences) using HiTrap

Heparin column (GE healthcare) with buffer A composition of 20mM Tris, 200mM

NaCl and 10mM βME at pH8.5 and buffer B of 20mM Tris, 2M NaCl and 10mM β

ME at pH8.5. The eluted protein was dialyzed in 20mM Tris, 200mM NaCl and

10mM βME at pH 7.5 and this was further subjected to gel filtration using HiLoad

16/60 superdex75 column. For NMR experiments NS5B protein was later exchanged in 10mM phosphate buffer pH 6.5 and also containing 10mM DTT. The recombinant proteins were veriﬁed by MALDITOF mass spectrometry.

3.2.6 Preparation of Isotope Labeled Proteins

For heteronuclear NMR experiments, 15 Nlabeled proteins were prepared following the similar expression and purification procedures except that E.coli cells were cultured in the M9 minimal medium (Na 2HPO 4•12H 2O 17.1g/L (J. T. Baker);

KH 2PO 4 3g/L (J. T. Baker); NaCl 0.5g/L; (15NH 4)2SO 4 1g/L (Cambridge Isotope

Laboratories Inc.); Glucose 4g/L (Sigma); MgSO 4 1mM (Merck); Thiamine 2mg/L

15 (Sigma); ampicillin 75mg/L). The (15NH 4)2SO 4 salt was used for N labeling and for

13 C labelling, labeled Glucose (2g/L) was used.

3.2.7 Determination of Protein Concentration by Spectroscopy

The protein concentration was measured at 280nm using the BeerLambert

Law:

A = ε ∗l∗C,

Where ε is the molar absorption coefficient (M1cm1), l is the path length (cm), and

C is the protein concentration (M). The absorption A of a protein at 280nm depends on the content of Trp, Tyr, and Cys (disulfide bonds).

3.2.8 Circular Dichroism Spectroscopy

CD experiments were performed on a Jasco J810 spectropolarimeter equipped with a thermal controller using 1mm path length cuvette and 0.1nm spectra resolution.

The farUV (190260 nm) CD experiment was done with a sample concentration of

20µM in 10mM phosphate buffer at pH 6.5 at 25°C and near UV (250360nm) with a sample concentration of 100µM also at 25°C. A final CD spectrum was obtained by adding and averaging data from three independent scans.

3.2.9 NMR experiments

The NMR samples of full length VAPC and its truncated constructs, VAPC43,

VAPC31, VAPC14 and VAPC mutant were prepared by dissolving the lyophilized powder in 450µl of 10mM phosphate buffer pH 6.5 containing 10 mM DTT. 50µl

D2O (10% of total protein sample) was later added to all NMR samples for NMR spin lock signal.

Onedimensional (1D) 1H NMR twodimensional (2D) 1H–15 N HSQC (Sattler

M. 1999) experiments for full length VAPC were followed by backbone assignment by HNCACB and CBCACONH experiments and all these were carried out on Bruker

Avance 800MHz NMR spectrometer at temperature of 298K. Twodimensional

NOESY (Jeener J. 1979) and TOCSY (Bax 1985) experiments were done for the backbone assignment of VAPC43.

For the titration experiments, the concentrated NS5B protein was added to

VAPC full length and its constructs to reach a molar ratio of 1:1.5. Kd values were calculated from titration experiments of VAPC43 and VAPC14 with NS5B with the final molar ratio of 1: 2.75 using Datafit Software. Spectra processing and analysis was carried out using NMRview software (Johnson 2004). Sequencespecific assignments for VAPC full length were achieved through identification of spin systems in the HNCACB spectra, combined with sequential connectivities in the

CBCACONH spectra, similarly sequencespecific assignments for the VAPC43 were achieved through identification of spin systems in the TOCSY spectra, combined with sequential NOE connectivities in the NOESY spectra (Wagner, Wuthrich 1982; wuthrich K 1986).

3.3 RESULTS AND DISCUSSION

3.3.1 Protein purification

VAPC constructs were first purified by affinity purification using Ni 2+ beads or GST beads after which protein was purified by RPHPLC. The RPHPLC profiles of different VAPC constructs are shown in Figure 3.3.1A. The yield of VAPC full length was less (from 1liter media 50µM protein was obtained in 500µl buffer) as compared to VAPC43 and VAPC14 (1liter media gave ~100 µM and ~250 µM of respective protein in 500 µl of buffer solution).

Figure 3.3.1A) HPLC profiles of Top) VAPC full length Middle) VAPC43 Bottom) VAPC14

As NS5B is a RNA binding protein, this protein was purified using heparin column after affinity purification with Ni 2+ beads. After heparin column purification

(Figure 3.3.1B Top) protein was further purified by Superdex200 column (Figure

3.3.1B Bottom) to get very pure protein. The proteins purified were further used for structure and binding experiments. Yield of 6litre media was 250µl of 10mg purified protein.

