Structural Study of the IFIT and the IFIT-RNA Complex

Hongyu Wang Department of Biochemistry McGill University Montreal, Quebec April, 2017

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science under the Faculty of Medicine

©HONGYU WANG 2017 260692719-Hongyu Wang

TABLE OF CONTENTS LIST OF ABBREVIATIONS...... iii LIST OF TABLES AND FIGURES...... v ACKNOWLEDGEMENTS...... vii ABSTRACT...... viii RÉSUMÉ...... ix

1. INTRODUCTION...... 1 1.1 The Immune System...... 1 1.2 Innate Immune System...... 5 1.3 Innate Immune Antiviral Response...... 7 1.4 -Induced Proteins with Tetratricopeptide Repeats...... 12 1.4.1 The IFIT Family...... 12 1.4.2 Structure and RNA Binding Activity of IFIT Proteins...... 14 1.4.2.1 Tetratricopeptide Repeat Motifs...... 14 1.4.2.2 Structural Basis of IFIT Proteins for RNA Binding...... 18 1.4.2.3 Mouse Ifit1b and Rabbit Ifit1b...... 22 1.4.2.4 Ifit1b Proteins Studied in This Research...... 25 1.4.3 The Role of IFIT Proteins in Antiviral Response...... 27 1.5 Project Goals...... 30 2. MATERIALS and METHODS...... 31 2.1 Construct Cloning...... 31 2.2 Transformation into Escherichia coli...... 31 2.3 Cell Growth and Expression...... 32 2.4 Cell Harvest...... 32 2.5 Protein Purification...... 33 2.5.1 Sonication...... 33 2.5.2 Nickel Column...... 34 2.5.3 Ion exchange Chromatography...... 35 2.5.4 Gel filtration...... 37 2.5.5 SDS-PAGE...... 38 i 260692719-Hongyu Wang 2.6 Crystallization...... 40 3. RESULTS...... 43 3.1 Mouse Ifit1b and its Mutant...... 43 3.1.1 Wild Type mIfit1b...... 43 3.1.2 mIfit1b (L453E)...... 46 3.2 Mouse Ifit1b2 and its Mutant...... 49 3.2.1 Wild Type mIfit1b2...... 49 3.2.2 mIfit1b2 (L456E)...... 51 3.3 Mouse Ifit1b3...... 54 3.4 Rabbit Ifit1b2 and its Mutant...... 56 3.4.1 Wild Type rIfit1b2...... 56 3.4.2 Truncated rIfit1b2...... 59 3.4.3 Truncated rIfit1b2 (L456E)...... 62 3.5 Rabbit Ifit1b...... 66 4. DISCUSSION...... 67 5. REFERENCES...... 69

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LIST OF ABBREVIATIONS 3D three dimensional μL micro liter AUC after ULP cleavage CO-IP coimmunoprecipitation CV column volume DAI DNA-dependent activator of interferon-regulatory factor DNA deoxyribonucleic acid dsRNA double-stranded RNA DTT dithiothreitol E.coli Escherichia coli eIF eukaryotic initiation factor His histidine HIV human immunodeficiency virus HP high performance IFIT interferon-induced proteins with tetratricopeptide repeats IFN interferon IFNAR IFNα/β receptor IPTG isopropyl β-D-1-thiogalactopyranoside IRF interferon-regulatory transcription factor ISG interferon-stimulated gene ISGF3 interferon-stimulated gene factor3 ISRE interferon-stimulated response element JAK janus kinase kDa kiloDalton LB lysogeny broth LGP2 laboratory of genetics and physiology2 LPS lipopolysaccharide LRD leucine-rich domain MDA5 melanoma differentiation associated gene5 mL milliliter iii 260692719-Hongyu Wang mRNA messenger ribonucleic acid MTase 2’-O methyltransferase Ni nickel NLR NOD-like receptors NOD -binding oligomerization domain OD optical density PAGE polyacrylamide gel electrophoresis PAMP pathogen-associated molecular pattern PCR polymerase chain reaction PDB PKR protein kinase R PP5 protein phosphatase5 PPP triphosphate PRR pattern recognition receptor RLR RIG1-like receptors RNA ribonucleic acid rpm rotations per minute SDS sodium dodecyl sulfate ssRNA single-stranded RNA STAT signal transducer and activator of transcription TLR toll-like receptor TPR tetratricopeptide repeat TYK tyrosine-protein kinase ULP ubiquitin-like-specific protease UTR untranslated region VSV vesicular stomatitis virus

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LIST OF TABLES AND FIGURES Figure 1.1 - Components of the immune system...... 2 Figure 1.2 - Innate and adaptive immune systems...... 2 Figure 1.3 - Cells in the immune system...... 4 Figure 1.4 - PAMPs and TLRs...... 6 Figure 1.5 - Chemical structures of 5’ modified RNA...... 8 Figure 1.6 - Production of Type I Interferon...... 9 Figure 1.7 - Mechanisms of Type I Interferon-mediated signaling...... 11 Figure 1.8 - Tree of the IFIT protein family...... 13 Figure 1.9 - Alignment of the IFIT gene locus from several mammalian genomes...... 14 Figure 1.10 - Perpendicular view of the three TPRs of Protein Phosphatase5...... 16 Figure 1.11 - Domain organization of IFIT5...... 17 Figure 1.12 - Comparison of gene sequences in IFIT proteins...... 18 Figure 1.13 - Crystal structure of IFIT2...... 19 Figure 1.14 - Structure of IFIT5...... 20 Figure 1.15 - Structure of IFIT5 protein binding with 5’-ppp mRNA...... 21 Figure 1.16 - Function of MTase in human...... 23 Figure 1.17 - Yeast assay with mouse Ifit1b and human IFIT1...... 24 Figure 1.18 - The role of IFIT1 in viral mRNA translation...... 28 Figure 1.19 - IFITs can bind to viral nuclei acid...... 29 Table 1 - Mouse and rabbit Ifit1b constructs...... 31 Table 2 - LB medium compositions...... 32 Table 3 - Compositions of the buffers used in the purification...... 33 Figure 2.1 - The standard curve of gel filtration achieved by proteins with known molecular weight...... 38 Table 4 - Compositions of SDS-PAGE gels...... 39 Table 5 - Components of buffers for SDS-PAGE...... 40 Figure 3.1 - The purification of wild type mIfit1b...... 45 Figure 3.2 - The purification of mIfit1b (L453E)...... 47 Figure 3.3 - 10mg/mL mIfit1b (L453E)+5’-ppp oligo-A RNA crystals...... 49 Figure 3.4 - The purification of wild type mIfit1b2...... 50 v 260692719-Hongyu Wang

Figure 3.5 - 10mg/mL mIfit1b2+5’-ppp oligo-A RNA crystals...... 51 Figure 3.6 - The purification of mIfit1b2 (L456E)...... 53 Figure 3.7 - 10mg/mL mIfit1b2 (L456E)+5’-ppp oligo-A RNA crystals...... 54 Figure 3.8- The purification of mIfit1b3...... 55 Figure 3.9 - 10mg/mL mIfit1b3+5’- ppp oligo-A RNA crystals...... 56 Figure 3.10- The purification of wild type rIfit1b2...... 58 Figure 3.11 - The purification of truncated rIfit1b2...... 60 Figure 3.12 - 10mg/mL truncated rIfit1b2+5’-ppp oligo-A RNA crystals...... 62 Figure 3.13 - The purification of the of truncated rIfit1b2 (L456E)...... 64 Figure 3.14 - 10mg/mL truncated rIfit1b2 (L456E)+5’-ppp oligo-A RNA crystals...... 65 Figure 3.15 - SDS-PAGE of rIfit1b Ni-purification...... 66

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ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Bhushan Nagar for providing me with the great opportunity to work in his lab and for providing suggestions and directions when I was confused and got into trouble.

Also, I would like to thank Yazan Abbas in our lab for giving me basic information about the the research I did and providing me lots of experience, which is very crucial to my work. As well, I would like to thank Alexie Gorelik and Ahmad Gebai for teaching me the basic skills of my work and providing me with encouragement throughout my Master’s work, which are very important for me thriving in the lab. In addition, I would like to thank to Katalin Illes, Jon Labriola and Heidi Olesen in our lab, for giving me advice on new experiments to try in order to get better results. All the members who have worked in Nagar’s lab, both past and present, are really helpful for me, they are kind, clever and always ready to give help. They make my life in the lab easy and enjoyable.

I would also like to thank my research advisory committee, composed of Dr. Bhushan Nagar, Dr. Albert Berghuis, and Dr. Jorg Fritz, for their support and suggestions. Finally, I would like to thank the Department of Biochemistry and my gorgeous school, McGill University, for providing me with the best environment and equipment to do research.

This is the best of times. Wish everybody a splendid future.

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ABSTRACT The innate immune system is very important to defend the host from viral infection. Interferon-Induced proteins with Tetratricopeptide repeats (IFITs) are the culmination of a signaling cascade that is found to restrict the viral infection. IFITs have many members in different species, and I’m working with the Ifit1b proteins from mice and rabbits.

IFIT proteins recognize viral single-stranded RNA from self RNA in different ways, and IFIT1b does this on the basis of RNAs’ 5’-modifications. That is, IFIT1b can only bind to the viral RNA that bears a triphosphate or that has an N-7 methylation but without 2’-O methylation of the first nucleotide. This binding can compete with eIF4E binding with mRNA, which is crucial for mRNA translation, and as a result eliminate viral infection.

