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Unveiling the neglected roles of nucleoprotein NLS2 and cellular vimentin during Influenza A virus infection Wu, Wei 2014

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Unveiling the neglected roles of nucleoprotein NLS2 and cellular vimentin during Influenza A virus infection    by Wei Wu B.Sc., Shandong University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2014  © Wei Wu, 2014  ii Abstract Influenza A virus exploits the cellular transport machinery during the early stages of infection. It enters cells by endocytosis and takes advantage of the endocytic trafficking to move towards the perinuclear region with the assistance of actin filaments and microtubules. A recent proteomic study identified vimentin as a putative interacting protein of influenza viral components. However, the role of vimentin during influenza A infection has not yet been determined. After endocytosis, the viral ribonucleopotein complexes (vRNPs), containing the RNA viral genome, the viral polymerases, and several copies of nucleoprotein, are released from late endosomes and enter the nucleus for replication. Two nuclear localization sequences (NLSs), NLS1 and NLS2, on nucleoprotein mediate the nuclear import of vRNPs. The function of NLS1 has been well studied, however, the role of NLS2 remains to be defined.  This thesis has two major aims: to characterize the function of NLS2 and to determine the role of vimentin during influenza infection. For the first aim, I use a chimeric protein (5GFP) fused to NLS2, in combination with RNAi of several importin  isoforms and biochemical assays, and found that NLS2 is able to mediate the nuclear import of 5GFP by interacting with importin 1, 3, 5, and 7. NLS2 contains only a single amino acid difference at position 17 between different strains of influenza A virus, which could be either lysine (K) or arginine (R). I found that NLS2K induces more nuclear accumulation of 5GFP than NLS2R. Using site-directed mutagenesis I demonstrated that NLS2K contains two independent functional basic clusters, while NLS2R only has one. Moreover, my study also revealed that inhibiting the function of NLS1 and NLS2 impairs the nuclear import of vRNPs and further inhibits viral infection.   iii For the second aim I followed influenza A virus infection in both vimentin null cells and vimentin RNAi knock-down cells, and found that vimentin plays a role in releasing vRNPs from endosomes. In summary, my work dissects the basic mechanisms involved in influenza A virus endocytic trafficking and nuclear import, which provide us with better insights into the viral-host interaction during influenza A virus infection. iv Preface My supervisor Dr. Nelly Panté and I designed all the experiments presented in this thesis. I conducted all the experimental procedures, quantified, and analyzed all data. Dr. Lixin Zhou assisted me in the GST-pull down assay and the Co-immunoprecipitation experiments (Figures 3-14A and 3-15A). Dr. Shelly Au helped me to obtain electron microscope images of negative stained influenza viruses generated by reverse genetics (Figure 4-1B).   The research presented in this thesis was approved by the UBC Bio-Safety Committee (Certificate B10-0057).  v Table of Contents Abstract ..................................................................................................................... ii Preface ...................................................................................................................... iv Table of Contents ...................................................................................................... v List of Tables ......................................................................................................... viii List of Figures .......................................................................................................... ix List of Abbreviations ............................................................................................... xi Acknowledgement ................................................................................................. xvi Chapter 1 Introduction ............................................................................................ 1 1.1 Nucleocytoplasmic transport ............................................................................................... 1 1.1.1 Nuclear pore complex (NPC) ........................................................................................... 2 1.1.2 Classical nuclear import pathway .................................................................................... 6 1.1.3 Nuclear localization sequence (NLS) ............................................................................... 9 1.1.4 Classical nuclear import receptors: importin  and importin  ..................................... 12 1.1.5 Non-classical nuclear import pathways .......................................................................... 19 1.2 Influenza A virus ................................................................................................................. 21 1.2.1 Introduction to influenza A viruses ................................................................................ 21 1.2.2 The structure of influenza A virus and its ribonucleoprotein complex (vRNP) ............. 22 1.2.3 The replication cycle of the influenza A virus ................................................................ 25 1.2.4 The involvement of the cytoskeleton during influenza infection ................................... 29 1.2.5 Influenza A virus vRNP nuclear import ......................................................................... 30 1.2.6 Nuclear localization signals on vRNP ............................................................................ 32 1.2.7 Current antiviral treatments for influenza A virus infection .......................................... 37 1.3 Novel functions of vimentin intermediate filaments ........................................................ 40 1.3.1 Overview of novel functions of vimentin ...................................................................... 44 1.3.2 The role of vimentin during viral infection .................................................................... 46 1.4 Research objectives ............................................................................................................. 49 The following are the rationales for these aims. ..................................................................... 50 1.4.1 Aim 1: To characterize the NLS2 of influenza A virus nucleoprotein and to identify its cellular binding partners .......................................................................................................... 50 1.4.2 Aim 2: To explore whether the NLSs of influenza A virus NP are good candidates for novel antiviral approaches ....................................................................................................... 51 1.4.3 Aim 3: To study the role of vimentin during influenza A virus infection ...................... 52 Chapter 2 Materials and Methods ........................................................................ 53 2.1 Cell culture .......................................................................................................................... 53 2.2 Reagents and antibodies ..................................................................................................... 53 2.3 Construction of recombinant plasmids ............................................................................. 57 2.4 Transfection of recombinant DNA and small interference RNA (siRNA) ..................... 61 2.4.1 Transfection of recombinant DNA ................................................................................. 61  vi 2.4.2 Small-interfering RNA (siRNA) transfection ................................................................ 61 2.5 Influenza A virus infection ................................................................................................. 62 2.6 Fluorescence microscopy .................................................................................................... 63 2.6.1 Indirect immunofluorescence microscopy ..................................................................... 63 2.6.2 Epidermal growth factor (EGF) uptake .......................................................................... 63 2.6.3 Dextran uptake ............................................................................................................... 64 2.6.4 Quantification of fluorescence images ........................................................................... 64 2.6.4.1 Quantification of nuclear import .......................................................................................... 64 2.6.4.2 Quantification of vesicle area .............................................................................................. 