Figure 3.3.1B) NS5B purification Top) by Heparin column Bottom) by Superdex200 column

3.3.2 Structure characterization of full length VAPC protein

Sequence analysis of VAPC shows (Figure 3.3.2 A) that it is abundant in amino acids P, S, R, K, E and Q which are found more frequently in unstructured proteins and are named as disorder promoting amino acids and is depleted in W, Y, F,

C, I, L and N residues, called as order promoting amino acids which are found more in the folded proteins (Dunker A.K. et al . 2001; Williams R.M. et al . 2001).This is in agreement to the CD profile of VAPC. As shown in Figure 3.3.2 B, the CD spectrum of VAPC in aqueous buffer (10mM Na 2HPO 4, 10mM DTT pH6.5) had a maximal negative peak at ~198 nm and had no significant positive signal at 192nm. In addition, there existed a very small negative shoulder signal at ~225nm. All of these indicated that VAPC possess a random coil structure but could have propensity (because of small negative shoulder at ~225nm) to attain some partial structure. pH titration ranging from pH 3 to pH 7.3 (Figure 3.3.2 B) shows that VAPC appeared to become more disordered as the amplitude of the maximal negative signal increased at ~200 nm.

The structural properties of the VAPC were further assessed by the NMR.

The 1D experiment (Figure 3.3.2 C) shows very narrow dispersion ranging from ~7.0 to 8.0ppm. Narrow dispersion is characteristic feature of unstructured proteins because they lack the hydrogen bond interactions at amide proton region and this results in severe overlapping in 2D [ 1H15 N]HSQC spectra. HSQC [ 1H15 N] experiment is a sensitive probe to tertiary packing and it is evident from the Figure

3.3.3 A that VAPC showed very narrow spectral dispersions over both dimensions

(~1ppm over 1H and ~10ppm over 15 N dimensions), indicating that VAPC lacks a

tight sidechain packing. In particular, the observed NMR cross peaks were only about 75, less than that expected for a 99residue protein, thus indicating that slow

Figure 3.3.2 A) Sequence of VAPC, residues with green color are found more frequently in unstructured proteins and in VAPC approx. 40% population is disorder promoting amino acids. B) Characterization of VAPC by CD spectrum; left panel shows the far UV spectra of VAPC and right panel depicts the pH titration CD spectrum of VAPC, where red color represents spectrum at pH 3.0 and blue color at pH 7.0 C) 1D 1H NMR of VAPC.

Figure 3.3.3 2D 1H15 N HSQC A) VAPC 2D shows narrow dispersion and shows amino acids specific clustering (represented with red circles) B) HSQC and sequential assignment of VAPC. The HSQC spectrum was acquired on an 800MHz NMR spectrometer in a 20mM phosphate buffer (pH6.5). The assigned residues are labeled in the spectra

conformational exchanges or/and fast exchange with H 2O existed over few regions of the protein. Usually, slow conformational exchange would result in significant line broadening for HSQC peaks and make these peaks undetectable. Consistent to the CD result, the HSQC spectrum above indicated that VAPC was unstructured. In addition to a lack of dispersion of the proton chemical shifts, 2D [ 1H 15 N]HSQC spectra of

VAPC exhibit characteristic amino acid type specific clustering (Figure 3.3.3A).

Backbone assignments of VAPC (full length) was achieved for nonproline residues with the analysis of a pair of tripleresonance experiments, CBCA(CO)NH and HNCACB, acquired on a 15 N/13 Clabeled VAPC sample at a protein concentration of 300 µM and the assigned HSQC spectra are shown in Figure 3.3.3B.

In total, 75% of the residues in the full length VAPC (1–99) were assigned (Cα and

Cβ).

Lack of well defined 3D structure has dramatic effect on carbon chemical shifts. The chemical shifts of Cα, Cβ, Co and Hα nuclei are highly sensitive to Φ/Ψ angle propensities of the backbone (Eliezer et al . 1998), and so to the secondary structure. The chemical shift index (CSI), or the difference between experimental chemical shifts and random coil chemical shifts (table 3.3.1), can be used to determine secondary structure in proteins (Szilagyi and Jardetzky 1989; Wishart and Sykes 1994). The Cα CSI is positive in an αhelix and negative in a βstrand and for VAPC Cα CSI it was found that it is closer to the random coil chemical shift values, again conforming that VAPC is a predominantly unstructured protein. As is evident from Figure 3.3.4, CSI of VAPC for most of the residues is even less than

0.2ppm. For few residues it reaches around 11.5ppm but that is insignificant as to have structure, at least 4 to 5 continuous residues should have CSI value of around

0.7ppm (Wishart and Sykes 1994b). All the experiments above confirm that VAPC is an intrinsically unstructured protein.