Human IFIT1 has been studied for a long time, but IFIT1b, which was first thought to be closely related to IFIT1, has not been structurally solved. In this study, I aim to crystallize Ifit1b protein and its complex with ppp-RNA in mice and rabbits, in order to study the binding core of Ifit1b and its nucleotide preferences. Since the 3D structures of human IFIT5, IFIT2, N-terminal IFIT1, and IFIT5 binding with triphosphate RNA have been solved, and the structures of human IFIT1 binding with capped-RNA and different 5’-modifications will be published soon, these structures have provided me with directive information on my research.

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RÉSUMÉ Le système immunitaire inné est très important pour défendre l'hôte contre l'infection virale. Les protéines induites par l'interféron avec des répétitions de tetratricopepetide (IFIT) sont le point culminant d'une cascade de signalisation qui se trouve à restreindre l'infection virale. Les IFIT ont nombreux membres dans différentes espèces, et je travaille avec les protéines Ifit1 et Ifit1b chez la souris et le lapin.

Les protéines IFIT reconnaissent l'ARN viral avec simple brin et l'ARN auto de différentes manières, et IFIT1b le fait sur la base de la 5'-modification des ARN. IFIT1b peut seulement se lier qu'à l'ARN viral qui porte un triphosphate ou qui a une méthylation N-7 mais sans méthylation 2'-O du premier nucléotide. Cette liaison peut entrer en compétition avec la liaison de eIF4E avec l'ARNm, ce qui est crucial pour la traduction d'ARNm et, par conséquent, éliminer l'infection virale.

L'IFIT1 humain a été étudié depuis longtemps, mais IFIT1b, qui était d'abord pensé être étroitement lié à IFIT1, sa structure n'a pas été résolue. Dans cette étude, je cherche à cristalliser la protéine Ifit1b et sa liaison avec l'ARN chez la souris et le lapin, afin d'étudier le noyau de liaison de Ifit1b et ses préférences nucléotidiques. Puisque chez humain, les structures 3D de l'IFIT5, IFIT2, IFIT1 N-terminal et IFIT5 se liant avec l'ARN triphosphate ont été résolues, et les structures de la liaison IFIT1 humaine avec l'ARN coiffé et différentes modifications 5 'seront publiées prochainement, ces structures ont m'a fourni des renseignements directeurs sur mes recherches.

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1. INTRODUCTION 1.1 The Immune System

Our bodies are surrounded by various pathogens, and cells experience canceration constantly. Thousands of cells decay and die everyday, but multicellular organisms still remain healthy. It is “immunity” that distinguishes self from non-self pathogen and helps keep homeostasis.

In order to control the invasion of varieties of foreign and dangerous infectious organisms, the body must develop a rapid and effective antiviral response. Through this series of steps which is called the immune response, the immune system attacks organisms and substances that invade body system and cause disease.

The immune system undertakes immunity function. From macro to micro description, it is comprised of immune organs (central immune organs and peripheral immune organs), immune cells (hematopoietic stem cells, lymphocytes, and other immune cells), and immune molecules (antibody, complement, cytokines).

These organs, cells and molecules work together to protect the body1-3 (Figure1.1).

In many species, the immune system is divided into subsystems: the innate immune system and the adaptive immune system4 (Figure 1.2).

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Figure 1.1 - Components of the immune system. Adapted from Lieff J. et al. (2012)

Figure 1.2 - Innate and adaptive immune systems. Adapted from Dranoff G. et al. (2004)

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The innate immune system, also called the non-specific immune system, is comprised of a series of cells and relative mechanisms, and it can defend viral infection non-specifically. Although it cannot provide long-lasting protective immunity5, it serves as a rapid antiviral tool among all the animals and plants. From an evolutionary perspective, the innate immune system is an earlier body defense mechanism which works in a general way6. It has a predominant position in most plants, fungi, insects and most primitive multiorganisms6.

The most important components of innate immune system in higher organisms are white blood cells, which are also known as leukocyte. Leukocytes include natural killer cells, mast cells, eosinophils, basophils, phagocytic cells (including macrophages and neutrophils), and dendritic cells7 (Figure 1.3). These cells help detect and exterminate possible viral pathogens.

In higher organisms, there is also an adaptive immune system, gradually built defense system after birth, that targets a specific pathogen or substance, so it is also called specific immune system. Its characteristics are specificity, immune memory, negative and positive response, and multi-cell participation.

The adaptive immune system is obtained from internal and external environmental stimuli after birth. It can recognize the same pathogen when re-exposed to it, and start a

3 260692719-Hongyu Wang corresponding immune reaction8. The adaptive immune system needs the cooperation of highly differentiated tissues and cells.

The adaptive immune system is comprised of immune organs (e.g, thymus, lymph node and spleen) and immune cells called lymphocytes (Figure 1.3), a type of leukocytes.

B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow (Figure 1.2). B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response.

Figure 1.3 - Cells in the immune system. Adapted from MedQuarterly. (2014)

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1.2 Innate Immune System

After the first few hours upon viral infection, the innate immune system reacts fast and provides protection. It serves as a physical and chemical barrier to infectious pathogens. It can produce several chemical factors including cytokines to gather the immune cells into infected or inflamed areas, and can also utilize specified leukocytes to detect and exterminate foreign substances in organs or blood.

Innate immune system relies on the fast response of Pattern Recognition Receptors

(PRRs) to distinguish self from foreign substances. PPRs are encoded by a limited number of germlines, and are very conserved in evolution. Thus, these receptors are essential for the survival of organisms. PRRs are mainly expressed on the surface of innate immune cells, and can recognize one or several Pathogen-Associated Molecular

Patterns (PAMPs). PAMPs are conserved components present in lower microorganisms, and can be found on their cell walls (e.g. lipopolysaccharide, muramyl dipeptide, etc.) or as cellular components (e.g. double-stranded RNA, 5’-triphosphate single-stranded RNA, unmethylated CpG-DNA and envelope glycoproteins9 (Figure 1.4)).

PRRs are divided into four types: Toll-Like Receptors (TLRs), RIG1-Like Receptors

(RLR), NOD-Like Receptors (NLR) and DNA-Dependent Activator of Interferon- regulatory factors (DAI)10. TLRs (Figure 1.4) and RLRs are the most studied and are the main receptors for recognizing PAMPs11. TLRs are Type I transmembrane proteins, and

5 260692719-Hongyu Wang are made of an extracellular domain, a transmembrane domain and a cytoplasmic domain.

The extracellular domain is formed by 19~25 Leucine-Rich Repeats (LRRs). It is highly conserved and it is the binding site of PAMPs12. TLRs in different locations and with different chemical characters can recognize different kinds of PAMPs. RLRs belong to

RNA helicase family with DExD/H domain13. There are currently three known RLR members, those are Retinoic acid-Induced Gene-I (RIG-I), Melanoma Differentiation

Associated gene5 (MDA5) and Laboratory of Genetics and Physiology2 (LGP2)14.

Figure 1.4 - PAMPs and TLRs. Adapted from Molecular Biology of the Cell. 4th edition.

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1.3 Innate Immune Antiviral Response

In mammalian cells, the fast and effective antiviral response is initiated by the

detection of single-stranded or double-stranded viral nucleic acids15. It is important for

the host to correctly distinguish self from non-self nucleic acids. Most cellular

cytoplasms have single-stranded mRNA with an N7 methylated guanosine cap linked via

a 5’-to-5’ triphosphate bridge to the first base, which is called a cap0 structure16

(Figure1.5 C). In higher eukaryotes, RNA is further methylated at the 2’O position of

the first nucleotide17-18, which is called a cap1 structure (Figure1.5 D), or it is even

further methylated at the 2’O position of the second nucleotide, which is called a cap2

structure17,19 (Figure1.5 E). However, many viruses can only form single-stranded

mRNA with a 5’-triphosphate, called a ppp-RNA (Figure1.5 B), or ssRNA with a cap0

structure lacking further methylation20-22.

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A B

C

D

E

Figure 1.5 - Chemical structures of 5’ modified RNA. Adapted from Daisy W. L et al. (2016) (A)

5’-OH RNA (B) 5-’ppp RNA (C) 5’-cap0 RNA (D) 5’-cap1 RNA (E) 5’-cap2 RNA.

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After recognizing and binding with specific viral PAMP, the conformations of

TLRs and RLRs will be changed, and this change will trigger a signaling cascade that lead to the expression of Type I (Type I IFNs) (Figure1.6)23, pro inflammatory cytokines, chemokines and a series of antiviral factors. This can help modulate antiviral immunity and restrict viral replication.

Figure 1.6 - Production of Type I Interferon. Adapted from Andrea K P et al. (2005)

Interferons are classified into IFN-α, IFN-β and IFN-γ, according to their structure, antigenicity and cell resource. IFN-α and IFN-β have similar biological

9 260692719-Hongyu Wang activity and bind to the same receptors on the cell surface, so they are collectively referred to as Type I IFNs24. Type I IFNs work on IFNα/β receptors (IFNARs) via Janus

Kinase1 (JAK1, which binds IFNAR-2 subunit) and non-receptor Tyrosine-protein

Kinase2 (TYK2, which binds IFNAR-1 subunit), this binding leads to the dimerization of

IFNAR-1 and IFNAR-2 subunits25. Then this dimerization makes TYK2 and JAK1 interactively phosphorylated and also activates JAK’s tyrosine-protein kinase activity.

