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 co-supervisor 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 ex-Ph.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 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 good work. This acknowledgment will be incomplete if I 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 house-mates 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. II TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS III SUMMARY VII LIST OF FIGURES VIII LIST OF TABLES X LIST OF SYMBOLS XI CHAPTER INTRODUCTION 1.1 Protein structure studies 1.2 Features of NMR spectroscopy 1.2.1 NMR for proteins 1.3 Principle of NMR 1.3.1 Larmor frequency 1.3.2 Chemical shift 1.3.3 Coupling 1.3.4 Free induction decay 1.3.5 Relaxation 1.3.5.1 Spin-spin relaxation time (T1) 1.3.5.2 Spin-spin relaxation time (T2) 1.3.5.3 NOE (Nuclear Overhauser Enhancement) 1.4 Structure details by NMR 1.4.1 1-Dimension NMR 1.4.2 The 1H-15N coupling for the heteronuclear NMR analysis 11 1.4.3 Sequential assignment 1.4.3.1 Homonuclear 1H-NMR 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 16-17 1.7 Protein-ligand interaction by NMR 17 1.7.1 Mapping of Chemical Shifts 1.8 Circular Dichroism 18-19 19-21 CHAPTER BIOLOGICAL SIGNIFICANCE OF VIPERIN 2.1 Introduction 2.1.1 Viperin sequence details 23-25 2.1.2 Viperin in Immune response 26-27 2.1.3 Viperin Induction and Action 27-28 2.1.4 Influenza virus inhibition by viperin 29-30 2.1.5 Radical SAM domain proteins 30-32 2.1.6 AIMS 33 2.2 MATERIALS AND METHODS 2.2.1 Vector Construction 35-36 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 [Fe4–S4] cluster in Viperin (45–361) 38-39 2.2.3 Media prepration for NMR sample 39 2.2.4 UV–visible Spectroscopy 39 2.2.5 Circular dichroism (CD) 39-40 2.2.6 NMR sample prepration 40 IV 2.3 RESULTS AND DISCUSSION 2.3 Structure details of Viperin 2.3.1 Structural characterization of the human viperin fragments 42-45 2.3.2 Reconstitution of the [4Fe–4S] cluster in Viperin (45–361) and viperin (45-361) mutant 2.3.3 Structural characterization of the buffer-insoluble Viperin (214–361) 45-49 2.3.4 Conclusion 2.3.5 Future work 49-51 52-53 53 CHAPTER BIOLOGICAL SIGNIFICANCE OF VAPC 3.1 Hepatitis 56 3.1.1 Acute and Chronic Hepatitis 56-57 3.1.2 HCV genotype 57-58 3.1.3 Life cycle of HCV 58-59 3.1.4 Genome organization 60-61 3.1.5 NS5B (RNA dependent RNA polymerase, RdRp) 61-63 3.1.6 VAP Proteins 63-65 3.1.7 Interaction of VAP proteins with HCV proteins 65-67 3.1.8 Therapeutics for HCV 67-69 3.1.10 AIMS 70 3.2 MATERIALS AND METHODS 3.2.1 Vector Construction 72 3.2.2 Codon optimization 72-73 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 V truncated constructs 3.2.5.2 Expression and purification of NS5B 73-74 74-75 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 79-80 3.3.2 Structure characterization of full length VAPC protein 81-85 3.3.3 Interaction of VAPC to HCV NS5B 86-88 3.3.4 VAPC C-terminal constructs 88-89 3.3.4.1 Structural characterization of VAPC43, VAPC31 and VAPC14 90-93 3.3.4.2 Interaction of VAPC43 with HCV NS5B 94-98 3.3.3.3 Interaction of VAPC14 with HCV NS5B 98-99 3.3.4 Determination of dissociation constant (Kd) through HSQC titration 100102 3.3.5 Discussion 103-105 3.3.6 Future work 105 CHAPTER 4. PERSPECTIVE 107 4.1 Association of Viperin and VAP proteins 107-108 4.2 Other Cellular proteins as target for antiviral drugs 108-109 REFERENCES 110-119 PUBLICATION 120 VI 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 up-regulation 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, interferon-inducible), 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 N-terminal domain and reconstitution of purified protein under anaerobic conditions. Another cellular protein, VAPC (vesicle-associated membrane proteinassociated 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 C-terminal half of VAPC is involved in binding with NS5B and the isolated C-terminal 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 VAPC-peptide 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 Figure 1.2 The spinning nucleus with a charge precessing in a magnetic field Figure 1.3 Dihedral angle (φ) Figure 1.4 The free induction decay (FID) 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 31-32 Figure 2.2.1 Secondary structure prediction and truncation representation 36 Figure 2.3.1 FPLC and DLS profiles of viperin 43-44 Figure 2.3.2 Far UV and Near UV CD and 2D NMR spectra of unreconstituted viperin 45-46 Figure 2.3.3 CD, UV and 1D characterization of viperin (45-361) 48-49 Figure 2.3.4 CD and NMR characterization of viperin C-terminal 50-51 Figure 2.3.5 2D HSQC for Viperin (SAM+C-terminal) 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 hVAP-C with hVAP-B and hVAP-C. 64 Figure 3.1.7 Model for the mechanism of formation of HCV replication complex on lipid raft 68-69 Figure 3.3.1 Purification profiles of