Table 3.3.1 Random Coil Chemical shifts (in ppm) for the 20 common amino acids in acidic 8 M urea (Adapted from Schwarzinger S. et.al . 2000)

VAPC Full lengthCα(obs ran) 2.5 2 1.5 1 0.5 0 CSD (ppm) CSD -0.5 MVVLQLRFVTKNDVKTPYRSIGISRAEAQFSWR -1 -1.5 -2 Residue Figure 3.3.4 Cα chemical shift values of VAPC for most residues collapse with the random coil values and so VAPC doesn’t have significant secondary structure.

3.3.3 VAPC forms a “fuzzy” complex with NS5B

Recent studies show that VAPC interacts with NS5B protein of HCV and inhibits the replication process of virus. For the interaction studies of VAPC with

NS5B, we purified the HCV NS5B protein and characterized it with CD and NMR.

The farUV CD (Figure 3.3.5 A) spectrum of NS5B shows characteristic of a well folded helixdominant protein, with two negative signals at ~209 and 222 nm, as well as a very large positive signal at ~193 nm. However, due to the short transverse relaxation time (T2) resulting from its very large molecular size, the NMR resonance peaks are very broad (Figure 3.3.5 B). But the presence of several highly upfield signals clearly demonstrate that NS5B is well folded, with a tight tertiary packing in solution.

Figure 3.3.5 A) The far UV CD spectra of NS5B shows to negative signals at ~209 and 222 nm respectively B) 1D 1H NMR of VAPC, the blue arrows points towards the upfield peaks. Subsequently, unlabelled NS5B was titrated with labeled VAPC and to characterize the binding interactions, twodimensional 1H15 N HSQC spectra of the

15 Nlabelled VAPC was acquired with a protein concentration of ~100M in the absence of NS5B followed by constant addition of NS5B in the molar ration of 1:0.5,

1:1 and 1:1.5 (VAPC:NS5B). The shifted residues of VAPC were assigned by superimposing the HSQC spectra in the absence and presence of the NS5B. The degree of perturbation was evaluated by an averaged chemical shift according to the formula [( 1H) 2+( 15 N/4) 2]1/2 (ppm). Addition of NS5B triggered the shifts of many

HSQC peaks of VAPC(Figure 3.3.5 C), indicating that VAPC was indeed able to bind to NS5B but most of the residues which show perturbation on interaction with NS5B are mainly located at the Cterminus of VAPC. It is also important to note that the

HSQC spectral dispersions of VAPC did not significantly increase even upon binding to NS5B, implying that VAPC remained largely flexible even in the complex (Figure

3.3.5 D).

0.07

0.06

0.05

0.04

0.03

CSI (ppm) CSI 0.02

0.01

0 MVVLQLRFVTKNDVKTPYRSIGISRAEAQFSWR Residue

Figure 3.3.5 C) Titration experiment of VAPC to HCV NS5B C) CSD calculation of VAPC amino acids upon binding with NS5B shows perturbance of most residues at C terminus

Figure 3.3.5 Titration experiment of VAPC to HCV NS5B D) VAPC 2D spectrum with perturbed peaks. Black peaks represent the VAPC spectrum without NS5B, blue peaks represent the VAPC spectrum with NS5B in molar ratio of 1:1.5 (VAPC: NS5B)

3.3.4 VAPC Cterminal constructs

Based on interaction of full length VAPC to HCV NS5B, we designed different VAPC constructs with truncation/removal of Nterminus. The constructs were named as VAPC43, VAPC31 and VAPC14 (Figure 3.3.6). VAPC43 consist of

Cterminal 43 residues, starting with asparagine57. Titration of full length VAPC with

NS5B (Figure 3.3.5 A) shows that the C–terminus of VAPC is mainly involved in binding with NS5B and the binding region begins from serine58 and so we truncated full length VAPC from residue 57 to get VAPC43. VAPC14 was designed based on titration of VAPC43 with NS5B (discussed later) and so the more tightly binding

residues were truncated to get VAPC14. But VAPC31 was not studied very extensively as initial experiments shows the aggregation behavior did not show very tight interaction of this construct with NS5B as compared to other two constructs.

Figure 3.3.6 Representation of VAPC cterminal constructs. Black box (1) represents cterminal 43 residues (VAPC43), red box (2) represents cterminal 31 residues (VAPC31) and green box represents 14 residues with further truncation of cterminus.