The activated JAK in turn, phosphorylates the conserved tyrosine residues within the recipient cell, and further provides Signal Transducer and Activator of Transcription1

(STAT1) and Signal Transducer and Activator of Transcription2 (STAT2) with docking sites. At the same time, STAT1 and STAT2 become the substrates of JAK. As a result, the STAT1-STAT2 heterodimer binds with Interferon-Regulatory Factor9 (IRF9) to form

Interferon-Stimulated Gene Factor3 (ISGF3). This JAK-STAT signaling cascade leads to the expression of a series of , including Mx, Interferon-Stimulated Gene (ISG) and

Protein Kinase26-29 (Figure 1.7).

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Figure 1.7 - Mechanisms of Type I Interferon-mediated signaling. Adapted from Lionel B. I et al. (2005)

Different members of ISG proteins have different functions in restricting viral translation, replication, assembly and spread, as well as modulating adaptive immunity.

For example, the ISG BST2 encodes tetherin that prevents HIV-1 infection30. Viperin is an another ISG that interacts with TLR7 and helps promote antiviral signalling31.

Among all the ISGs, IFITs (Interferon-Induced proteins with Tetratricopeptide

repeats) are among the most potently expressed type and are found to work against

different kinds of viruses32. 11 260692719-Hongyu Wang 1.4 Interferon-Induced Proteins with Tetratricopeptide Repeats

1.4.1 The IFIT Gene Family

Among all the ISGs, Interferon-Induced proteins with Tetratricopeptide repeats

(IFITs) family is found very early. In general, the expression of the IFIT family molecules is low in most cells32, but previous studies show that upon viral infection, IFN or LPS stimulation, the mRNA levels of IFITs can increase 100- to 1000-fold33, and they target a broad range of viruses34-36.

IFIT proteins have been widely found in various mammals, such as human, mouse and rat, in some birds, reptiles and amphibians. However, the compositions and numbers of IFIT family members vary in different species (Figure 1.8). For example, humans have five intact IFIT genes (IFIT1, IFIT1B, IFIT2, IFIT3, and IFIT5), while rabbits have four intact IFIT genes (Ifit1, Ifit1b, Ifit2, and Ifit3) 37-38 (Figure 1.9). IFIT proteins are structurally related, although some of them are still unclear39. Human IFIT1 is the first

IFIT gene to be found; it is a 56kDa protein and is highly expressed in the cytoplasm upon

IFN stimulation40-41.

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Figure 1.8 - Tree of the IFIT protein family. Adapted from Zhou et al. (2013)

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Figure 1.9 - Alignment of the IFIT gene locus from several mammalian genomes. Adapted by Mattew D.D et al. (2016)

1.4.2 Structure and RNA Binding Activity of IFIT Proteins

1.4.2.1 Tetratricopeptide Repeat Motifs

All the IFIT proteins are 54 to 56 kiloDaltons cytoplasmic proteins, and they are comprised of multiple tetratricopetide repeat (TPR) domains. A TPR is a 34-amino acid repeating domain with helix-turn-helix motif. TPRs are found in lots of prokaryotic and eukaryotic cells42, and they are engaged in various biological activities by mediating

14 260692719-Hongyu Wang protein-protein interaction, such as cell cycle regulation, transcription restriction, protein transportation, stress response, RNA cleavage and protein folding.

Usually 3 to 16 TPR motifs are arranged in parallel in a protein. The amino acid compositions and arrangements vary in different motifs, but certain residues in specific positions are highly conserved. For example, the 8th, 20th and 27th positions are often small, uncharged amino acids like glycine and alanine, while the 21st and 28th positions are often aromatic amino acids like tyrosine and phenylalanine. These conserved amino acids and the distances between them are important for the conformation and function of the protein43.

Repeating TPR motifs can form a super helix. For example, in Protein Phosphatase 5

(PP5) (Figure 1.10), there are 3 TPR motifs in parallel, and each motif has a pair of anti- parallel α-helices, called helix A and B. The adjacent α-helices in different motifs form an anti-parallel structure43. The TPR motifs form into a regular super helix, with the inner surface is comprised of α-helices A from each TPR motif, and the outer surface comprised of the α-helices B in each TPR motif. The inner and outer surfaces in this unique structure allow the protein to bind to various of ligands44.

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Figure 1.10 - Perpendicular view of the three TPRs of Protein Phosphatase5. It shows the helix and the loop region. Adapted from Das, A.K, et al. (1998)

All the IFIT proteins identified to date have 8-12 TPRs with intervening α-helices, and Figure1.1117 shows the TPR arrangement in human IFIT5. Since only 9 residues in certain positions of these TPR motifs show limited conservation, the gene sequences and chemical structures vary in different species or different members within the same species. In cats, for example, the gene sequences of Ifit1 and Ifit1b are 86% identical, with 97% identity within the N-terminus and only 63% identity within the C-terminus.

When comparing the same gene from different species, Ifit1 from ferrets and cats show

83% identity (Figure 1.12)45. It is thus necessary to determine the variations in IFITs from within a single or different species, in order to figure out their functional differences

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Figure 1.11 - Domain organization of IFIT5. Adapted from D.W. et al. (2016)

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Figure 1.12 - Comparison of gene sequences in IFIT proteins. Adapted from Daugherty et al. (2016) (A) Comparison of different IFIT genes in the same species. (B) Comparison of the same gene in different species.

1.4.2.2 Structural Basis of IFIT Proteins for RNA Binding

In IFIT proteins, different TPR domains form a positively charged binding pocket.

Until now, the crystal structures of human IFIT5, IFIT5 with triphosphate RNA, IFIT2, and the N-terminal region of IFIT1 have been solved, and all the IFIT structures to date have a significant basic patch that can accommodate ssRNA17. IFIT proteins specifically

targets 5 ’-modified single-stranded RNA34. The crystal structure of the IFIT2 dimer shows that each monomer has 9 TPR domains. The C-terminus of IFIT2 folds into a

18 260692719-Hongyu Wang super helix, and the inner surface of this super helix has a positively charged nucleotide- binding channel, which helps IFIT2 specifically bind to AU-rich RNA (Figure 1.13)46.

Figure 1.13 - Crystal structure of IFIT2. Adapted from Pichlmair A. (2011) This shows the front and top views of an IFIT2 dimer. One monomer is shown in grey, and in the other monomer, N-terminal region, domain-swapped region, C-terminal region are shown in blue, grey and yellow, respectively.

The crystal structure of IFIT5 shows it also has 9 TPR domains that form three subdomains (I, II, and III). there is a clamp structure with a gap inside, narrow and deep, about 28A × 15A, and it is positively charged inside (Figure 1.14)47. A very similar subdomain I is also found in the N-terminus of IFIT148.

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Figure 1.14 - Structure of IFIT5. Adapted from Abbas Y.M et al. (2013) (a) TPR domains and subdomains of IFIT5. (b) 3D structure with helices of IFIT5. (c) Surface charge of IFIT5. Blue shows positively charged region and red shows negatively charged region.

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It has been shown that after Type I IFN stimulation, IFIT1, 2 and 3 can bind with each other and form a 150-200kDa complex. However, the three IFITs involved in this complex weakly interact with IFIT549. Also, after construction of molecular fragments and coimmunoprecipitation (co-IP), Stawowczyk et al found that the binding of IFIT2 and 3 needs the participation of the first TPR in IFIT2, while any fragment of IFIT2 can bind to IFIT149.

Since we already know that there is a positively charged pocket in all the IFITs for single-stranded RNA accommodation, it is necessary to figure out different RNA preferences for different IFITs. Researches have shown that IFIT5 and IFIT1 can directly recognize and bind 5’-ppp RNA sequence (Figure 1.15), and that this binding is non- sequence specific50. Additionally, IFIT1 can bind cap0 RNA50.

Figure 1.15 - Structure of IFIT5 protein binding with 5’-ppp mRNA. (A) The structure of human IFIT5 binding with 5’-ppp RNA, the RNA is colored in red. Adapted from Liu Y et al. (2002) (B) Cross-section of IFIT5 with 5’-ppp RNA, showing an arrow pocket that binds four . Adapted from Gregory, I. V, et al. (2014)

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1.4.2.3 Mouse Ifit1b and Rabbit Ifit1b

There exists a confusion about IFIT spelling, that it should be spelled in all capital letter IFIT, or should be only one capital letter Ifit. For convenience and clearness, I spell human IFIT in all capital letters, and spell mouse and rabbit Ifit in one capital letter.

At first, it was thought that mouse Ifit1 and human IFIT1 were closely related.

However, recent studies show that when comparing the sequence identity of these two proteins, human IFIT1 is as different from mouse Ifit1 as it is from human IFIT2, and mouse Ifit1 is actually more similar to human IFIT1B than to human IFIT1 (57% versus

53% pairwise amino acid identity)45. Also, the evolution research on the IFIT locus among a wide panel of mammalian genomes showed that mouse Ifit1 and human IFIT1 are paralogs (caused by a gene duplication event) rather than orthologs (caused by a speciation event)45.

The most important difference between mouse Ifit1 and human IFIT1 is their different methods of recognizing 5’-modified single-stranded RNA.

As mentioned before, viral RNA has a 5’-ppp or a 5’-cap0 structure, while cellular

RNA has a 5’-cap0 or a 5’-cap1 structure. In mammalian cells, there is an enzyme called cap-1 methyltransferase (MTase) which can add a methyl group to the first ribose sugar of the RNA molecule to produce a 5’-cap1 structure (Figure 1.16)51. However, many viruses have evolved in a way that they can imitate this methylation by encoding a

22 260692719-Hongyu Wang methyltransferase to have their own cap1 structure, allowing them to evade the antiviral response51.