65 2.6.4.3 Quantification of lysosome distribution ............................................................................... 65 2.6.4.4 Quantification of colocalization of NP and EEA1/Rab7 ..................................................... 66 2.6.4.5 Quantification of endosome acidification using phrodo EGF .............................................. 66 2.7 Western blot analysis .......................................................................................................... 66 2.8 Generation of influenza A virus by reverse genetics ........................................................ 67 2.9 Plaque assay ........................................................................................................................ 68 2.10 Electron microscopy ......................................................................................................... 69 2.11 Co-IP .................................................................................................................................. 69 2.12 Expression and purification of importin s ................................................................... 70 2.13 GST-pull down assay ........................................................................................................ 71 2.14 RNA extraction and cDNA synthesis ............................................................................... 72 2.15 Real-time qPCR ................................................................................................................ 72 Chapter 3 Characterization of the NLS2 of Influenza A Virus Nucleoprotein and Identification of its Cellular Binding Partners ............................................ 74 3.1 Introduction ......................................................................................................................... 74 3.2 Results .................................................................................................................................. 76 3.2.1 Nuclear import function of NLSs from influenza A virus nucleoprotein ....................... 76 3.2.2 One basic amino acid difference changes the nuclear import function of NLS2 ........... 78 3.2.3 Both clusters of basic residues in NLS2K are important for efficient nuclear import ... 81 3.2.4 The N-terminal cluster of basic residues in NLS2R is the main contributor for nuclear import ...................................................................................................................................... 83 3.2.5 The linker of NLS2R also contributes to the NLS2R function ...................................... 85 3.2.6 Threonine at position 18 contributes to the function of NLS2K but not to the function of NLS2R ................................................................................................................................ 88 3.2.7 Identification of the binding partners of NLS2 during nuclear import .......................... 92 3.2.7.1 Importin 3 is important for NLS2K-mediated nuclear import........................................... 92 3.2.7.2 Importin 3 mediates nuclear import of NLS2R-containing proteins ................................. 93 3.2.7.3 Importin 1, 3, 5, and 7 bind to NLS2K and NLS2R ................................................ 101 3.3 Discussion .......................................................................................................................... 105 Chapter 4 NLSs of Influenza A Virus Nucleoprotein as Novel Antiviral Candidates ............................................................................................................. 111 4.1 Introduction ....................................................................................................................... 111 4.2 Results ................................................................................................................................ 113 4.2.1 NLS1 or NLS2 mutations on NP lead to the production of non-infectious influenza A viruses by reverse genetics .................................................................................................... 113 4.2.2 Competing with the NLS1 or NLS2‟s function impair nuclear import of vRNP during influenza A virus infection .................................................................................................... 118  vii 4.3 Discussion .......................................................................................................................... 126 Chapter 5 Characterization of the Role of Vimentin During Influenza A Virus Infection ................................................................................................................. 131 5.1 Introduction ....................................................................................................................... 131 5.2 Results ................................................................................................................................ 133 5.2.1 Vimentin is required for efficient influenza A virus infection ...................................... 133 5.2.2 The distribution and morphology of late endosomes and lysosomes are altered in both vimentin-/- cells and vimentin depleted HeLa cells. .............................................................. 138 5.2.3 Vimentin is required for the escape of the influenza genome from late endosomes .... 148 5.2.4 Lack of vimentin affects the endosomal pH ................................................................. 155 5.3 Discussion .......................................................................................................................... 160 Chapter 6 General Discussion and Future Perspectives .................................. 163 6.1 Towards dissecting the function of NLS2 ....................................................................... 163 6.2 Role of importin  in mediating the nuclear import of NLS2-containing proteins .... 168 6.3 NLSs of NP as potential antiviral targets ....................................................................... 170 6.4 Role of vimentin during influenza A virus infection ...................................................... 173 6.5 Proposed model of influenza A virus early infection steps. ........................................... 177 6.6 Future directions ............................................................................................................... 179 6.6.1 Does any post-transcriptional modifications affect the function of NLS2K? .............. 179 6.6.2 Characterization of the importin  interaction with NLS2K and NLS2R ................... 180 6.6.3 Drug screening for potential compounds targeting NLS1 and NLS2 from influenza A virus NP ................................................................................................................................. 180 6.7 Concluding remarks ......................................................................................................... 181 References ............................................................................................................. 183 Appendix A ............................................................................................................ 207  viii List of Tables Table 1-1: Consensus sequences and examples of different types of nuclear localization sequences.。 ............................................................................................................................ 11 Table 1-2: Structural basis of NLS binding by importin . ........................................................... 17 Table 1-3: Importin  isoforms and their alternate names. ........................................................... 18 Table 1-4: Details of the 9 influenza A virus strains that contain a NP with obpNLS. ................. 36 Table 1-5: Identified IF family members and their tissue distribution. ......................................... 42  Table 2-1: List of antibodies used in this thesis. ........................................................................... 55 Table 2-2: List of primers used to generate recombinant plasmids in this thesis. Primers were purchased from Integrated DNA Technologies. ...................................................................... 59  Table 3-1: Summary of importin  depletion experiments. ........................................................ 100                 ix List of FiguresFigure 1-1: Structure and composition of the nuclear pore complex (NPC). ................................. 5 Figure 1-2: Overview of the classical nuclear import pathway and the RanGTPase cycle. ........... 8 Figure 1-3: Structure of importin . .............................................................................................. 13 Figure 1-4: Binding affinity of importin  for cNLS cargo and its autoinhibitory mechanism. .. 14 Figure 1-5: Schematic diagram of the structure of the influenza A virus and its vRNP complex. 24 Figure 1-6: Schematic diagram of the influenza A virus life cycle. .............................................. 