3.3.4.1 Structural characterization of VAPC43, VAPC31 and VAPC14

The structural properties of all VAPC truncated constructs (VAPC43, VAPC31 and VAPC14) were first investigated by CD spectroscopy. The CD spectrum of all the constructs was collected in buffer with 10mM Na 2HPO 4 pH 6.5 at protein concentration of ~20µM. All the spectrums (Figure 3.3.7 A) showed the same pattern, with a maximal negative peak at ~202nm and no signal at any other wavelength.

Implying, like full length VAPC, all the constructs are without any folded structure.

The same observation was made for NMR experiments. The 1D spectrum for

VAPC43, VAPC31 and VAPC14 had narrow dispersion from ~7.0 to 8.0ppm and as explained in the previous section this narrow dispersion is the characteristic of unstructured proteins.

Figure 3.3.7 Top panel shows the CD spectrum of VAPC43, VAPC31 and VAPC14, all have similar kind of spectrum with negative peak at ~202nm. Bottom panel represents the 1D spectrum of three constructs. Both kind of experiments confirms unstructured nature of VAPC constructs.

Figure 3.3.8A) HSQC and sequential assignments of VAPC43. The spectra were acquired on an 800MHz NMR spectrometer in a 20mM phosphate buffer (pH6.5). The assigned residues are labeled in the spectra.

Figure 3.3.8B) HSQC and sequential assignments of VAPC14. The spectra were acquired on an 800MHz NMR spectrometer in a 20mM phosphate buffer (pH6.5). The assigned residues are labeled in the spectra.

To gain residuespecific insights into the solution conformation of VAPC43, we collected a pair of threedimensional heteronuclear NMR spectra, namely 15 Nedited

HSQCTOCSY and HSQCNOESY, and subsequently achieved its sequential assignment . The sequencespecific backbone assignment for VAPC14 and VAPC31 was achieved by comparing the almost identical resonance dispersion of VAPC43 on the 1H15 N HSQC spectra. The proton [ 1H] chemical shift values of VAPC43 were used to calculate the chemical shift index (CSI) of VAPC43 by subtracting the random coil proton [ 1H] chemical shift values (table 3.3.1). Hα(observed)

Hα(random coil) ssp (secondary structure propensity) scores of VAPC43 are shown in Figure 3.3.8. The Hα CSI is negative in an αhelix. For VAPC43 although Hα CSI was negative but its value was not substantial. As is evident from Figure3.3.9, CSI of

VAPC43 [ 1Hα] for all the residues was very low. It was closer to the random coil values hence suggesting the unstructured nature of VAPC43.

Based on characteristic NOEs patterns (Figure3.3.9) including dNN(i, i +

2), dαN(i, i +3), and dαN(i, i + 4), as well as αH conformational shifts , it was very clear that VAPC43 does not have any well defined and folded structure. The existence of the loop or helicallike segments was supported by characteristic NOEs (Figure

3.3.10) over the VAPC43 sequence defining the helical conformation, including dNN(i, i+1) , d NN(i, i+2) , d αN(i, i+2) , and d αN(i, i+3) . However, since no d αN(i, i+4) NOEs were observed, the helical conformation in VAPC43 appeared to be mainly dynamic 3 10 helix/loop, rather than αhelix, consistent with the above CD result.

Structure modeling of VAPC43 using CYANA shows that the protein had no folded structure. Figure 3.3.11 shows ten superimposed structures calculated from

CYANA. Six out of ten structures did not show any helix conformation. But four of the structures showed a small helix at residues 79, 80, 81 and 82 and one of the

structures out of these four also had a small helix at 60, 61, 62 and 63. Moreover, the calculated structure was found to be very much flexible at the Cterminal region.

Figure 3.3.9 Hα CSD (Chemical shift deviation) plot of VAPC43 shows the values in close proximity to the random coil values.

Figure 3.3.10 NOE pattern representation for VAPC43.

Figure 3.3.11 Structure of VAPC developed by modeling software CYANA

3.3.3.2 Interaction of VAPC43 with HCV NS5B

Monitoring the perturbations of 15 N and 1H chemical shifts of HSQC spectra upon binding to a ligand is commonly used to map residues involved in interaction. So to characterize the binding interactions of VAPC43 with NS5B, two dimensional 1H15 N HSQC spectra of the 15 Nlabelled VAPC43 was acquired with a protein concentration of ~100M in the absence of NS5B, and unlabeled NS5B was added stepwise up to a final molar ratio of 1:2.75 (VAPC43:NS5B), with each step monitored by acquiring 2D 1H15 N HSQC spectrum. The shifted residues of VAPC constructs were assigned by superimposing the HSQC spectra in the absence and presence of the NS5B. Figure 3.3.12 A shows the superimposition of the 1H15 N