Figure 1.16 - Function of MTase in human. Adapted from Jamie J.H, et al. (2016) Human cap1-methyltransferase converts cap0-mRNA to cap1-mRNA by adding a methyl group to the first ribose sugar.

We already know that yeast produces only cap0 RNA45. In a yeast assay (Figure

1.17)45 with mouse Ifit1b and human IFIT1, we can see that when mouse Ifit1b was expressed in the yeast, it targeted cap0 RNA to restrict yeast growth. However, when

MTase was co-expressed with mouse Ifit1b, cap0 RNA was transformed into cap1 RNA, and the restriction was rescued. But human IFIT1 was not affected by this change, since it can restrict both yeast cap0 and cap1 RNA. Thus, IFIT1b proteins distinguish self from foreign RNA by 5’-modification, while IFIT1 proteins must rely on a different way.

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Without human cap1-methyltransferase co-expression

With human cap1-methyltransferase co-expression

Figure 1.17 - Yeast assay with mouse Ifit1b and human IFIT1. Adapted from Jamie J.H, et al. (2016)

Evolutionary speaking, IFIT1B duplicated early and this duplication led to the divergence of its 3’end. Mice lost all copies of Ifit1 but generated duplicated copies of

Ifit1b. So the three mouse Ifit1 paralogs, which were first named mouse Ifit1, Ifit1b and

Ifit1c, have been renamed to mouse Ifit1b, Ifit1b2 and Ifit1b3 to reflect the copies of the gene. The evolution is the same for rabbit, so the two rabbit Ifit1 paralogs, which were first named rabbit Ifit1 and Ifit1b have been changed to rabbit Ifit1b and Ifit1b217.

IFIT1B can bind capped mRNA like IFIT1, but it has a different antiviral activity and a potentially different sensitivity to RNA methylation, RNA sequence, and RNA structure17. IFIT1B selectively inhibits the replication of a virus expressing cap0 mRNA, so it can inhibit a variety of viruses that are unable to encode a cap1-methyltransferase

24 260692719-Hongyu Wang and unable to snatch a cap from the host. For example, mIfit1b cannot target some cytoplasmic viruses like poxviruses, flaviviruses, coronaviruses, and rhabdoviruses because they have a virally-encoded cap1-methyltransferase17. Rabbit Ifit1b also shares this cap0 structure recognition and targets viruses lack MTase17.

The structure of human IFIT1 with capped-RNA and different 5’-modifications have been successfully solved, but human IFIT1B cannot be expressed, so I want to solve the structures of mouse and rabbit Ifit1b paralogs, to determine their structural basis for RNA binding.

1.4.2.4 Ifit1b Proteins Studied in This Research

Mouse Ifit1b was first called mouse Ifit1. it is a 463-amino acid protein with a molecular weight of 53737. Its theoretical pI is 7.24. The amino acid sequence is shown below.

MGENADGDQVMENLLQLRCHFTWKLLFENNDIPDLEVRISEQVQFLDIKNPLGM HNLLAYVRHLKGQQDEALQSLKEAEALIQSEQLSKRSLATWGNCAWLHYHRGS LAEAQIYLDKVEKVCKEFSSPFRYRLECAEMDCEEGWALLKCGGGNYKQAMAC FAKALKVEPENPEYNTGYAVVAYRQDLDDNFISLEPLRKAVRLNPEDPYLKVLL ALKLQDLGEHVEAEAHIEEALSSTSCQSYVIRYAAKYFRRKHRVDKALHLLNRA LQASPSSGYLHYQKGLCYKQQISQLRTSRNRQPRRQDNVQELAQQAIHEFQETL KLRPTFEMAYVCMAEVQAEIHQYEEAERNFQKALNNKTLVAHIEQDIHLRYGRF LQFHKQSEDKAITLYLKGLKVEEKSFAWRKLLTALEKVAERRVCQNVHLVESTS LLGLVYKLKGQEKNALFYYEKALRLTGEMNPAF

25 260692719-Hongyu Wang

Mouse Ifit1b2 was first called mouse Ifit1b. it is a 466-amino acid protein with a molecular weight of 54065. Its theoretical pI is 6.41. The amino acid sequence is shown below.

MEQLLSPSNMSEESHKSHIHDSLDELRCHFTWELDIKDKHIHDLEIKISETEFRDPI YSIGMHNLLAYVRHLKGQQDEALQSLKEAEALIQSEQLSKRSLATWGNCAWLH YHRGSLAEAQVYLDKVEKVCKEFSSPFRYRLECAEMDCEEGWALRKCGSQNYT RAMACFERALKVEPENPEYNAGYADVAYHLDYYDGNSLQPLKKAVSVKPEDPY LKVLLALKLQDLRKTDEAEKHIKEATLTISSQNNIFGYVAKFYRRKGCVEEALGF LKKALETKPSSPYLHFQIGLCHKTQFFQMKKATSRENRKRADQSCHLAICHFKKT LELKPTYDRAYIDLAEVYAKNHQQKEAEDNFQEVLSMSNLGDYMQQEIHFRYG NFQQYYKKSEEAAITHYLKGLKIEVTSHYRDKLLKALEELAEGRKEDHVLESLSL LGLVCRLRGDTSEAMSCYEKALRLTGAVNPEF

Mouse Ifit1b3 was first called mouse Ifit1c. it is a 470-amino acid protein with a molecular weight of 54477. Its theoretical pI is 8.4. The amino acid sequence is shown below.

MEQLLSPSNVSAKSHSCLIYDSLVELRCHFTWKLVIEKVDMPDLEVRISETEFFDA SYSIGMHNLLAYVRHLKGQQEEALQSLKEAEALIQSEQLSKRRLVTWGNCAWL HYHRGSLAEAQVYLDKVEKVCKEFSSPFQYRLECAEMDCEEGWALLKCGIQNY KGAMACFAKALKVEPENPEYNAGYAVVAYRLDHIDGTSLQHLQKAVSVKPEDP YLKVLLALKLQDLHKLEEAEKHIEETLPRISSQPYVFGYVAKFYRRKGLVKEALE FLGRALQKQPCSTFLHFQIGLCHKKRLIQIKKASNMQPRGEDRKRADQSIHLAICH FKRTLELKPTYVMAYVTLAEMYIEKNQLKEAEDNFQKLLNMSNLEDHIQQEIHF RYGNFQQYYKKSEEAAITHYLKGLKIEVTSHYRDKPLKALEKLAKRRKEDHVLE NLGLLGFVYKLKGNTSEAMSCYERALRLTGAVNPEF

Rabbit Ifit1b was first called rabbit Ifit1. it is a 479-amino acid protein with a molecular weight of 55134. Its theoretical pI is 6.19. The amino acid sequence is shown below.

26 260692719-Hongyu Wang

MSERAEEHPLKDRLQKLRCHFTWGLLIEDTGLPDLEDRILEEIQFLDTEHKVGIYN LLAYVKHLQGKHEDALENLKEAEEVVQGDQADHSDVRSLVTWGNYAWVYYH MGRLADAQTYLDKVENTCQKSANPSLYRMQCPEMDCEEGWALLKCGGKNYER AKACFEKALEADPENPEFNTGYAITVYRLDYPAKRPCDVSDAFSLQPLRKAVRL NPQDAYLKALLALKLQDVGQKAEGRECLEEALAHTSSQTYVFRYAAMFFRRQG RVDEALKYLKMALKATPSSAILHHQIGLCYREQMIQIKKTTHLQPTEQDRDNVD RLVQLVIFHFEYAVKQKPTFEFAYIHLAHMYITAGDLEKAEDTFQKVLCMTPARE HILQDIHFHYGQFQQFQKKSEVDAITHYLKAIKIGKDSYARDKSINASKQLASKK LKTNAFDLEGWSLQGLVHRLKGELKEALECYEQALRLAADCDNKVACGP

Rabbit Ifit1b2 was first called rabbit Ifit1b. it is a 521-amino acid protein with a molecular weight of 60542. Its theoretical pI is 6.59. The amino acid sequence is shown below.

MQSCLETPSIYKHRGCLRSPRPLWTNLGGAASQTDLSPEKHLLPCSTMSEKSHGY QINDRLVQVRCHFTWELLIEDIEMPDLENRIWEEIQFLDTEHKVGYNLLAYVKHL QGKHEDALENLKEAEEVVQGDQADHSDVRSLVTWSNYAWVYYHMGRLADAQ TYLDKVENTCQKSADPSRYRMECPEMDCEEGWALLKCGRKNYERAKACFEKA LEADPENPEFNTGYAITVYRLDYPAKRPYDVSDAFSLQPLRKAIRLNPQDAYLKA LLALKLQDLGEEAEGRECMEEALAHTSSQTYVFRYAAKFFRRQGRVDEALKYL KRALKATPRSVFLHHQIGLCYREQMIQIKNATHMQPRGRDRENVDRLVQLAINE FQKASVLKPTFELAHVHLAEMYAEIRQYKEAEEHFQKALCIPNSDDHIQQEIHYS YGNFLAYHWKSEDKAITQYLKGLKIEKVSYAREKLLKALERLAERRVNRNVQV VESTALLGLIHKLRGEVSKALLCYEKALRLAADLNSMF

1.4.3 The Role of IFIT Proteins in Antiviral Response

Viruses depend on eukaryotic initiation factor4E (eIF4E) for mRNA translation52. eIF4E is a cap binding protein and can specifically recognize the 5’-cap of mRNA. eIF4E

27 260692719-Hongyu Wang binds with eIF4A and eIF4G to form an eIF4F complex, which then binds to the 5’-cap of mRNA. eIF4E then leads eIF4A to the 5’-cap of mRNA, which opens the secondary structure of mRNA via its helicase activity53. This is essential for viral mRNA translation.