28 Figure 1-7: Schematic representation of the NLS on the vRNP proteins NP, PA, PB1 and PB2. 35  Figure 3-1: Fusion of either NLS1 or NLS2 to 5GFP induces nuclear import of the chimera protein. ..................................................................................................................................... 77 Figure 3-2: Fusion of either NLS2K or NLS2R to 5GFP induces nuclear import of the chimera proteins. ................................................................................................................................... 80 Figure 3-3: Both the N-terminal and the C-terminal basic clusters of NLS2K mediate the nuclear import of 5GFP-NLS2K. ......................................................................................................... 82 Figure 3-4: Mutations in the N-terminal basic cluster of basic amino acids of NLS2R reduce the nuclear accumulation of 5GFP-NLS2R. ................................................................................. 84 Figure 3-5: The linker of NLS2R also contributes to the NLS2R function. ................................. 87 Figure 3-6: Substituting threonine with lysine at position 18 reduces the nuclear accumulation of 5GFP-NLS2K but not that of 5GFP-NLS2R. ......................................................................... 90 Figure 3-7: Mutation of threonine to alanine in the C-terminal cluster reduces the nuclear accumulation of 5GFP-NLS2K, but not that of 5GFP-NLS2R. ............................................. 91 Figure 3-8: Depletion of importin α1 does not affect the nuclear accumulation of 5GFP-NLS2K. ................................................................................................................................................. 94 Figure 3-9: Knockdown of importin α3 affects the 5GFP-NLS2K nuclear accumulation. .......... 95 Figure 3-10: Depletion of importin α5 does not affect the nuclear accumulation of 5GFP-NLS2K. ................................................................................................................................................. 96 Figure 3-11: Knockdown of importin α1 reduces the nuclear accumulation of 5GFP-NLS2R. ... 97 Figure 3-12: Knockdown of importin α3 does not affect the nuclear accumulation of 5GFP-NLS2R. ......................................................................................................................... 98 Figure 3-13: Depletion of importin α5 does not affect the 5GFP-NLS2R nuclear accumulation. 99 Figure 3-14: Detection of the interactions between NLS2R and NLS2K with importin α1, α3, α5, and α7 using GST pull-down assay. ...................................................................................... 103 Figure 3-15: NLS2K and NLS2R bind importin 1, 3, 5, and 7 detected by Co-IP. .......... 104  Figure 4-1: Wild type and NLS mutant viruses were successfully generated by reverse genetics. ............................................................................................................................................... 116 Figure 4-2: Mutations of NLS1 or NLS2 inhibit the infectivity of influenza A virus................. 117 Figure 4-3: 5GFP-NLS1, NLS2K, and NLS2R do not affect the nuclear import of DsRed-cNLS. ............................................................................................................................................... 121 Figure 4-4: DsRed-cNLS does not inhibit the nuclear import of influenza A virus NP. ............. 122 Figure 4-5: Transfection of 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R plasmids inhibits nuclear import of influenza A virus. ...................................................................................... 125   10 Figure 5-1: Viral M1 mRNA expression decreased in vimintin null cells.. ................................ 135 Figure 5-2: The viral M1 and NP protein expression was attenuated in vimentin null cells. ..... 136 Figure 5-3: Influenza viral progeny production was significantly decreased in vimentin-/- cells. ............................................................................................................................................... 137 Figure 5-4: The distribution of late endosomes and lysosomes, but not early endosomes is modified in vimentin null cells. ............................................................................................. 142 Figure 5-5: Knockdown of vimentin modifies late endosomal and lysosomal distribution, but not early endosomal distribution. ................................................................................................ 147 Figure 5-6: Sequential subcellular events of NP during influenza A virus infection in vimentin+/+ MEFs. .................................................................................................................................... 151 Figure 5-7: Sequential subcellular events of NP during influenza A virus infection in vimentin-/- MEFs. .................................................................................................................................... 153 Figure 5-8: Colocalization analysis of NP and EEA1 or Rab7 during influenza A virus infection. ............................................................................................................................................... 154 Figure 5-9: The acidification of EGF-containing endosome is inhibited in vimentin-/- fibroblast cells. ....................................................................................................................................... 158 Figure 5-10: The enlargement of endo/lysosomal compartment during dextran uptake in vimentin-/- fibroblast cells. .................................................................................................... 159  Figure 6-1: Proposed scheme of the early steps of infection of influenza A virus. ..................... 178  Figure A-1: Alanine scanning of the linker of NLS2K/R. .......................................................... 207 Figure A-2: NP and EEA1 staining during influenza A virus infection in vimentin+/+ MEFs.. .. 209 Figure A-3: NP and EEA1 staining during influenza A virus infection in vimentin-/- MEFs...... 211 Figure A-4: NP and Rab7 staining during influenza A virus infection in vimentin+/+ MEFs. ..... 213 Figure A-5: NP and Rab7 staining during influenza A virus infection in vimentin-/- MEFs. ...... 215    xi List of Abbreviations Amino acids and its corresponding one-letter code:  A  Ala  Alanine E  Glu  Glutamate/Glutamic Acid F  Phe  Phenylalanine G  Gly  Glycine H  His  Histidine K  Lys  Lysine L  Leu  Leucine M  Met  Methionine P  Pro  Proline Q  Gln  Glutamine R  Arg  Arginine S  Ser  Serine T  Thr  Threonine Y  Tyr  Tyrosine  Other abbreviations: ARM: armadillo repeats AcMNPV: Autographa californica multiple nucleopolyhedrovirus  AFSV: African swine fever virus AP-3: adaptor protein complex 3 BSA: bovine serum albumin CAS: cellular apoptosis susceptibility  xii CaM: calmodulin  CIC: chloride channel cNLS: classical nuclear localization sequence CNS: central nerve system CO2: carbon dioxide Co-IP: Co-immunoprecipitation  DMEM: Dulbecco‟s modified Eagle medium DNA: deoxyribonucleic acid ECM: extracellular matrix EBV: Epistein-Barr virus E. coli: Escherichia coli EDTA: Ethylenediaminetetraacetic acid EE: early endosome EEA1: early endosome antigen 1 EGF: epidermal growth factor EM: electron microscopy ER: endoplasmic reticulum FBS: fetal bovine serum FDA: Food and Drug Administration  FG: phenylalanine glycine FG-Nups: nucleoporins containing phenylalanine glycine repeats GAPDH: glyceraldehyde 3-phosphate dehydrogenase GTP: guanine triphosphate GFAP: glia fibrillary acidic protein  xiii GST: Glutathione S-transferase HA: hemagglutinin HEAT: helical-repeat motifs HEK: human embryonic kidney HIV-1: human immunodeficiency virus type 1 hnRNP: heterogeneous ribonucleoprotein particle HTLV-1: human T-lymphotropic virs type 1 IBB: importin  binding domain IF: intermediate filament Imp: importin kDa: kilo Dalton LAMP1: lysosomal-associated membrane protein 1 LE: late endosome MDCK: Madin-Darby canine kidney MEF: mouse embryonic fibroblast  MOI: multiplicity of infection MT: microtubule  MTOC: microtubule organization center mRNA: messenger ribonucleic acid NA: neuraminidase NE: nuclear envelope NEP: nuclear export protein NF: neurofilament  NLS: nuclear localization sequence  xiv NP: nucleoprotein NPC: nuclear pore complex NS1: non-structural protein 1 NTF2: nuclear transport factor 2 Nups: nucleoporins obpNLS: overlap bipartite NLS PA: polymerase protein A PB1: polymerase protein B1 PB2: polymerase protein B2 PBS: phosphate-buffered saline PFA: paraformaldehyde PFU: plaque-forming unit p.i.: post infection PKA: protein kinase A PTHrP: parathyroid hormone-related protein PVDF: polyvinylidene difluoride Rab7: Ras-related GTP-binding protein 7 RanBP2: Ran binding protein 2 RanBP5: Ran binding protein 5 RanGDP: Ran guanine diphosphate RanGEF: Ran guanine exchange factor RanGTP: Ran guanine triphosphate RCC1: regulator of chromosome condensation 1 RNA: ribonucleic acid  xv RNPs: ribonucleoproteins SDS: sodium dodecyl sulphate S.E.M: standard error of the mean siRNA: small interfering ribonucleic acid snRNP: small nuclear ribonucleic particle  SPEBP-2: sterol regulatory element-binding protein 2 sumoylation: attachment of a small ubiquitin-like modifier (SUMO) to a protein SV40 Tag: simian virus 40 large T antigen TE: Tris-EDTA buffer Vim: vimentin vRNA: viral ribonucleic acid vRNP: viral ribonucleoprotein WGA: wheat germ agglutinin WT: wild-type xvi Acknowledgement First of all, I want to take this opportunity to express my deepest appreciation to my supervisor Dr. Nelly Panté, who not only provided me with valuable guidance during my PhD study, but also taught me to learn and grow as a scientist and also as a person. Moreover, she gave me strong support during my hardest time in my PhD career and taught me proper scientific writing in English. This thesis could not be completed without her patience and constant revisions after revisions.   