HSQC spectrum of free protein [VAPC43 shown in red] and protein in complex with

HCV NS5B (shown in blue). A series of titrations with corresponding molar ratios

(protein:ligand) 1:0, 1:0.25, 1:0.5, 1:0.75, 1:1, 1:1.25, 1:1.50, 1:1.75, 1:2.0, 1:2.50 and

1:2.75 were carried out to obtain chemical shift changes at different ligand concentrations for VAPC4:NS5B. The degree of perturbation was then evaluated by an averaged chemical shift according to the formula [( 1H) 2+( 15 N/4) 2]1/2 (ppm). By carefully analyzing the spectra we observe that the saturation point was reached at around 1.5 to 1.75 molar ratio of NS5B meaning that the peaks didn’t show any further perturbation. Almost all the peaks showed the shift but the most perturbed peaks for VAPC 43 spectrum were I61, R73, W74, E78, E77, D80, S81, A81, W94,

E95 and E98. Although VAPC43 is highly unstructured in solution, but the HSQC titration demonstrates its binding affinity with NS5B.

Figure 3.3.12 A) 2D [1H15 N] HSQC of VAPC43 on titration to HCV NS5B, red color represents the HSQC spectrum of VAPC43 without NS5B, green represents the spectrum with VAPC43:NS5B molar ratio of 1:1.0 and blue represents VAPC:NS5B ratio of 1:2.75. The satellite picture shows example of peaks (R73 & W74) which get perturbed on addition of NS5B, with black line represents the distance between the unperturbed and perturbed peaks.

However, even at a molar ratio of 1:2.5 (VAPC43:NS5B) where the peak shifting of many residues were largely saturated (Figure 3.12 B), the HSQC spectral dispersions still remained largely unchanged. This implies that like the fulllength

VAPC, VAPC43 also remains largely flexible without any tight tertiary packing in

the complex with NS5B. A vigilant observation of CSD graph suggests that VAPC43 has three binding pockets through which it binds to NS5B. It has an Nterminal pocket which consists of amino acid I61, Cterminal pocket with residues W94, E95 and R98 and a central pocket with R73, W74, E78, E79, D80, S81 and A82 residues.

Figure 3.3.12B) CSD chart for VAPC43, where blue bar represents titration point at VAPC43:NS5B molar ratio of 1:0.5, green bar at 1:1 and red bar at 1:2.75. The CSD data shows the presence of three binding pockets: small pockets at Nterminal and C terminal with one binding pocket at the centre. To identify the importance of second binding pocket of VAPC43 in binding to

NS5B, we mutated four residues (Arg73, Trp74, Glu78 and Asp80) into Ala. The mutated construct (pGEX4T1VAPC43Mut) was successfully expressed and purified. Like VAPC43, the VAPC43 mutant appeared to be highly unstructured without any wellformed secondary structures as judged from its farUV spectrum

(Figure 3.3.12 C), and lacked any tight tertiary packing as demonstrated by the very narrow HSQC spectral dispersions (Figure 3.3.12 D). The intensities of its HSQC peaks of VAPCmutant were not uniform, i.e. some were very strong but some very weak. This possibly could be because of some dynamic aggregation, or/and conformational exchanges on µs to ms timescale (Figure 3.3.12 D). Titration result of

labeled VAPC43 mutant with unlabeled NS5B shows the importance of second pocket of VAPC43 in binding to NS5B as, even at a molar ratio of 1:2.5 (VAPC43 mutant:NS5B), no significant shift of the HSQC peaks was observed (Figure 3.3.11

D), which clearly demonstrates that this second region is indeed critical for binding with NS5B.

Figure 3.3.12 C) Representation of far UV CD spectra of VAPC43mutant shows that VAPC43mutant is unstructured. D) 2D HSQC of VAPC43: blue peaks represents free VAPC43mutant whereas pink and red are in the presence of NS5B in the ratio of 1:1.5 and 1:2.5 respectively.

We also studied VAPC31 construct for its binding with NS5B but for this construct peak intensities were weak which could be because of dynamic aggregation or fast conformational exchange (Figure 3.3.12 D) and so this construct was not included for further studies.