IFIT proteins can compete with eIF4E to bind with 5’-cap0 or 5’-ppp mRNA, and as a result restrict viral mRNA translation (Figure 1.18).

Figure 1.18 - The role of IFIT1 in viral mRNA translation. Adapted from Henzy, E.J, et al. (2016)

IFIT proteins can recognize viral RNA from cellular RNA, and they target numerous viruses. For example, Vesicular Stomatitis Virus (VSV) and Type A flu virus can generate single-stranded RNA with 5’-ppp structure after they infect cells. IFIT1 and

28 260692719-Hongyu Wang

IFIT5 can directly recognize and bind with this 5’-ppp single-stranded

RNA17(Figure1.19).

IFIT1 can also work on capped RNA (Figure1.19). The loss of IFIT1 can make more viruses replicate.

Also, upon viral infection or IFN stimulation, IFIT1, 2 and 3 can interact with each other and form a complex, which can restrict viral replication or facilitate viral RNA degradation. IFIT1 can bind the viral cap0 RNA directly, and leads to a translation inhibition, and as a result restricts virus infection.

Figure 1.19 - IFITs can bind to viral nuclei acid. Adapted from Habjan M et al. (2013)

29 260692719-Hongyu Wang

1.5 Project Goals

IFIT proteins are known to bind the 5’-cap of viral mRNA, and different IFITs

may have their own preferences for different 5’-modified RNAs. The aim of this research

project is to determine the different chemical structures and biological functions of Ifit1b

paralogs in mouse (mouse Ifit1b, Ifit1b2 and Ifit1b3) and rabbit (rabbit Ifit1b and Ifit1b2),

and also their antiviral preferences for certain viruses. Mutagenesis is used to change the

protein dimers or trimers into monomers, ion exchange and gel filtration analysis are

operated to purify the proteins, and crystallization of the proteins is tried to determine the

3D crystal structures of the Ifit1b protein and its complex with RNA.

30 260692719-Hongyu Wang

2. MATERIALS and METHODS

2.1 Construct Cloning

Ifit1b paralogs in mouse and rabbit were cloned into a pSMT3 vector with a SUMO-

His tag followed by a ULP cleavage site.

Restriction N-Terminal Construct Residues Vector sites Fusion Tag mIfit1b 1~463 pSMT3 BamHI-Not His6-SUMO mIfit1b2 1~466 pSMT3 BamHI-Not His6-SUMO mIfit1b3 1-470 pSMT3 BamHI-Not His6-SUMO rIfit1b 1~479 pSMT3 BamHI-Not His6-SUMO truncated 48~510 pSMT3 BamHI-Not rIfit1b2 His6-SUMO

Table 1 - Mouse and rabbit Ifit1b constructs

2.2 Transformation into Escherichia coli

Plasmid of interest (100ng) was transformed into 50µL E.coli BL21 (star) (for

mouse Ifit1b paralogs) or E.coli Rosetta2 (Plys2) (for rabbit Ifit1b paralogs). The

competent cells were incubated with plasmid on ice for 30 minutes, and were then put

into 42°C water bath for 45 seconds for heat shock, followed by incubation on ice for 2

minutes. Then 500µL LB liquid medium (Table 2) was added to the cells, and they

were incubated at 37°C shaker for 1 hour. After that, 20µL of the final culture was

added on LB-Agar plates (kanamycin resistant), and the plates were incubated at 37°C

overnight (about 16 hours).

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medium component Mass(per 1L water) NaCl 10g LB liquid medium Yeast extract 5g Tryptone 10g LB-Agar plates Agar 15g

Table 2 - LB medium compositions

2.3 Cell Growth and Protein Expression

Single colonies on the kanamycin plates were picked and incubated in LB medium with kanamycin in 37°C incubator overnight (about 16 hours) to grow starter cultures.

The following morning, the starter cultures were added into 1L LB medium with

kanamycin to a final OD600nm=0.1. Then the rabbit Ifit1b paralogs transformed into E.coli

Rosetta2 (Plys2) were incubated in 30°C for 3 hours, and the mouse Ifit1b paralogs transformed into E.coli BL21 (Star) were incubated in 37°C for 2 to 2.5 hours, both until

OD600nm reached 0.8. After this they were induced with 1mM isopropyl β-D-1- thiogalactopyranoside (IPTG) overnight (about 16 hours), with rabbit Ifit1b paralogs in

18°C and mouse Ifit1b paralogs in 16°C, for protein expression.

2.4 Cell Harvest

Cells were harvested by the centrifuge, at a speed of 5000rpm for 12 minutes in 4°C.

The supernatant was thrown away, and NiA buffer (Table 3) was used to dissolve the pellet.

32 260692719-Hongyu Wang

Buffer Component Concentration NaCl 500mM Tris-HCl(pH7.8 at 4°C ) 20mM Ni A buffer Glycerol 10% Imidazole 20mM NaCl 500mM Tris-HCl(pH7.8 at 4°C ) 20mM Ni B buffer Glycerol 10% Imidazole 1M Tris-HCl(pH7.8 at 4°C ) 20mM Q-A buffer DTT 1mM Tris-HCl(pH7.8 at 4°C ) 20mM Q-B buffer DTT 1mM NaCl 1M NaCl 500mM Gel filtration buffer Tris-HCl(pH7.8 at 4°C ) 20mM DTT 1mM

Table 3 - Compositions of the buffers used in the purification

2.5 Protein Purification

2.5.1 Sonication

For rabbit and mouse Ifit1b paralogs, sonication was performed to break the cells, with 70% amplitude, 10 seconds on, 40 seconds off and a total sonication time of 1 minute.

After sonication, cells were pelleted by the centrifuge, at a speed of 20000rpm for 25 minutes in 4°C, then soluble proteins I want were in the supernatant, while DNAs, cell debris, and other insoluble proteins were in the pellet.

33 260692719-Hongyu Wang

2.5.2 Nickel Column

All the proteins I purified were in the SUMO plasmid, which has a His-tag in N-

Terminus. This His-tag can bind to the HisPur Ni2+-NTA Agarose Resin (Thermo

Scientific). So a nickel column can be used to separate Ifit1b proteins with other proteins.

1st Nickel was operated first. The supernatant after sonication was combined with nickel beads, with the principle of 0.5mL of nickel beads per 1L bacteria culture. And then the mixture was incubated and shaken at 4°C for 1 hour. The mixture was loaded on a gravity column and was first washed with 5 to 10 column volume (CV) of NiA buffer with 20mM imidazol for 3 times. 3 CV of the Elution buffer (65% of NiA buffer and

35% of NiB buffer) with 350mM imidazol was then loaded onto the column, and the elution containing Ifit1b proteins was collected. 1 CV of Elution buffer was loaded 3 more times to make sure that all the Ifit1b proteins flow through. Sample of this elution was named “Ni1” in SDS-PAGE. Finally, 5 to 10 CV of NiB buffer was loaded to wash all the proteins off the nickel beads. Sample was taken and named “Ni1NiB”.

The ULP enzyme was added into the elution I collected with a mass ration of 1:100, and it was put into a dialysis membrane with a 14kDa cut-off, after which it was incubated in 1L NiA buffer with 1mM DTT overnight in 4°C, to cut the His-tag off and get rid of the high imidazol concentration.

34 260692719-Hongyu Wang

The following day, a 2nd nickel was applied, in order to separate the cleaved protein from His-tag, before which an “AUC (after ULP cleavage)” sample was taken for SDS-

PAGE. The protein solution was loaded onto the nickel column, and flow through with cleaved protein was collected and a “Ni2” sample was taken for SDS-PAGE. The column was washed with 1 CV of NiA buffer 3 times to wash off all the cleaved proteins. And the

His-tag was then washed off with 5 to 10 CV of NiB buffer. The sample of this portion was named “Ni2NiB”.

2.5.3 Ion exchange Chromatography

After nickel column, further purification should be applied using ion exchange chromatography. This was performed by Q HP column, S HP column and Mono Q column via ÄKTApurifier.

Q HP column and Mono Q column are positively charged so they can bind with negatively charged proteins. S HP column is negatively charged so it can bind with proteins with positive charge. Since the surface of IFIT proteins is randomly positively and negatively charged, so different ion exchange chromatographies were used to further purify them.

An S HP column was applied to mouse Ifit1b and its mutant, mouse Ifit1b2, truncated rabbit Ifit1b2 and its mutant, and a Q HP column was used to purify mouse

35 260692719-Hongyu Wang

Ifit1b2 mutant and mouse Ifit1b3. The column was equilibrated with 5 CV of Q-B buffer and then 5CV of Q-A buffer. Then the protein solution was loaded onto the column at a rate of 1mL/min with Q-A buffer, and flow through was collected . An “S flow through” or “Q flow through” sample was taken from this flow through. After loading, the percentage of Q-B buffer was set to 15% in an S HP column with the salt concentration of 150mM, and 20% in a Q HP column with the salt concentration of 200mM. The fractions eluted during this salt concentration were collected, and the sample for SDS-

PAGE was called “S column” or “Q column” for convenience. Finally, 100% of Q-B buffer with a salt concentration of 1M was applied to wash off all the proteins that combined to the S or Q HP column, and an “S100%QB” or “Q100%QB” sample was taken for SDS-PAGE.