I would also like to express my sincerest appreciation to the members of my doctoral supervisory committee, Dr. Ninan Abraham, Dr. Linda Matsuuchi, and Dr. Calvin Roskelly for their patient instructions, valuable ideas, and critical feedbacks over the years.    I would like to thank every current and former laboratory members. In particular, thank you to Dr. Lixin Zhou for your help with my project and your precious ideas and discussions over the years. Thank you to Dr. Winco Wu for your patient training and great help at the beginning of my graduate study. Thank you to my lovely friends and lab mates, Dr. Shelly Au and Maria Acevedo, for all your support, encouragement, and precious discussion about science and life over the last six years. I would not be here without you two in the lab. Thanks too to Nikta Fay, who brought me to explore the vimentin world, I really appreciate your useful advice about my project. Thank you to Dr. Pierre Garcin for your suggestions about my project. I learned a lot from you as a hard working scientist who have strict principles for science and life. And Thanks to Dr. Sarah Cohen for all her great work and contributions to the lab, you are always my role model.     xvii In addition, I would like to thank my parents, Gang Wu and Baohui Xu, who supported me to take the opportunity to study in Canada. Thank you dad and mom for your love and encouragement. This was not an easy journey. Thanks for your understanding in the occasions that I had bad temper and to hear my complaining over the years. I would also like to express my deepest memorial to my grandmother, Xinping Zhang, who brought me up and taught me to work hard. She was really happy to know there will be finally a PhD in the family when I transfer to the PhD program. I did it grandma!   Bo, I would not be here without you. Over the years, you kept me sane and supported me unconditionally. Thank you for walking together though all the hard time with me and encourage me to pursue what I want. You are the inspiration of my life. Thank you for knowing I can, when I think I cannot.    1 Chapter 1 Introduction The threat of influenza A virus has greatly increased over the past several years with the emergence of the high virulent avian H5N1 virus and the pandemic H1N1 virus. Studying the infection process of influenza A virus may not only provide us with a better understanding of the biology of influenza A virus infection, but also give us new insight toward development of new anti-viral drugs. In order to achieve a successful infection, influenza A virus hijacks the host cellular machineries to deliver its genome into the host cell nucleus for transcription and replication. During infection, the viruses-containing endosomes use the cytoskeleton (actin filaments and microtubules (MTs)) to travel towards the nucleus. After uncoating, the influenza A viral genome gains access to the cell nucleus using the nuclear import machinery. In this thesis, I investigate the function of nuclear localization sequences that mediate the nuclear import of the influenza A viral genome. I also explore the role of vimentin intermediate filaments during influenza A virus infection because vimentin was recently identified in a proteomics-based screen as a cellular factor associated with influenza A viral ribonucleoproteins (vRNPs). This introduction will begin with background information about the structure of the nuclear pore complex (NPC) and nucleocytoplasmic transport. Then, I will introduce the life cycle of influenza A virus, followed by a literature review about the role of the cytoskeleton and nuclear import during influenza A virus infection. Finally, I will briefly introduce novel functions of vimentin intermediate filament and its interaction with other viruses during infection.   1.1 Nucleocytoplasmic transport Eukaryotic cells have highly defined compartmentalized biological systems. The nucleus contains  2 genetic materials and the cellular transcriptional machinery, whereas the cytoplasm retains the translational machinery and metabolic systems. The nuclear envelope (NE), a double membrane, is the nucleus-cytoplasm boundary that selectively regulates the bidirectional transport between these two compartments. Material traffics across the NE through large, multiple protein complexes called nuclear pore complexes (NPCs). Molecules smaller than about 40 kDa or 9 nm in diameter can freely diffuse through the NPC, whereas large molecules with diameter up to 39 nm are actively transported through the NPC (Pante and Kann, 2002). Regulation of the bidirectional transport through the NPC is critical for the cell in controlling gene expression and keeping nuclear identity.   1.1.1 Nuclear pore complex (NPC) The NPC is a huge structure of around 125 MDa in vertebrates and 60 MDa in yeast (Reichelt et al., 1990; Rout and Blobel, 1993). It spans the outer and inner nuclear membranes, providing a physical barrier to molecular trafficking in and out the nucleus. The number of NPCs per cell varies between species. The mammalian nucleus contains 1,000 to 5,000 NPCs, whereas the yeast nucleus usually contains 100 to 200 NPCs (reviewed by Grossman et al., 2012). The large nucleus from Xenopus oocyte is characterized with the highest NPC density and contains up to 5107 NPCs (reviewed by Grossman et al., 2012). Thus, it has been used as a model system to study the structure of the NPC. The three dimensional structure of the NPC is highly conserved, although size differences have been observed among evolutionary distant species. The morphology and structure of the NPC has been examined using transmission electron microscopy, cryo-electron microscopy, scanning electron microscopy, and atomic force microscopy. In general, a NPC has eight fold rotational symmetry, containing a NE-embedded ring around a central  3 channel, a cytoplasmic ring, and a nuclear ring (Figure 1-1). The 70 nm cytoplasmic ring connects with eight 35-50 nm cytoplasmic filaments on the cytoplasmic side. The nuclear ring attaches to a nuclear basket that is formed by eight 50-100 nm nuclear filaments, and these nuclear filaments join at the distal end by an about 30 nm diameter ring. The size of the NE-embedded ring is 120 nm in diameter and 70 nm in height. The size of the hourglass-shaped NPC central channel is about 60-70 nm in diameter near its cytoplasmic and nuclear periphery and around 45 nm in diameter at the middle-plane (Beck et al., 2004; Beck et al., 2007; Maimon et al., 2012).   Using mass spectrometry analysis of biochemically purified NPCs, researchers have identified around 30 different proteins, called nucleoporins (Nups), which constitute the NPC (Cronshaw et al., 2002). The majority of Nups are symmetrically distributed near the middle-plane of the NPC. There are small subsets of Nups that are located asymmetrically at either the nuclear or cytoplasmic side of the NPC (reviewed by Wente and Rout, 2010). Nups are grouped into three different classes based on their secondary structure and localization (reviewed by Grossman et al., 2012). The first class is transmembrane Nups, which span the nuclear membranes and anchor the NPC to the NE. The second class is scaffold Nups, which form the outer and inner rings and comprise the NPC core scaffold. The barrier Nups, the third class of Nups, are located at the inner most layer of the NPC, and contain multiple copies of phenylalanine glycine (FG) sequence motifs that bind to nuclear transport factor and facilitate active transport (reviewed by Grossman et al., 2012; Onischenko and Weis, 2011). The major component of the NPC cytoplasmic filaments is the nucleoporin RanBP2/Nup358, which facilitates nuclear entry by interacting with import cargo at the NPC cytoplasmic side. The cytoplasmic ring is enriched with Nup214 and the nuclear basket is enriched with Nup153. The major proteins of the NPC central channel are  4 Nup62, Nup58, and Nup54, which are also termed the Nup62-complex (reviewed by Wente and Rout, 2010). All these Nups together play important roles in transporting and releasing cargo during nuclear entry. Taken together, Nups are important structure elements of the NPC, and their major function is to mediate nucleocytoplasmic exchange of proteins and RNAs through the NPC.    5  Figure 1-1: Structure and composition of the nuclear pore complex (NPC). A schematic representation of the NPC with its structural components and their dimensions. Positions of different classes of Nups are shown. Adapted from Cohen et al. (2012).    