Figure 3.3.12 D) 2D [1H15 N] HSQC of VAPC31 on titration to HCV NS5B, black color represents the HSQC spectrum of VAPC31 without NS5B and blue represents VAPC:NS5B in molar ratio of 1:2.0

3.3.3.3 Interaction of VAPC14 with NS5B

Based on the titration results of VAPC43wild type and VAPC43mutant, another construct, VAPC14 was subjected to titration experiments with NS5B. Again, even at a molar ratio of 1:2.5 (VAPC14:NS5B), the HSQC spectral dispersions still remained largely unchanged (Figure 3.3.13 A). This indicates that like the fulllength VAPC and VAPC43, even in the complex with NS5B, VAPC14 remained largely flexible without any tight tertiary packing. Although, all the residues showed perturbation but the observation of CSD graph (Figure 3.3.13 B) shows that the most perturbed peaks were D77, E78, E79, D80, E83 and Q85. Removal of N and Cterminals affects the binding affinity with HCV NS5B. This can be observed from the dissociation constant values for VAPC43 and VAPC14 which is discussed in the next section.

Figure 3.3.13 A) 2D [1H15 N] HSQC of VAPC14 on titration to HCV NS5B, black color represents the HSQC spectrum of VAPC14 without NS5B and blue represents VAPC:NS5B in molar ratio of 1:2.5

Figure 3.3.13 B) The CSD representations of VAPC14 on binding to NS5B at different molar ratios of 1:0.5 (blue), 1:1 (green) and 1:2.5 (red).

3.3.4 Determination of dissociation constant (K d) through HSQC titration

Our attempts to measure the binding constant of VAPC with NS5B failed, which could probably be due to the complex binding mode, and/or the fact that many

VAPC residues still remain largely flexible even in the complex. So as to quantitatively access the binding between VAPC43/VAPC14 and NS5B, the dissociation constant (K d) was calculated by fitting the chemical shift values from different titration points into the equation below and it was done using software

Datafit 7.1 (Oakdale).

δ = δmax × {1/2+0.5X + Kd/[P] [1/4 +(0.5X + Kd/2[P]) 2 + Kd/2[P] – 0.5X] 1/2 }

δmax and Kd are separate variables that are adjusted to produce the best fit of calculated to observed data (Macomber R. S. et. al . 1992). A binding isotherm may be calculated with knowledge of the complex stoichiometry, this forms the principle of curve fitting methods.

Fitting data was generated for the residues which showed high degree of perturbation as compared to other residues. Figure 3.3.14 A represents the fitting curve for Nterminal (I61) and Cterminal (E95) binding pockets of VAPC43 and

Figure 3.3.13 B represents the fitting curves for the central binding pocket of

VAPC43. Fitting curve for VAPC14 is represented in Figure 3.3.14. The Kd values obtained by fitting in the Datafit software are shown in table 3.3.2. The average Kd value for VAPC43 was 18.29 µM and for VAPC14 it was 49.13 µM. Interestingly, comparing the Kd values shows that the deletion of first and third binding pocket resulted in only 3fold reduction of binding affinity with NS5B.

100

VAPC43 VAPC14

Residues Kd (µM) ± SD Kd (µM) ± SD

I61 1.269 ± 19.536

R72 161.318 ± 63.857

R73 41.397 ± 19.536

W74 19.932 ± 5.371

D77 17.18 ± 5.76

E78 0.01 ± 0.07 18.032 ± 3.315

E79 5.526 ± 2.058 1.41 ± 1.34

D80 4.348 ± 2.152 68.877 ± 18.928

A82 0.01±0.22 27.34 ± 12.63

E83 18.62 ± 3.32

Q85 0.11 ± 0.2

E95 20.265 ± 7.632

Average 18.29 ± 7.35 49.13 ± 17.64

Table 3.3.2 Kd values with SD (standard deviation) of shifted residues from VAPC43 and VAPC 14. (underlined Kd values have large errors, and thus were not included for calculating the average Kd values)

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Figure 3.3.14 Shows the final fitting curves of most perturbed residues in the three pockets of VAPC43 A) Fitting curve for N (I61) and Cterminal (E95) pocket (B) Fitting curve for the centre pocket residues.

Figure 3.3.13 C) Show the final fitting curves of most perturbed residues in VAPC14.

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3.3.5 Discussion

Most of the RNA viruses, such as HCV form the replication complexes associated with the host membrane (Johnson B.A et.al . 1994). Currently knowledge is very less about the structure of the complexes but it is anticipated that studies of RNA virus replication machineries will have a critical impact on antiviral drug development due to their specific roles in virus replication (Johnson B.A. et.al . 1994). In vivo studies show a cellular protein VAPC is the negative regulator for HCV replication and it does so by inhibiting the RNA dependent RNA polymerase (RdRp) activity.

Although it is been more than a decade now but till date no invitro data is available for the structure as well as for the binding characterization of VAPC and NS5B. By studying the structure of VAPC along with its interaction with NS5B using NMR as a tool, we for the first time provide the detailed study of VAPC and HCV NS5B interaction at the residue level.