For rIfit1b2, Q HP column, S HP column and Mono Q column were all applied to purify it. Mono Q column can separate proteins better than a Q HP column. After loading the protein, percentage of Q-B buffer was set from 0 to 60%, with salt concentration from

0 to 600mM in 90 minutes. And after collecting the elutions, salt concentration was set to

1M to wash off all the proteins on the column.

36 260692719-Hongyu Wang

2.5.4 Gel filtration

Gel filtration chromatography is used to separate the molecules in solution on the basis of their size, and molecular weight. It was used for all the Ifit1b paralogs after ion exchange chromatographies. It was operated by a Hiload 16/60 Superdex200 prep grade column (GE Healthcare) via either the ÄKTApurifier (GE Healthcare) or the ÄKTApure.

After ion exchange chromatographies, protein solution was concentrated to 0.5mL and then loaded to the column with a maximum amount of 10mg. The rate was set to

0.5mL/min. Proteins eluted with gel filtration buffer, and fractions were collected, also samples for SDS-PAGE were taken and named “gel filtration”.

Some proteins with known molecular weight were applied to Superdex 200 of the

ÄKTApure, and according to their elution volumes and molecular weights, a standard curve was made (Figure 2.1). Then according to the IFIT protein elution volume, an approximate molecular weight can be inferred compared with this standard curve, in order to see whether the proteins exist as monomers or dimers.

37 260692719-Hongyu Wang

Figure 2.1 - The standard curve of gel filtration achieved by proteins with known molecular weight. The X-axis shows their elution volume, and the Y-axis shows their molecular weight. An equation was calculated from this curve and can be used as a reference for IFIT proteins molecular weight according to their elution volumes.

2.5.5 SDS-PAGE

Protein solutions after gel filtration were concentrated by Amicon Ultra-15

Centrifugal Filter Units (EMD Millipore) with a molecular weight cut-off of 30kDa, at a speed of 4000rpm at 4°C. SDS PolyAcrylamide Gel Electrophoresis (SDS-PAGE) was then used to check the purity of the fractions in each purification process. 12% SDS-

PAGE gels were prepared with components in Table 4, which can be used to make 8 gels.

38 260692719-Hongyu Wang

Component Running gel Stacking gel

ddH2O 8.4mL 11.6mL

1.5M Tris-HCl (pH=8.8) 6.25mL

0.5M Tris-HCl (pH=6.8) 5mL

10% (w/v) SDS 250uL 200uL

30 % (29:1) 10mL 3.2mL Acrylamide :bisacrylamide

10 % (w/v) APS 125uL 120uL

TEMED 12.5uL 30uL

Table 4 - Compositions of SDS-PAGE gels

2μL of 5×SDS loading dye (Table 5) was added into 8μL of protein sample, and they were boiled in 95°C for 5 minutes. Then the samples were loaded on the gel in electrophoresis buffer at 250V for 50 minutes. After that, SDS gel was stained in

Coomassie Blue solution for 1 hour and then destained in destaining buffer for 1 hour. In this way the bands were clearly shown on the gel.

Buffer Component Concentration Tris-HCl (pH 6.8 ) 60mM Glycerol 30% Saccharose 10% SDS loading dye SDS 5% β-mercaptoethanol 3% Bromphenolblue 0.02%

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Coomassie Blue 0.1% Coomassie Blue solution Methanol 40% Acetic acid 10% Acetic acid 10% destaining buffer Methanol 40%

Table 5 - Components of buffers for SDS-PAGE

2.6 Crystallization

After all the steps of purification, vapor diffusion was used for crystallization.

Classics I, Classics II, JCSG+, PEGs and SM2 screens were used to set up 96-well plates. Mouse and rabbit Ifit1b proteins were concentrated to 10mg/mL and were mixed with 2x excess 5’-ppp oligo-A RNA. Crystallization trays were set up with 3 drops per condition, those are 10mg/mL protein with 2x excess 5’-ppp oligo-A RNA, 5mg/mL protein with 2x excess 5’-ppp oligo-A RNA and 10mg/mL protein only, in both room temperature and 4°C.

No crystals appeared in mouse Ifit1b 96-well plates after one month. And for mouse

Ifit1b mutant, native crystals in 10mg/mL protein with 2x excess RNA grew with the condition Classics1 G2 (0.2M Calcium chloride, 0.1M HEPES PH7.5, 28% PEG 400), and Classics2 F8 (0.2M Ammoniumm sulfate, 0.1M HEPES PH7.5, 25% PEG3350) at

4°C. 24-well plates were applied to optimize the conditions. 0.5µL droplet of 10mg/mL protein with 2x excess RNA was mixed with 0.5µL of a certain buffer, changing slightly the precipitations and pHs around the conditions where crystals grew in the 96-well plates,

40 260692719-Hongyu Wang and this mixture was put in 1mL of large reservoir with the same buffers. Crystals grew with 28% PEG350, 0.2M Calcium chloride, 0.1M Tris PH7.5 at 4°C.

For mouse Ifit1b2 in 96-well plates, native crystals in 10mg/mL protein with 2x excess RNA grew with ClassicsI G2 (0.2M Calcium chloride, 0.1M HEPES sodium salt

PH7.5, 28% PEG400) at 4°C. Further optimizations such as 24-well plates, crystal seeding with human IFIT1 crystals and additive screen with heavy ions were also attempted for mIfit1b2. In the 96-well plates of its mutant 10mg/mL mIfit1b2 (L456E) with RNA, crystals grew with 0.1M HEPES pH7.5, 10% PEG8000, 8% Ethylene glycol at 4°C and 0.2M Magnesium chloride, 0.1M Sodium cacodylate pH6.5, 50% PEG200 at room temperature. Crystal seeding with human IFIT1 crystals was also tried around these two conditions.

10mg/mL mIfit1b3 with RNA was crystallized under the condition of 0.2M

Potassuin formate, 20% PEG3350 and 0.2M di-sodium tartrate, 20% PEG3350 at 4°C.

Then 24-well plates around these two conditions and additive screen were applied to optimize the crystals.

10mg/mL truncated rabbit Ifit1b2 with RNA was crystallized with JCSG+ D10

(0.2M Calcium acetate, 0.1M MES pH6.5, 40%PEG300) and SM2 D3 (0.2M Calcium acetate, 0.1M Sodium cacodylate pH6.5, 45% Glycerol) at 4°C. In 24-well plates, crystals for 10mg/mL truncated rabbit Ifit1b2 with RNA grew under the condition of 0.1M

41 260692719-Hongyu Wang

Sodium cacolylate pH6, 0.2M Calcium acetate, 47.5% glycerol and 0.1M Sodium cacolylate pH6.5, 0.2M Calcium acetate, 50% glycerol at 4°C. For its mutant, rIfit1b2

(L456E), in 96-well plates, crystals for 10mg/mL protein with RNA grew with 0.2M

Calcium chloride, 0.1M Bis-Tris pH6.5, 45% MPD at room temperature. And in 24-well plates optimization, crystals grew under the condition of 0.2M Calcium chloride, 0.1M

Bis-Tris pH7, 47.5% MPD at room temperature.

42 260692719-Hongyu Wang

3. RESULTS 3.1 Mouse Ifit1b and its Mutant

3.1.1 Wild Type mIfit1b

Wild type mIfit1b was cloned and expressed. To crystallize mouse Ifit1b, we first look at its purification. After the 1st Ni-purification and ULP cleavage, His-tag was cut off, but pure protein was not obtained after 2nd Ni-purification (Figure 3.1A). Also the

A260/A280 value of the protein sample was about 1.0, which means that the mIfit1b sample was contaminated with bacteria RNA. To separate RNA-free from RNA- contaminated mIfit1b, the protein sample was applied onto an S column (Figure 3.1D).

RNA-free protein with an A260/A280 value of 0.56 eluted from the column at a salt concentration of 150mM. At 1M salt concentration, RNA-contaminated mIfit1b with

A260/A280 value of 1.2 was purified (Figure 3.1B). RNA-free mIfit1b was then applied onto a size-exclusion chromatography column for further purification (Figure 3.1E).

Three peaks eluted from the gel filtration: the first peak (~7mL) represented aggregated protein eluting in the void volume of the column; the middle peak (~12mL) was a mixture of mIfit1b and bacterial impurities; the third peak (13.4mL) represented mostly pure RNA-free mIfit1b (Figure 3.1C). The third peak eluted at 13.4ml, according to the standard curve of Superdex 200 (Figure 2.1), this volume represents for an around

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110kDa-protein. In this case, wild type 54kDa-mIfit1b eluted as a dimer. After purification, no crystals were observed in all the 96-well plates.

A B

C

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D

E

13.4mL(110kD)

Figure 3.1 - The purification of wild type mIfit1b. (A) SDS-PAGE of the 1st and 2nd nickel purification. One distinct band was observed for the full-length His-tagged mIfit1b (~68kDa) after 1st Ni-purification and another distinct band was observed for the full-length mIfit1b (~54kDa) after ULP cleavage (AUC). Also there were some other bands indicating the protein was not pure. (B) SDS-PAGE of the S column elution. One distinct band was observed for the full-length mIfit1b (~54kDa) with some impurities. (C) SDS-PAGE of the gel filtration elution. (D) S column purification. RNA-free mIfit1b eluted at 150mM salt concentration and RNA-contaminated mIfit1b eluted at 1M salt concentration. (E) Wild type mIfit1b eluted as a dimer from the Superdex 200 gelfiltration.

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3.1.2 mIfit1b (L453E)

Since the mIfit1b dimer didn’t crystallize, I wanted to attempt crystallizing its monomer instead. Our lab has worked on human IFIT1, and the mutagenesis of its

Leucine 464 into Glutamate can change it from a dimer into a monomer.