6 1.1.2 Classical nuclear import pathway The active transport of macromolecules between the cytoplasm and the nucleus is facilitated by NTFs called “karyopherins” (Radu et al., 1995), with those participating in nuclear import termed “importins”(Gorlich et al., 1994), and those involved in nuclear export called “exportins” (Stade et al., 1997). Importins and exprotins recognize nuclear localization signals (NLSs) and nuclear export signals (NESs) in proteins and direct them in and out of the nucleus, respectly. Currently, there are 19 known human karyopherins and 14 yeast karyonphrins (Chook and Suel, 2011). Among these, importin  (karyopherin 1) is the most common karyopherin involved in nuclear import of cargoes.     In the classical nuclear import pathway, a cytoplasmic cargo containing a classical NLS is first recognized by importin , which associates with importin  via its N-terminal importin  binding (IBB) domain. Once the trimeric import complex (cargo-importin -importin ) is formed, importin β interacts with FG-Nups and docks the cargo to the NPC (reviewed by Stewart, 2007) (Figure 1-2A). The direction of nuclear transport is determined by the Ran-GTP/GDP concentration gradient: Ran-GTP is abundant in the nucleus, while Ran-GDP is concentrated in the cytoplasm (Gorlich et al., 1996) (Figure 1-2B). The differential compartmentalization is maintained by Ran guanine exchange factor (RanGEF; also termed regulator of chromosome condensation 1 (RCC1)) in the nucleus, which catalyses the exchange of GDP for GTP on Ran (Bischoff and Ponstingl, 1991), and Ran GTPase-activating protein (RanGAP) in the cytoplasm, which stimulates GTP hydrolysis of RanGTP (Bischoff et al., 1994). In the cytoplasm, the presence of Ran-GDP allows the formation of the cargo-importin α-importin β complex. When this complex is transported into the nucleus, RanGTP binds to importin  and triggers the  7 dissociation of importin  and  (Gorlich et al., 1996). The importin -cargo complex then attaches to Nups at nuclear basket, such as Nup153 and Nup50 (Makise et al., 2012; Matsuura and Stewart, 2005; Ogawa et al., 2012). This interaction breaks the binding between importin  and the NLS, and releases the cargo into the nucleus. Importin  and  then recycles back to the cytoplasm. This recycling is mediated by RanGTP. In the nucleus, RanGTP mediates the attachment of cellular apoptosis susceptibility (CAS) protein to importin , which helps it releasing from Nup50 and recycles back to the cytoplasm (Pumroy et al., 2012). RanGTP also mediates importin -recycling to the cytoplasm (Matsuura and Stewart, 2004). Finally, after importin  and  are transported back to the cytoplasm, RanGAP stimulates the RanGTPase, generating RanGDP, which dissociates from the importins and releases them for another nuclear import cycle (Figure 1-2A) (reviewed by Yarbrough et al., 2014).    8  Figure 1-2: Overview of the classical nuclear import pathway and the RanGTPase cycle. A) During the classical nuclear import, an import complex is formed in the cytoplasm between the cargo that contains a cNLS, importin α, and importin β. This import complex is translocated into nucleus through the NPC. The binding of RanGTP to importin β in the nucleus dissociates importin β from importin α. The cNLS-containing cargo is then displaced from importin α and CAS exports importin α back to the cytoplasm. B) Cytoplasm RanGDP is imported into the nucleus by NTF2, where RanGEF catalyzes the exchange of GDP to GTP on Ran and generates RanGTP. RanGTP then binds to importin β and it is exported back to the cytoplasm, where RanGAP stimulates GTP hydrolysis of RanGTP. 9 1.1.3 Nuclear localization sequence (NLS) Macromolecules destined for active transport into the nucleus contain amino acids targeting sequences called NLSs. The best characterized NLS is the classical NLS (cNLS) (Table 1-1), first identified by Kalderon et al. (1984). These authors demonstrated that simian virus 40 (SV40) large T antigen contains a short basic sequence, PKKKRKV, which promotes nuclear import of cytoplasmic proteins, such as -galactosidase and pyruvate kinase. A few years later, Dingwall et al. (1988) found that a 16-amino-acids sequence, KRPAATKKAFQAKKK, at the C-terminal end of the Xenopus laevis protein nucleoplasmin mediated the nuclear import of pyruvate kinase. The NLSs from SV40 large T antigen and nucleoplasmin are now considered the prototypes for classical monopartite and bipartite NLSs respectively. Monopartite NLSs contain a single cluster of basic residues, and are further divided into two groups: pattern 4 (pat4) NLS and patter 7 (pat7) NLS (Kosugi et al., 2009). The Pat4 NLS (also classified as class 1) is defined as a continuous stretch of four basic amino acids (K or R) or 3 basic amino acids with a fourth amino acid being either histidine (H) or proline (P) (K-K/R-X-K/R), exemplified by SV40 large T-antigen NLS (PKKKRK) (Table 1-1). The Pat7 NLS (class 2) starts with P and follow within 3 residues by a basic segment containing 3 basic residues out of 4, exemplified by c-Myc NLS (PAAKRVKLD) (Table 1-1). The classcical bipartite NLS consists of two basic residues separated by a spacer region of 10-13 amino acids. The putative sequence of a classical bipartite NLS is defined as (K/R)(K/R)X10-13(K/R)3/5, where X indicates any amino acids and (K/R)3/5 indicates that on the C-terminus of the classical bipartite NLS at least three out of five amino acids are either lysine or arginine (Kosugi et al., 2009). Recently, both Lange et al. (2010) and Krebs et al. (2010) revealed that the linker within a classical bipartite NLS could contain more than 13 amino acids, as Ty1 integrase contains a linker of 29 amino acids and transcription factor  10 DNA-binding-with-one-finger contain a linker of 17 amino acids. These findings suggest the consensus of a classical bipartite NLS could be expanded to include a linker region containing up to 29 amino acids. Bioinformatics analysis results show that, in yeast, 57% of nuclear proteins use classical nuclear import, with 27.2% containing monopartite cNLS and 21.9% containing bipartite cNLS (Lange et al., 2007).  Using a large-scale screen of random peptide libraries, Kosugi et al. (2009) identified three other classes (class 3, 4, and 5) of noncanonical monopartite NLSs that could bind to yeast, plant, or human importin s in vitro (Table 1-1). Class 3 and 4 NLSs have KRX(W/F/Y)XXAF and (P/R)XXKR(K/R) sequences respectively, which are found in yeast, mammals, and plants. Class 5 NLSs have a LGKR(K/R)(W/F/Y) core sequence and is specific only to plants.  Besides importin -dependent NLSs, there are a number of NLSs that directly bind to proteins members of the karyopherin  family (Table 1-1). NLSs that are rich in arginine can bind to importin  (karyopherin 1) (Gu et al., 2011). Importin-5/Kap121 (karyopherin 3) recognizes lysine-rich sequences, which are usually longer (>25 amino acids) and more complex compared to the lysine-rich cNLSs (Chook and Suel, 2011). There are also NLSs that can be recognized by tranportin-1 (karyopherin 2). These are termed proline-tyrosine NLSs (PY-NLSs) and consist of a N-terminal hydrophobic or basic motif and a C-terminal R/H/KX(2-5)PY consensus sequence (Zhang and Chook, 2012). The first PY-NLS was identified in the mRNA-binding protein hnRNP A1 and was originally called M9. Transportin-3 was shown to interact with a wide range of arginine/serine (RS) domain-containing proteins, the majority of which are involved in mRNA metabolism (Maertens et al., 2014).   11 Table 1-1: Consensus sequences and examples of different types of nuclear localization sequences.。  NLS Type Consensus sequence Protein Name NLS Amino Acid Sequence Class 1 monopartite NLS (Pattern 4) KR(K/R)R, K(K/R)RK SV40 large T-antigen PKKKRKV Class 2 monopartite NLS (Pattern 7) (P/R)XXKR(∧DE)(K/R) Human c-myc PAAKRVKLD Class 3 noncanonical monopartite NLS KRX(W/F/Y)XXAF Human Nucleolar RNA helicase II KRSFSKAF Class 4 noncanonical monopartite NLS (P/R)XXKR(K/R) (∧DE) SUMO E3 ligase PIASy NLS2 PRPKRRCPFQF Class 5 NLS LGKR(K/R)(W/F/Y) DNA cross-link repair protein SNM1 LGKRRR  Class 6 bipartite NLS (K/R)(K/R)X(10-13)(K/R)3/5 Xenopus nucleoplasmin KRPAATKKAFQAKKK Arginine rich NLS None HIV-1 Rev protein NLS RQARRNRRRWR Lysine rich NLS None Saccharomyce cerevisiae Spo12p KKSTSNLKSSHTTSNLVKKTMFKRDLLKQDPKRKL  PY-NLS Central hydrophobic/basic+ C-terminus R/H/KX(2-5)PY Human hnRNP A1 FGNYNNQSSNFGPMKGGNFGGRSSGPY RS domain containing NLS None Human SR protein ASF/SF2 Residule 198- 248  Sequence representation is as follow: (∧DE), any amino acids except Asp or Glu; X, any amino acid; X(10-13), any 10-13 amino acids;X(2-5), any 2-5 amino acids. Table adapted from Chook and Suel (2011); Marfori et al. (2011). 