For VAPC, very limited knowledge is available about its functional roles particularly no structure study data have been reported for this protein till date. The human VAPC is a 99residue splicing variant of the 243residue VAPB, with the N terminal 70 residues being identical in both proteins. Protein sequence analysis shows that VAPC contained high percentage of disorder promoting amino acids. The absence of a welldefined structure was confirmed by CD and NMR spectroscopy and by use of these techniques; we provide here the first residuespecific evidence that

VAPC is in fact highly unstructured in solution, lacking any stable secondary structure and tight tertiary packing. Upon binding to NS5B, there is no observed conformational change in VAPC. The three binding pockets remain connected by the flexible loops. As a consequence, our study suggests that VAPC is a new member of socalled ‘‘intrinsically unstructured protein (IUP)’’ family which represents an

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expanding category of proteins of being biologically active but highly unstructured under physiological conditions. The recent discovery of IUPs (intrinsically unstructured proteins) have challenged the decades old paradigm that to be functionally active a protein needs to have a stable, 3D structure. It is now recognized that a large portion of proteins encoded by the eukaryotic genome (>30%) are IUPs

(Tompa P. 2002; Dyson H.J et.al. 2005). Highly flexible and randomcoil conformation is the native and functional state of many proteins and same is the situation for VAPC.

NMR characterization allows the identification of VAPC residues critical for binding to NS5B. The results show that VAPC interacts with NS5B, but through c terminal. Interestingly, it appears that three discrete VAPC regions are involved in binding with NS5B. The first (Nterminal binding pocket) and third (Cterminal binding pocket) regions are relatively small and are centered at Ile62 and Glu95 respectively, while the second region is much longer, spanning residues from Arg73

Ala82. Interestingly, the sequence analysis shows that only the first binding pocket is located within the identical sequence of VAPC and VAPB, while the second and third are both within the VAPC unique sequence. The importance of the second region was further confirmed by characterizing a VAPC43 mutant which dramatically lost binding ability to NS5B. The dissociation constant (Kd) value for VAPC43 upon binding with NS5B is 18.29µM. Removal of Nterminal binding pocket or Cterminal binding pocket or both have negative effect on the interaction with NS5B. VAPC14 is the construct without N and Cterminal binding pockets and its Kd value is 49.13µM.

Although the dissociation constant value for VAPC14 is thrice as much as compared to VAPC43, but still it is in the moderately tight binding range. This small VAPC fragment can be used for further studies and can be a target for drug design against

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HCV. In conclusion, VAPC14 may be useful for exploring the biological functions of VAPC in vivo , and also represents a promising starting point to develop potent peptide inhibitors for interfering with the VAPCNS5B interface in the treatment of

HCV infection by use of NMRguided approaches.

3.3.6 Future Directions

VAPC14 can be good target for drug design since the interaction surface is not only typically smaller but is also more easily defined. For future work, VAPC14 can be used to cocrystallize with HCV NS5B, so as to check the exact location of NS5B residues which are involved in binding with VAPC. As it is reported that VAPC inhibits NS5B activity, it is necessary to do the RNA dependent RNA polymerase assay (RdRp) for NS5B and check the inhibitory activity of VAPC as well as its truncated constructs (VAPC43 and VAPC14). Functional study of these fragments can also give a clear picture of VAPCNS5B interaction pattern. Moreover, VAPC is an unstructured protein and it is highly possible that it is involved in some other interactions with host or viral proteins, so it will be quite interesting as well as challenging to find out the interacting partners of VAPC and their functional impact.

VAPC is the hopeful target and further work can help to develop it as an effective drug against HCV, which will be resistant to mutagenesis and will be effective against entire HCV genotypes.

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Chapter – 4 PERSPECTIVE

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4.0 Perspective

Viral infection result in millions of deaths each year. Millions of dollars are spent annually to better understand how virus infect their hosts and to identify potential targets for therapeutics. An important aspect of host virus system is the mechanism by which a virus is able to invade a host cell. Within these complex systems, proteinprotein interactions (PPIs) between surface proteins form the foundation of communication between a host and a virus and play a vital role in initiating infection. The study of the interactions among viral and host factors provide the data to create novel therapeutic strategies and the development of new antiviral drugs (Huang L. et. al . 1998). The two proteins studied, viperin and VAPC also have been known to inhibit virus through proteinprotein interactions.

4.1 Association of Viperin and VAP proteins

Viperin and VAPC are recently discovered antiviral proteins. Since its discovery in 1997 viperin has constantly been observed to have antiviral properties against multiple viruses ranging from DNA virus to RNA virus (Chapter 2.1).