Alignment of mIfit1b with human IFIT1 helped identify the potential orthologous mutation required to disrupt the mouse Ifit1b dimer. The same mutation as hIFIT1 was applied to mIfit1b. Leucine453 in mIfit1b was changed into Glutamate.

mIfit1b

After Ni-purification and S column purification (Figure 3.2 A), a single peak eluted from gel filtration at a volume of 13.4ml (Figure 3.2 B), indicating a 110kd dimer. It showed that the mutation was not strong enough to disrupt the dimerization of the mouse

Ifit1b protein, likely because the dimerization interface is stronger than in the human

IFIT1 dimer.

46 260692719-Hongyu Wang

A

B UV absorption(mAU)

13.4ml(110kd) C

54kd

Volume(mL)

Figure 3.2 - The purification of mIfit1b (L453E). (A) S column purification. RNA-free mIfit1b (L453E) eluted at 150mM salt concentration and RNA-contaminated mIfit1b (L453E) eluted at 1M salt concentration. (B) mIfit1b (L453E ) still eluted as a dimer. The peak was observed at 13.4mL. (C) SDS-PAGE of the gel filtration elution. One distinct peak was observed at around 54kDa.

47 260692719-Hongyu Wang

96-well plates were set up for crystallization, which yielded very small crystals

(Figure 3.3 A) and some very big crystal clusters (Figure 3.3 B). 24-well plates were tried to optimize these conditions, but only very small crystals were obtained (Figure 3.3

C).

A

B

C

48 260692719-Hongyu Wang

Figure 3.3 - 10mg/mL mIfit1b (L453E)+5’-ppp oligo-A RNA crystals. The pictures to the left are bright field image of the crystals, and the pictures to the right are fluorescence of the crystal upon exposure to UV light indicates that the crystal is likely composed of protein. The crystals are shown by the red arrows. (A) Crystals obtained in condition G2 in screen Classics1 at 4°C: 0.2M Calcium chloride, 0.1M HEPES PH7.5, 28% PEG 400. (B) Crystals obtained in condition F8 in screen Classic2 in 4°C: 0.2M Ammoniumm sulfate, 0.1M HEPES PH7.5, 25% PEG3350. (C) Crystals obtained in solution with 28% PEG350, 0.2M Calcium chloride, 0.1M Tris PH7.5 at 4°C

3.2 Mouse Ifit1b2 and its Mutant

3.2.1 Wild Type mIfit1b2

After Ni-purification, mIfit1b2 was applied onto an S column (Figure 3.4 A). The

RNA-free protein was then concentrated and applied onto a size-exclusion chromatography column for gel filtration (Figure 3.4 B). The elution volume (12.8mL) of the peak from gel filtration indicated that mIfit1b2 eluted as a dimer as well. Pure mIfit1b2 was obtained after gel filtration(Figure 3.4 C).

A

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B C

Figure 3.4 - The purification of wild type mIfit1b2. (A) S column purification. RNA-free mIfit1b2 eluted at 150mM salt concentration and RNA-contaminated mIfit1b2 eluted at 1M salt concentration. (B) mIfit1b2 eluted as a dimer. The peak was observed at 12.8mL. (C) SDS-PAGE of the S column and gel filtration elution. One distinct peak was observed at around 54kDa in gel filtration elution.

96-well plates were set up for crystallization of mIfit1b2. Small needles were obtained in the condition containing 0.2M Calcium chloride, 0.1M HEPES sodium salt

PH7.5, 28% PEG400 at 4°C(Figure 3.5). 24-well plates and additive screens were attempted for optimization but these only yielded crystal clusters, similar to the clusters obtained from mIfit1b (L453E).

50 260692719-Hongyu Wang

Figure 3.5 - 10mg/mL mIfit1b2+5’-ppp oligo-A RNA crystals. The pictures to the left are bright field image of the crystals, and the pictures to the right are fluorescence of the crystal upon exposure to UV light indicates that the crystal is likely composed of protein. The crystals are shown by the red arrows. Crystals were obtained in the condition containing 0.2M Calcium chloride, 0.1M HEPES sodium salt PH7.5, 28% PEG400 at 4°C

3.2.2 mIfit1b2 (L456E)

Mutagenesis was attempted to get crystals for mIfit1b2. It was aligned with human

IFIT1. Leucine456 was changed into Glutamate for mIfit1b2.

After Ni-purification, the protein was applied on a Q column (Figure 3.6 A) in order to separate RNA-free and RNA-contaminated mIfit1b2 (L456E). RNA-free protein with an A260/A280 value of 0.54 eluted from the column at a salt concentration of 200mM. At

1M salt concentration, RNA-contaminated mIfit1b2 (L456E) with A260/A280 value of

1.0 was purified. RNA-free protein was then applied onto a size-exclusion chromatography column (Figure 3.6 B). One sharp peak eluted from the gel filtration at

51 260692719-Hongyu Wang

15.3mL, corresponding to a 45kDa-protein according to the standard curve. This showed that mutated mIfit1b2 (L456E) was changed from a dimer into a monomer. mIfit1b2

(L456E) was obtained after gel filtration purification.

A Percentage of Q-B buffer UV absorption(mAU)

Volume(mL) ‘'

52 260692719-Hongyu Wang

D

Figure 3.6 - The purification of mIfit1b2 (L456E). (A) Q column purification. RNA-free mIfit1b2 (L456E) eluted at 200mM salt concentration and RNA-contaminated mIfit1b2 (L456E) eluted at 1M salt concentration. (B) mIfit1b2 (L456E) eluted as a monomer. The peak was observed at 15.3mL. (C)The peak shift between the gel filtration of mIfit1b2 and its mutant. mIfit1b2 (L456E) eluted from 12.9mL to 15.3mL after mutagenesis. (D) The SDS-PAGE for the main purification of mIfit1b2 (L456E). One distinct single band was observed after gel filtration.

96-well plates were set up for mIfit1b2 (L456E) for crystallization. Small crystals grew in the drop of 10mg/mL protein with ppp-RNA, under the condition of 0.1M

HEPES pH7.5, 10% PEG8000, 8% Ethylene glycol at 4°C (Figure 3.7 A) and 0.2M

Magnesium chloride, 0.1M Sodium cacodylate pH6.5, 50% PEG200 at room temperature

(Figure 3.7 B). Crystal seeding with hIFIT1 crystal yielded in similar very small crystals

(Figure 3.7 C).

53 260692719-Hongyu Wang

A B C Figure 3.7 - 10mg/mL mIfit1b2 (L456E)+5’-ppp oligo-A RNA crystals. The crystals are shown by the red arrows. A. The small crystals I got under 0.1M HEPES pH7.5, 10% PEG8000, 8% Ethylene glycol at 4°C. B: The cluster I got under 0.2M Magnesium chloride, 0.1M Sodium cacodylate pH6.5, 50% PEG200 at room temperature. C. The small crystals I got with human IFIT1 crystal seeding under 0.2M Magnesium chloride, 0.1M Sodium cacodylate pH6.5, 50% PEG200 at room temperature

3.3 Mouse Ifit1b3

The same Ni purification, Q column (Figure 3.8 A) and gel filtration (Figure 3.8 B) as mIfit1b2 (L456E) were applied for mIfit1b3. Two peaks were seen after gel filtration .

SDS-PAGE showed that the peak at 13.2mL, representing a molecular weight of approximately 118 kDa, indicating that mIfit1b3 also purifies as a dimer.

A UV absorption(mAU) Percentage of Q-B buffer

Volume(mL)

54 260692719-Hongyu Wang

B

Figure 3.8- The purification of mIfit1b3. (A) Q-column purification. RNA-free mIfit1b3 eluted at 200mM salt concentration and RNA-contaminated mIfit1b3 eluted at 1M salt concentration. (B) mIfit1b3 eluted as a dimer. The peak was observed at 13.2mL.

96-well plates were set up for mIfit1b3 for crystallization. Small crystals grew in the drop of 10mg/mL protein with ppp-RNA, under the condition of 0.2M Potassuin formate,

20% PEG3350 at 4°C (Figure 3.9 A) and 0.2M di-sodium tartrate, 20% PEG3350 at 4°C

(Figure 3.9 B).

55 260692719-Hongyu Wang

A

B

Figure 3.9 - 10mg/mL mIfit1b3+5’- ppp oligo-A RNA crystals. The pictures to the left are bright field image of the crystals, and the pictures to the right are fluorescence of the crystal upon exposure to UV light indicates that the crystal is likely composed of protein. The crystals are shown by the red arrows. A. Small crystals got under 0.2M Potassuin formate, 20% PEG3350 at 4°C. B: Small crystals got under 0.2M di-sodium tartrate, 20% PEG3350 at 4°C

The mutagenesis to change mIfit1b2 from a dimer into a monomer didn’t seem to

help with crystallization, so the same mutagenesis was not applied to mIfit1b3.

3.4 Rabbit Ifit1b2 and its Mutant

3.4.1 Wild Type rIfit1b2

After Ni-purification (Figure 3.10 A), rIfit1b2 required further purification so it

was applied to a Q column (Figure 3.10 B). However, a small amount of protein was

obtained in the 0-200mM salt concentration gradient, as most protein was bound to

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RNA and eluted at higher salt concentration. Mono Q column (Figure 3.10 C) was attempted after Ni-purification instead for further separating the proteins. RNA-free protein with an A260/A280 value of 0.62 eluted from the column at a salt concentration from 0 to 400mM gradient. RNA-contaminated rIfit1b2 with A260/A280 value of 1.3 eluted at 1M salt concentration. RNA-free protein was then applied onto a size- exclusion chromatography column (Figure 3.6 D). Every fractions in the main peak were collected and samples were ran on the SDS-PAGE, and no distinct band for rIfit1b2 was observed on SDS-PAGE (Figure 3.6 E), indicating that most rIfit1b2 degraded.