12 1.1.4 Classical nuclear import receptors: importin  and importin   Nuclear import pathways are mediated by -karyopherins. The family of -karyopherins share weak sequence homology in general but their N-terminal sequences are highly conserved and are essential for the RanGTP binding (Figure 1-3). As described in Section 1.1.2, in the classical nuclear import pathway, importin  (also known as karyopherin 1 or importin 1) does not bind to the cNLS directly, but binds to importin , which serves as an adaptor between the cNLS-containing cargo and importin .   Structure and biochemical analysis of importin  revealed three functional regions within importin  (Figure 1-3): An N-terminal region that binds to importin  (the importin  binding (IBB) domain), a central region containing two NLS binding sites and the C-terminal region. Moroianu et al. (1996) demonstrated that the N-terminal IBB domain of importin  contains a cluster of basic residues (KRR) similar to a cNLS (Table 1-2). The function of this sequence in the IBB domain is to regulate and compete for binding of cNLS cargo to importin  (Kobe, 1999), thus it is termed the autoinhibitory sequence. Based on competition assays, in the cytoplasm, cNLS-containing cargo preferentially bind to the importin / complex rather than to free importin  (Goldfarb et al., 2004). Once inside the nucleus, the binding of RanGTP to importin  triggers the dissociation of importin / complex. When released from importin , the IBB domain competes with cNLS of the cargo and facilitates cargo release from importin  in the nucleus (Figure 1-4). 13  Figure 1-3: Structure of importin . Importin  contains three functional regions: an N-terminal importin  binding domain (IBB), a central ARM repeats region, which contains the major (ARM repeats 2-4) and minor (ARM repeats 6-8) NLS binding sites, and a C-terminal region.  14  Figure 1-4: Binding affinity of importin  for cNLS cargo and its autoinhibitory mechanism. In the cytoplasm, importin  binds to importin  through its IBB domain and binds to cNLS cargo through its cNLS-binding pocket. The binding affinity of importin  to a cNLS cargo is strong because there is no competition between the IBB domain with a cNLS cargo for the cNLS binding pocket. Once the import complex is translocated into the nucleus, RanGTP binds to importin , which releases the IBB domain of importin . This allows the autoinhibitory motif within the IBB domain to compete with cNLS for the cNLS binding pocket on importin , which results in the dissociation of the cNLS cargo from importin . 15 The central region of importin  contains a tandem series of 10 armadillo (ARM) repeats that form a NLS-binding sites (Conti et al., 1998). There are two specific NLS binding sites located in this central region of importin  (Figure 1-3): a major site spans ARM repeats 1-4 that can interact with class 1 and 2 monopartite NLS and the larger basic cluster of classical bipartite NLS, and a minor site spans ARM repeats 6-8 that can bind to class 3 and 4 monopartite NLS and the shorter basic cluster of classical bipartite NLS (Table 1-2) (Chook and Blobel, 2001; Kosugi et al., 2009; Xu et al., 2010). The critical amino acids binding to the minor site and the major site are termed P1‟ to P4‟ and P1 to P5, respectively (reviewed by Marfori et al., 2011). A lysine-arginine (KR) motif is always present in position P1‟ and P2‟ to bind to the minor binding site on importin  (reviewed by Chang et al., 2013). For the major binding site, a lysine at P2 position on NLS is critical for its binding affinity with importin , followed by P3 and P5 occupied by lysine or arginine (Table 1-2).   It has been shown that all organisms have a single gene encoding for importin  (Chi et al., 1995). In contrast, the human importin  family consists of at least 7 isoforms (Table 1-3), which fall into 3 phylogentically subfamilies (1, 2, and 3) based on sequence homology (Goldfarb et al., 2004). Subfamily 1 includes importin 5, 6, and 7. Subfamily 2 includes importin 1 and the recently reported importin 8 (Hu et al., 2010; Kelley et al., 2010). Subfamily 3 includes importin 3 and importin 4. Importin 1 subfamily is found in all eukaryotes, while importin 2 and 3 subfamilies are only found in metazoan animals suggesting that they appeared during the evolution of multicellular organism (reviewed by Marfori et al., 2011). The identity of members within a importin  subfamily share at least 80% in amino acid sequence similarity, whereas members of different subfamilies have around 50% sequence identity (Lange et al.,  16 2007). Although most importin  isoforms are expressed in all tissues, it has been shown that importin 1, 2, and 3 subfamilies could also exhibit unique expression patterns among different tissues, with the exception of importin 6, which has only been found in testis (Kamei et al., 1999; Kohler et al., 2002; Quensel et al., 2004). In addition, certain NLSs selectively bind to a specific importin  isoform for nuclear import, while other NLSs could bind to all importin  isoforms (Kohler et al., 1999), which indicate that importin s exhibit a broad functional redundancy. 17 Table 1-2: Structural basis of NLS binding by importin .   Protein Name Minor pocket binding site          linker         Major pocket binding site  P1’P2’P3’P4‟                                    P1 P2 P3 P4 P5 Linker length SV40 Tag                                               P PK K K R K V  IBB domain                                          D E Q M L K K R N V S   Murine p53                                             P P Q P K K K P L D G E   NF-B p65                                                E E K R K R  Nucleolar RNA helicase II   G Q K R S F S K A F  PIASy NLS2  P R P K R R C P F Q F  Nucleoplasmin   A V K R             P A A T K K A G Q A          K K K K L D  10 N1N2      R K K R K T         E E E S P L K D K A         K K S K  11 Human P53       K R             A L P N N T S S P Q P         K K K P  11 Human IL-5       K K             Y I D G Q K K K C G E E      R R R V N Q 12   Abbreviations: SV40 Tag, SV40 T-antigen; N1N2, Xenopus phosphoprotein N1N2; Human IL-5, Human Interleukin-5. Bold letters indicate the basic amino acid residues require for the nuclear import. The monopartite and bipartite cNLSs are aligned based on the interaction with importin . Table adapted from Marfori et al. (2011). 18 Table 1-3: Importin  isoforms and their alternate names.  Protein Name Approved Symbol Alternate Names Importin 1 Karyopherin Subunit alpha-2 (KPNA2) SRP1alpha, IPOA1, QIP2, NPI-3 Importin 3 KPNA4 SRP1gamma, SRP4, hSRP1, IPOA4 Importin 4 KPNA3 SRP3, QIP1, IPOA3 Importin 5 KPNA1 SRP1, RCH2, NPI-1, IPOA5, MGC11217 Importin 6 KPNA5 SRP6, IPOA6 Importin 7 KPNA6 IPOA7, MGC17918 Importin 8 KPNA7   19 1.1.5 Non-classical nuclear import pathways  Importin  also participates in non-classical nuclear import pathways that are independent of importin . One of these pathways uses another adaptor protein that contains an IBB like domain. During nuclear import of splicosomal ribonucleoproteins containing UsnRNAs (UsnRNPs), the m3G-cap on the RNA acts as an NLS to bind to the adaptor protein, snurportin1 (Fischer et al., 1993). The N-terminal of snurportin1 contains a 40-amino acids domain that shares large homology with the IBB domain of importin  (Huber et al., 1998). Therefore, the snurportin1 IBB-like domain binds to importin  to facilitate nuclear import of UsnRNPs.   Besides using adaptor proteins to interact with the cargo, importin  also participates in nuclear import by direct interaction with the cargo. A range of cargo proteins has been identified to bind to importin  directly. These proteins include the parathyroid hormone-related protein (PTHrP) (Roth et al., 2011), the sterol regulatory element-binding protein 2 (SREBP-2) (Nagoshi et al., 1999), the viral protein HIV-1 Rev (Arnold et al., 2006), and human T-lymphotropic virs type 1 (HTLV-1) Rex (Tsuji et al., 2007). All of these proteins contain an arginine-rich NLS that binds directly to importin .   Other proteins of the -karyopherin family have also been identified to mediate nuclear import. For example, transportin-1 (also know as karyopherin 2 in yeast) is a nuclear import factor for many mRNA-binding proteins, which are transported into the nucleus to facilitate the nuclear export of mRNA. The heterogeneous ribonucleoprotein A1 (hnRNP A1) is the best-characterized protein that uses tranportin-1 for nuclear import. Its NLS is the PY-NLS (Table 1-1), which  20 consists of 38 amino acids that binds to the large region in the C-terminal half of transportin-1 to translocate hnRNP A1 into the nucleus. Using bioinformatics analysis, Chook and colleagues identified 81 cargo-bearing PY-NLS that could interact with transportin-1 during nuclear import in human (Chook and Suel, 2011; Lee et al., 2006).   