Mechanism of action of viperin against most of these viruses is still not known. But its activity against HCV seems to be associated with hVAP33 (VAPA) (Helbig K.J. et.al. 2011). Just like VAPC, viperin also inhibits HCV by disrupting the formation of replication complex. Viperin inhibits HCV replication by interfering with the association of NS5A with lipid droplets. Recent studies show the interaction between viperin and hVAP33. As discussed before hVAP33 is a host protein and is required for the formation of HCV RNA replication complex on lipid droplets. The HCV protein NS5A bind to the coiledcoil domain and NS5B bind to the MSP domain of hVAP33 during the formation of replication complex.NS5B cannot anchor itself into

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the lipid raft membrane and is therefore dependent on the interaction of hVAP33 with NS5A. Interaction of viperin with hVAP33 disrupts hVAP33 interaction with

NS5A and thus results in the inhibition of formation of HCV replication complex.

This suggests that Viperin binds to NS5A whereas VAPC binds to NS5B to disrupt the replication complex formation and thus lead to inhibition of HCV. So viperin along with VAP proteins could be an effective target for hepatitis c virus.

There are lot more host cell factors which are under study for hepatitis c. Some of them have been identified and have been proposed to use as targets for antiviral development. Because HCV variants with one or two nucleotide substitutions are generated every day, which implies that resistanttotreatment mutants could be generated daily. In this respect, host cell factors have emerged as a promising alternative. They reduce the risk of development of antiviral resistance and they increase the chance for broad spectrum activity, by covering all HCV genotypes. The search, identification and characterization of host proteins involved in virus life cycles, have become a hot spot in virology during the last years. The few important targets identified for HCV, other than VAP and Viperin are cyclophilins (Fisher G. et. al 1989), FK506binding proteins (FKBP) (Siekierka JJ et.al . 1989a; 1989b) and parvulins (Rahfeld JU et.al. 1994) and the secondary amide peptide bond cis/trans isomerase (APIase) (SchieneFischer C et.al . 2002).

4.2 Other Cellular proteins as target for antiviral drugs

Viral infection of mammalian cells results in the activation of a number of viral recognition pathways triggered by replication intermediates and/or viral proteins that ultimately induce innate defenses to limit viral replication (Helbig K.J. et.al .

2011). Pivotal to this antiviral response is the induction of IFN. Interferon forms the

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first line of defense in viral infection. The interferon response is capable of controlling most virus infections, even in the absence of an adaptive immune response. The type I

IFNs (IFNα and β) are essential for immune defenses against viruses and, after binding to the type I IFN receptor, induce the expression of hundreds of interferon stimulated genes (ISGs), many of which act to limit viral replication. ISGs may have direct or indirect antiviral action (Sen G.C 2001). Although the structure study of these proteins along with their binding partners is a very challenging work but it will have a great impact upon drug design and drug discovery field. Some of the proteins which are known to have antiviral activities and can be studied in future are, ISG15

(IFNstimulated protein of 15 kDa) (Osiak A. et.al . 2005), the GTPase Mx1

(myxovirus resistance 1) (Melén K et. al . 1994), ribonuclease L (RNaseL) and protein kinase R (Takizawa T et. al . 2002) (PKR; also known as EIF2αK2). Some additional

ISGs that probably also have important roles in antiviral activities include: the deaminases ADAR1 (adenosine deaminase, RNAspecific 1) and APOBEC

(apolipoprotein B mRNAediting enzyme, catalytic polypeptide) protein; the exonuclease ISG20; members of the tripartitemotif containing (TRIM) proteins such as TRIM19 (also known as PML); and the highly IFNinduced translation regulators

IFIT1 (IFNinduced protein with tetratricopeptide repeats 1) and IFIT2. All of these

ISGs have been reported to function as antiviral proteins in vitro but the complete spectrum of antiviral ISGs and their mechanisms of action remain to be elucidated.

Further investigation using the appropriate gene knockout mouse models is needed for a better understanding of their relative importance in the antiviral response.

By understanding at the molecular level how viruses counteract the host immune response and understanding the mechanism of interactions between viral and host factors, new medicines and new ways of combating infections may be developed.

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G. Shaveta , J.Shi, V.T. Chow, J.Song. (2010) Structural characterization reveals that viperin is a radical SadenosylLmethionine (SAM) enzyme. Biochem. Biophys. Res. Commun. 391 :13901395.

Shaveta Goyal, Garvita Gupta, Haina Qin, Megha Haridas Upadya, Yee Joo Tan,

Vincent T.K. Chow and Jianxing Song. VAPC, an Human Endogenous Inhibitor for

Hepatitis C Virus (HCV) Infection, is Intrinsically Unstructured but Forms a “Fuzzy

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