VAPC and NS5B 79-80 Figure 3.3.2 VAPC sequence, CD and 1D spectra representation 82 Figure 3.3.3 2D-1H 15N HSQC 83 Figure 3.3.4 VAPC full length Cα (observed-random) 85 Figure 3.3.5 Far UV CD spectra,1D NMR and CSD calculation of VAPC 86-88 Figure 3.3.6 Representation of VAPC c-terminal 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 95-98 Figure 3.3.13 VAPC14 on titration to HCV NS5B 99 Figure 3.3.14 Fitting curves for VAPC43 and VAPC14 102 IX HCV. In conclusion, VAPC-14 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 VAPC-NS5B interface in the treatment of HCV infection by use of NMR-guided 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 co-crystallize 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 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 VAPC-NS5B 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. 105 Chapter – PERSPECTIVE 106 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, protein-protein 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 protein-protein 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 hVAP-33 (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 hVAP-33. As discussed before hVAP-33 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 coiled-coil domain and NS5B bind to the MSP domain of hVAP-33 during the formation of replication complex.NS5B cannot anchor itself into 107 the lipid raft membrane and is therefore dependent on the interaction of hVAP-33 with NS5A. Interaction of viperin with hVAP-33 disrupts hVAP-33 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 resistant-to-treatment 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), FK506-binding 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) (Schiene-Fischer 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 108 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 (IFN-α and β) are essential for immune defenses against viruses and, after binding to the type I IFN receptor, induce the expression of hundreds of interferonstimulated 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 (IFN-stimulated 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, RNA-specific 1) and APOBEC (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide) protein; the exonuclease ISG20; members of the tripartite-motif containing (TRIM) proteins such as TRIM19 (also known as PML); and the highly IFN-induced translation regulators IFIT1 (IFN-induced 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. 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Communicated. 120 [...]... 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. .. It measures the heteronuclear coupling between 1HN and 15N in one residue and the coupling across 13 C to the 13 Cα and 13 Cβ in the preceding residue HNCaCb: This particular 3-D experiment measures the one bond coupling between 1 HN and 15 N and the one and two bond coupling between 15N and one residue and also records the coupling across 13 C to the 13 13 Cα and Cα and 13 Cβ in 13 Cβ in the preceding... 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 B0, where γ is the gyromagnetic ratio and is constant for all nuclei of. .. residue The combined analysis of these 2 spectra helps in identification of the 13Cα and 13Cβ in the same residue and the preceding residue and thus helps to solve the backbone assignment (Figure 1.8) 13 ation Figure 1.8 HNCACB and CBCACONH correlation Magnetization is transferred 1 1 13 13 13 from Hα and Hβ to Cα and Cβ, respectively, and then from Cβ to 13Cα From here it is transferred first to13CO, then... using all of the secondary shifts from the Cα, Cβ and CO nuclei with an output of +1 for beta-strand, 0 for random coil and -1 for α-helix (Figure 1.10) strand, 14 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... angström apart, and in the β-strand the distance is only 2.2(Å) angström 1.4.3.2 Heteronuclear sequential assignment Along with 2D [1H,15N] 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... sensitive to the spin orientations of its neighbors It is considered the fingerprint of the protein H-N bond is present in every amino acid residue except the N-terminal 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... 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... based on the 1H-15N HSQC, which is a 2-D 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... 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 If the complex binds tightly, Kd . 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. 3.3.4.1 Structural characterization of VAPC4 3, VAPC3 1 and VAPC1 4 90-93 3.3.4.2 Interaction of VAPC4 3 with HCV NS5B 94-98 3.3.3.3 Interaction of VAPC1 4 with HCV NS5B 98-99 3.3.4 Determination of. 2.3 RESULTS AND DISCUSSION 2.3 Structure details of Viperin 2.3.1 Structural characterization of the human viperin fragments 42-45 2.3.2 Reconstitution of the [4Fe–4S] cluster in Viperin (45–361)