Some proteins do not require karyopherin  for their translocation through the NPC. The direct interaction between a cargo protein and Nups offers another mechanism by which the NPC promotes nuclear import of cargo in a karyopherin -independent manner. For example, it has been reported that Nup153 could interact with several proteins that undergo nuclear import receptor-independent via its FXFG-rich C-terminal region. These proteins include HIV-1 integrase (Woodward et al., 2009), transcription factor PU.1 (Zhong et al., 2005), STAT1 (Marg et al., 2004), and kinase ERK2 (Whitehurst et al., 2002). Other Nups have also been identified to interact directly with the cargo to facilitate their nuclear entry, including Nup214 (Marg et al., 2004; Whitehurst et al., 2002) and Nup62 (Leng et al., 2007).   Finally, cellular cytoskeleton systems have recently been discovered to be implicated in the nuclear import of many proteins and viruses. Specifically, the cytoskeleton can accelerate the cargo transport through the cytoplasm towards the nuclear periphery, where importin proteins take over and translocate the cargo into the nucleus. For example, parathyroid hormone-related protein (PTHrp) contains a region that overlaps with the importin -recognized NLS region, which interacts with microtubules (MTs) (Klemmt et al., 2007). This region promotes PTHrP movement along MT to the perinuclear region where importin  displace MT to mediate PTHrP transport into the nucleus (Roth et al., 2007; Roth et al., 2011). Viruses also hijack the  21 cytoskeleton to enter the nucleus. For example, the baculovirus Autographa california multiple nucleopolyhedrovirus (AcMNPV) contains a viral Wishkott-Aldrich syndrome protein (WASP)-like capsid protein, P78/83, that serves as a nucleation-promoting factor that activates actin polymerization by host Arp2/3 (Goley et al., 2006). The actin forming comet tail has been observed to push AcMNPV through the NPC into the nucleus using live cell fluorescence microscopy (Ohkawa et al., 2010).   Taken together, many mechanisms are involved in controlling the transport of materials between the cytoplasm and the nucleus, including direct diffusion, classical nuclear import, alternative carriers facilitated transport, nuclear import through binding to Nups, and transport enhanced or facilitated by the cytoskeleton. All these mechanisms work together to enable the cell to have an appropriate and efficient way to exchange materials between the cytoplasm and the nucleus, which is critical for eukaryotic cell‟s function.  1.2 Influenza A virus 1.2.1 Introduction to influenza A viruses Influenza viruses are important human pathogens, frequently causing widespread disease and a significant loss of life. Influenza viruses are members of the orthomyxovirus family, which consists of three genera: influenza A, B, and C virus (Kawaoka, 2006). Influenza viruses contain seven (influenza C) or eight (influenza A and B) segmented genomes composed of negative-sense single-stranded RNA. In humans, only influenza A and B viruses are of epidemiological interest. The type A influenza viruses are the most virulent human pathogens among the three influenza  22 types, and mostly are responsible for seasonal epidemics and global pandemics. Research on the mechanism of influenza A virus transport within the host cell, will not only assist us to understand the transmission and infection of this virus, but also will provide us with important background for influenza infection control.   1.2.2 The structure of influenza A virus and its ribonucleoprotein complex (vRNP) Influenza A virus is an enveloped virus with pleomorphic morphology that can appear to be small spherical particles, 80-120 nm in diameter, or long filamentous particles, 100 nm in diameter and up to 20 m in length (Ada et al., 1958; Bourmakina and Garcia-Sastre, 2003; Calder et al., 2010). Three proteins are embedded in the viral membrane: the 58 kDa type I transmembrane glycoprotein, hemagglutinin (HA), the 50 kDa type II transmembrane glycoprotein, neuraminidase (NA), and a 11 kDa proton ion channel, M2 protein (Figure 1-5A). The viral matrix protein (M1) underlies the lipid bilayer and associates with the viral ribonucleoproteins (vRNPs). This 28 kDa multifunctional protein is the most abundant protein in the virus and each virion is estimated to have 3000 copies (Harris et al., 2001). The non-structural protein NS2 and the nuclear export protein (NEP) can bind to vRNP via M1.  The genome of influenza A virus consists of eight segmented, single-stranded viral RNAs (vRNAs), which are packaged into the virions as vRNPs (Figure 1-5A). In addition to the viral RNAs, each vRNP contains a single copy of the RNA-dependent RNA polymerase (consisting of the subunits PB1, PB2, and PA) and multiple copies of nucleoprotein (NP; about 56 KDa) (Elton, 2006) (Figure 1-5B). Each NP is associated with 24 nucleotides of the vRNA (Ortega et al., 2000). Because the length of the vRNA varies from 890 to 2341 nucleotides (Lamb and Krug, 2013),  23 there are 37-97 copies of NP in each vRNP. Electron microsopy study revealed that vRNPs are in long helical rode shapes and their length varies from approximately 30 to 110 nm (Compans et al., 1972). The vRNPs are enclosed in the viral envelope in a distinct pattern with one vRNP in the middle circled by seven other vRNP (Noda et al., 2006).   The influenza A viral genome also encodes a 26 kDa RNA binding protein, NS1, which is not packaged into mature virion in the influenza A virus. NS1 is the interferon α/ß antagonist and reside strictly in the host cell (Garcia-Sastre, 2001).    24  Figure 1-5: Schematic diagram of the structure of the influenza A virus and its vRNP complex. A) Cartoon of the influenza A virus with labeled key viral structure and proteins. The virus contains an outer membrane envelope and an inner matrix protein layer that encloses eight-segmented RNAs. B) Cartoon of the influenza A virus vRNP. Yellow spheres represent NP proteins and the blue ovals represent the viral polymerase proteins (PA, PB1 and PB2). The RNA molecule is shown by the red curve that wrapped around the NP proteins.     25 1.2.3 The replication cycle of the influenza A virus The replication cycle of the influenza A virus is well characterized in the host cell (Das et al., 2010). As illustrated in Figure 1-6, the influenza A virus first attaches to the host cell membrane by HA-sialic acid glycoprotein association (Figure 1-6, step 1) (Skehel and Wiley, 2000). It then enters the host cell via receptor-mediated endocytosis or macropinocytosis (Figure 1-6, step 2) (de Vries et al., 2011; Matlin et al., 1981; Sieczkarski and Whittaker, 2002). The acidic environment of the late endosome then triggers HA to undergo a conformational change, which exposed the N-terminus HA2 subunit. The HA2 subunit inserts into the endosome membrane, resulting in the fusion of the viral envelope with the endosome membrane (reviewed by Hamilton et al., 2012; Sun and Whittaker, 2013). The M2 ion channel allows the flow of ions from the late endosome to the virion interior, leading to the dissociation of vRNPs from M1, which then move into the cytoplasm (Figure 1-6, step 3) (Doms et al., 1985). The vRNPs are then transported in the cytoplasm, and gain access to the nuclear import machinery of the cell (e.g., NPCs and importins). NLSs on NP of vRNPs recognize importin , which binds with importin . This complex is then tranlocated through the NPC to nucleus by the classical nuclear import pathway. (Figure 1-6, step 4) (Martin and Helenius, 1991a; Wu et al., 2007a). Incoming M1, on the other hand, enters the nucleus by passive diffusion. Inside the nucleus, the RNAs on vRNPs serve as templates to produce two forms of positive-sense vRNA: the complementary RNAs (cRNAs) and viral messager RNA (mRNA). The viral RNA transcription is initiated by the viral RNA polymerases, PA, PB1, and PB2 (Deng et al., 2006b). After being produced, the mRNAs are then exported into the cytoplasm to participate in viral protein synthesis, and the cRNA remains in the nucleus to produce large amount of vRNA (Figure 1-6, step 5) (Megan L. Shaw, 2013a; Nagata et al., 2008). After viral protein synthesis, HA, NA, and M2 are processed in the endoplasmic reticulum (ER),  26 glycosylated in the Golgi aparatus, transported to the plasma membrane, and associated with lipid rafts (Figure 1-6, step 9) (Leser and Lamb, 2005; Lin et al., 1998; Zhang et al., 2000). NP and the polymerase subunits are imported back into the nucleus. M1 and NEP/NS2 (which function in nuclear export of newly assembled vRNPs) are also imported into the nucleus (Figure 1-6, step 8) (Megan L. Shaw, 2013b). The vRNPs are then assembled with vRNA, NP, and polymerase subunits (Figure 1-6, step 10). The assembled vRNPs associate with M1, which then binds to NEP/NS2. The nuclear exports signal (NES) on NEP/NS2 initiates nuclear export of vRNP-M1-NEP/NS2 via the interaction with Crm1, a nuclear export receptor. (Figure 1-6, step 11). The eight vRNP segments are then directed to regions of the host plasma membrane for packaging into virions (Figure 1-6, steps 12 and 13). Mature virions are released from the host cell after NA cleaves the sialic acids on host glycoproteins (Figure 1-6, step 14) (Dowdle et al., 1974). 