A B

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C

D

E

marker Gel filtration factions

Figure 3.10- The purification of wild type rIfit1b2. (A) SDS-PAGE of Ni-purification. An obvious band appeared at 56kDa with some impurities after 2nd nickel. (B) Q column purification. Few RNA-free protein eluted during the salt concentration gradient. (C) Mono Q column purification. RNA-free protein eluted during the salt concentration from 0 to 400mM gradient. (D) The gel filtration graph of the rIfit1b2. The main peak was observed at 17.5mL. (E) SDS-PAGE of gel filtration purification. No distinct band was observed.

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3.4.2 Truncated rIfit1b2

The alignment with human IFIT1 showed that rIfit1b2 has an elongated N-terminus.

In order to stabilize the protein and avoid degradation, the first 47 amino acids of the N- terminus were truncated.

rIfit1b2

After Ni-purification and S column (Figure 3.11 A), and RNA-free protein was applied on gel filtration (Figure 3.11 B). One sharp peak was observed at 13mL, indicating an around 156kDa protein, suggesting a possible rIfit1b2 trimer, since it appeared pure on SDS-PAGE (Figure 3.11C).

A UV absorption Percentage of Q-B buffer

volume

59 260692719-Hongyu Wang

B C

Figure 3.11 - The purification of truncated rIfit1b2. (A) S column purification. RNA-free protein eluted at the salt concentration of 150mM, and RNA-contaminated protein eluted at 1M salt concentration. (B) Truncated rIfit1b2 eluted as a trimer. A peak was observed at 13mL. (C) SDS-PAGE for gel filtration purification. One distinct band was observed after gel filtration.

96-well plates were set up for truncated rIfit1b2. Crystal clusters grew under 0.2M

Calcium acetate, 0.1M MES pH6.5, 40%PEG300 at 4°C (Figure 3.12 A) and 0.2M

Calcium acetate, 0.1M Sodium cacodylate pH6.5, 45% Glycerol at 4°C (Figure 3.12 B).

24-well plates were attempted to optimize the conditions to get crystals. Similar crystal clusters grew under 0.1M Sodium cacolylate pH6, 0.2M Calcium acetate, 47.5% glycerol at 4°C (Figure 3.12 C) and 0.1M Sodium cacolylate pH6.5, 0.2M Calcium acetate, 50% glycerol at 4°C (Figure 3.12 D).

60 260692719-Hongyu Wang

A

B

C

D

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Figure 3.12 - 10mg/mL truncated rIfit1b2+5’-ppp oligo-A RNA crystals. The pictures to the left are bright field image of the crystals, and the pictures to the right are fluorescence of the crystal upon exposure to UV light indicates that the crystal is likely composed of protein. The crystals are shown by the red arrows. A. The crystal clusters grew in 96-well plates under 0.2M Calcium acetate, 0.1M MES pH6.5, 40%PEG300 at 4°C. B:The crystal clusters grew in 96-well plates under 0.2M Calcium acetate,0.1M Sodium cacodylate pH6.5, 45% Glycerol at 4°C. C:The crystals clusters grew in 24-well plates under 0.1M Sodium cacolylate pH6, 0.2M Calcium acetate, 47.5% glycerol at 4°C. D: The crystal clusters grew in 24-well plates under 0.1M Sodium cacolylate pH6.5, 0.2M Calcium acetate, 50% glycerol at 4°C.

3.4.3 Truncated rIfit1b2(L456E)

Truncated rIfit1b2 was aligned with human IFIT1. Leucine456 was changed into

Glutamate to change it into a monomer.

rIfit1b2

Ni-purification, S column (Figure 3.13 A) and Q column (Figure 3.13 B) was applied to truncated rIfit1b2 (L456E) in turn. Then RNA-free protein was loaded on the gel filtration (Figure 3.13 C). During gel filtration, a sharp peak eluted at 14.5ml, corresponded to the size of a monomeric rIfit1b2 according to a standard curve. The peak shift after mutagenesis (Figure 3.13 D) indicated that the mutation changed truncated rIfit1b2 into a monomer.

62 260692719-Hongyu Wang

A Percentage of Q-B buffer UV absorption(mAU)

Volume(mL)

B UV absorption(mAU) Percentage of Q-B buffer

Volume(mL)

63 260692719-Hongyu Wang

C UV absorption(mAU)

14.5ml(47kd)

Volume(mL) D UV absorption(mAU)

13ml(156kd) 14.5ml(47kd)

Volume(mL) Figure 3.13 - The purification of the of truncated rIfit1b2 (L456E). (A) S column and its SDS-PAGE. RNA-free proteins eluted at 150mM salt concentration. (B) Q column and its SDS-PAGE. RNA-free proteins eluted at 200mM salt concentration. (C) Truncated rIfit1b2 (L456E) eluted as a monomer. A peak was observed at 14.5mL. (D) Peak shift between truncated rIfit1b2 and its mutant.

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96-well plates were set up for truncated rIfit1b2 (L456E). Crystal clusters grew for

10mg/mL protein with ppp-RNA under the condition of 0.2M Calcium chloride, 0.1M

Bis-Tris pH6.5, 45% MPD at room temperature (Figure 3.14 A). During 24-well plates optimization, similar crystal clusters grew under the condition of 0.2M Calcium chloride,

0.1M Bis-Tris pH7, 47.5% MPD at room temperature (Figure 3.14 B).

A

B

Figure 3.14 - 10mg/mL truncated rIfit1b2 (L456E)+5’-ppp oligo-A RNA crystals. The pictures to the left are bright field image of the crystals, and the pictures to the right are fluorescence of the crystal upon exposure to UV light indicates that the crystal is likely composed of protein. The crystals are shown by the red arrows. A. The crystal cluster grew under 0.2M Calcium chloride, 0.1M Bis-Tris pH6.5, 45% MPD at room temperature. B:The crystal clusters grew under 0.2M Calcium chloride, 0.1M Bis-Tris pH7, 47.5% MPD at room temperature.

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3.5 Rabbit Ifit1b

Full-length rabbit Ifit1b was transformed into E.coli Rosetta2 (Plys2) for expression, but on the SDS-PAGE analysis of the protein sample after Ni-purification showed that the protein degraded (Figure 3.15 A). So E.coli BL21 (Star) was used instead to express the cell but the protein still degraded (Figure 3.15 B). The pET system is usually stable, but maybe some toxin proteins that express recombination can cause plasmid loss. Or the protein may be degraded by protease.

A B

63kDa 75kDa 63kDa 48kDa 48kDa

35kDa 35kDa

25kDa 25kDa 20kDa 20kDa 17kDa 17kDa 11kDa

Figure 3.15 - SDS-PAGE of rIfit1b Ni-purification. (A) The gel of 1st Ni-purification of rIfit1b expressed in E.coli Rosetta2 (Plys2). Lots of bands were observed, showing that the proteins degraded (B) The gel of 1st Ni-purification of rIfit1b expressed in E.coli BL21 (Star). The protein also completely degraded.

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4. DISCUSSION

The IFIT proteins play an important role in innate immunity, because it can directly bind viral single strand mRNA and restrict viral replication. In order to better understand its function in antiviral response, it is necessary to know its structural basis for RNA recognition and binding mechanism. Also, by identifying possible nucleotide preferences for certain IFIT family proteins, it would be easier to infer their abilities to specifically work against certain viruses.

Ifit1b proteins in mice (mouse Ifit1b and its mutation, mouse Ifit1b2 and its mutation, mouse Ifit1b3) and rabbits (rabbit Ifit1b, rabbit Ifit1b2, truncated Ifit1b2 and its mutation) were studied here, with the goal of solving the crystal structures of these proteins.

Most IFIT proteins bind bacteria RNA, even after RNase was added and lysis buffer with high salt concentration was used during purification. This can be seen from the high

A260/A280 value after nickel purification. It is thus necessary to use ion exchange columns to separate RNA-free and RNA-contaminated proteins.

After the initial nickel purification, followed by ion exchange column and size exclusion gel filtration analysis, mouse Ifit1b, Ifit1b2, Ifit1b3 and truncated rabbit Ifit1b2 proteins formed dimers. A mutagenesis of leucine in C-terminus into glutamate can change mouse Ifit1b2 and truncated rabbit Ifit1b2 into a monomer, but it didn’t work with

67 260692719-Hongyu Wang mouse Ifit1b. This is probably because the binding interface between mouse Ifit1b monomers is much more stronger than that of other Ifit proteins. Also, a deletion construct (48-510) was used to save rabbit Ifit1b2 from degradation, since it can stabilize the protein,

Crystallization was attempted for the Ifit1b proteins and the complex of them binding with 5’-ppp oligo-A RNA , but the crystals obtained for both for Ifit1b dimers and monomers were not suitable for further studies. Different pHs, precipitants, salts and temperatures didn’t help with crystallization. There were either no crystals or only crystal clusters and small crystals that cannot be used for crystal structure studies. Ifit1b protein with cap0 RNA complex should also be crystallized to study the 3D structure.

Although no crystal structures were determined from this Ifit1b protein study, this research still provides us with some useful experience and information that may help further studies on crystallization of IFIT protein.

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