27     28 Figure 1-6: Schematic diagram of the influenza A virus life cycle. Influenza A virus adheres to its host cell by using its surface HA glycoprotein to recognize sialic acid (1). The virus then enters the host cell through endocytosis (2). Fusion of the viral envelope with the endosomal membrane occurs to release vRNPs to the cytoplasm (3). The vRNPs then are imported into the nucleus (4) for viral transcription and replication (5). mRNA is then exported to the cytoplasm (6) for viral protein synthesis (7). Newly synthesized viral proteins are either secreated through the Golgi apparatus onto the cell surface (9) or transported back into the nucleus (8) to bind with vRNAs and form new vRNPs (10). The vRNPs are then exported to the cytoplasm (11) and directed to the host plasma membrane (12) for packaging into virions (13 and14).    29 1.2.4 The involvement of the cytoskeleton during influenza infection Upon infection, influenza A virus binds to sialic acids on the cell surface to initiate infection. After the virus is internalized by either endocytosis or macropinocytosis, the virus is localized to early endosomes and then reaches late endosomes where the virus fuses its membrane with the endosome membrane to release viral vRNPs. In the cytoplasm, the presence of organelles, cytoskeleton, and molecular crowding restricts free diffusion of moleculars larger that 500 kDa (Luby-Phelps, 2000). Therefore, influenza A virus has to use an active mechanism for transport through the cytoplasm. Active cytoplasmic transport depends on a complex network of three cytoskeleton filaments: MTs, intermediate filaments, and microfilaments (reviewed by (Sodeik, 2000)). Using fluorescently labeled virion, it has been demonstrated that actin filament and MT are involved during endosomal trafficking of influenza A virus (Lakadamyali et al., 2003). The virus was visualized first to internalize to early endosomes at the cell periphery in an actin-dependent manner and then the virus-bearing endosomes transport via a dynein-dependent movement on MT to the perinuclear region where the endosome further undergoes a maturation process and turns into late endosome (Figure 1-3, steps 2 and 3) (Lakadamyali et al., 2003). By treating cells with cytochalasin D, an actin filament-disruption drug, or jasplakinolide, a filamentous actin stabilizer, Sun and Whittaker (2007) further showed that influenza A virus entry into polarized cells was inhibited. However, influenza A virus could still enter and infect non-polarized cells after these cells were treated with cytochalasin D or jasplkinolide (Sun and Whittaker, 2007), which suggested a role for actin filament during influenza A virus entry into polarized cells that is not shared with non-polarized cells.   At later stages of influenza A virus infection, vRNPs assemble inside the nucleus, are exported to  30 the cytoplasm, and travel to the cell periphery for viral exit. Using GFP-labeled vRNPs, Amorim et al. (2011) suggested that vRNPs are associated with Rab11 on recycling endosomes through its PB2 polymerase and travel along the MT network towards the cell periphery and plasma membrane (Figure 1-3, step 12). Later, a protein called YB-1 was identified to directly associate with vRNP and facilitate vRNP binding to MTs, which leads the travel of vRNP onto Rab11 positive recycling endosomes (Kawaguchi et al., 2012).   Although the role of actin filaments and MTs during influenza virus infection is well established, the involvement of intermediate filaments is less defined. A previous study showed that during influenza A virus infection, the intermediate filament, vimentin, was rearranged around the nucleus, and by 16 hours post infection, the vimentin network was completely collapsed around the nucleus (Arcangeletti et al., 1997). Using acrylamide, a drug that disrupts the vimentin intermediate filament network, it was shown that the viral production was significantly decreased in acrylamide-treated cells infected with influenza A virus (Arcangeletti et al., 1997). These results suggested a role of vimentin during influenza A virus infection. At which step of the influenza infection cycle vimentin is involved and why would infection be impaired in cells with disrupted vimentin network are some of the questions that I address in this thesis.   1.2.5 Influenza A virus vRNP nuclear import Unlike most RNA viruses that replicate in the cytoplasm, influenza A virus replication occurs in the nucleus. Babcock et al. (2004) showed that after using wheat germ agglutinin to inhibit translocation across the NPC, microinjected vRNPs did not enter the nucleus, indicating that vRNP translocate from the cytoplasm to the nucleus through the NPC. The vRNP complex is  31 about 20 nm in diameter and 30-100 nm long (reviewed by Compans et al., 1972; Pons et al., 1969). Since the size limit for passive import into the nucleus is 9 nm, vRNPs must be actively transported into nucleus through the NPC.   Although all four proteins (NP, PA, PB1, and PB2) on the vRNP contain NLSs (Figure 1-7), previous studies indicate that the interaction of vRNP with the nuclear import machinery occurs via the NLSs on NP. Several studies have shown that NP binds to importin 1, 3, and 5 in vitro (Melen et al., 2003; O'Neill et al., 1995). More recently, Gabriel et al. (2011) found that importin 7 can not only promote nuclear import of NP but is also important for replication of influenza A virus, as viral production was reduced in importin 7 RNAi silenced cells. Aida and her colleges recently reported that NLS1 can bind to importin 1, 3, and 5 using an in vitro pull-down assay, and NLS1 has a preferential binding to importin 3 via serine (S) at position 9 (Sasaki et al., 2013). In order to determine the necessity of cytosolic components during nuclear import of NP or vRNP, an import assay in digitonin-permeablized cells was used (Adam et al., 1990). In this system, after permeablization of the cell membrane with digitonin, the contents of the cytoplasm were washed away leaving the nucleus and the nuclear envelope intact. Using this system, O'Neill et al. (1995) defined the cellular components required for nuclear import of NP or an in vitro-formed NP-vRNA complex. In the presence of importin 1, importin , RanGTP, and nuclear transport factor 2 (NTF2), NP was imported into the nucleus of digitonin-permeabilized cells, whereas vRNAs could not be imported into the nucleus of these cells without NP. These results indicate that NP is responsible for the nuclear import of vRNP through the classical importin /importin  pathway. In addition, a recent genome-wide RNAi screen study identified transportin-3 as a host factor required for influenza virus replication (Konig et al., 2010),  32 suggesting that other nuclear import pathways may play a role during nuclear import of influenza A vRNP. But these could be involved in the nuclear import of any of the viral proteins that are rewuired to reach the nucleus for successful replication of the virus.  1.2.6 Nuclear localization signals on vRNP At least three NLSs have been found on NP (Figure 1-7): an unconventional NLS (NLS1) located at the N-terminus (amino acids 1-13) (Neumann et al., 1997; Wang et al., 1997), a bipartite NLS (NLS2) at amino acids 198-216 (Weber et al., 1998), and an overlapping bipartite NLS (obpNLS) at amino acids 90-121 (Ketha and Atreya, 2008). Several studies have shown that NLS1 is the major sequence not only for the nuclear import of NP but also for the nuclear import of vRNP. Using alanine substitution of basic amino acids on NLSs of NP, O‟ Neil and his colleges found that mutation of NLS1 impaired the nuclear import of NP (O'Neill et al., 1995). They also found that NLS1 peptide could inhibit the nuclear import of in vitro-formed NP-vRNA complexes, which indicates that NLS1 might be the dominant NLS during nuclear import of influenza A vRNPs (O'Neill et al., 1995). Using an antibody inhibition assay as well as peptide competition studies, Wu et al. (2007a) showed that the NLS1 on NP is the main contributor to the nuclear import of native vRNPs isolated from influenza A virus. These authors then determined that NLS1 is the main contributor for nuclear import because NLS1 has higher surface exposure on the vRNPs compare to NLS2 (Wu et al., 2007b).   The interaction of NLS1 with importin  isoforms has also been characterized. Previous studies have discovered that three different importin  isoforms can associate with NLS1. Using a yeast-two-hybrid assay, Wang et al. (1997) found that NLS1 binds to importin 1 through amino  33 acids 3SxGTKRSYxxM13 and also binds to importin 5 through amino acids 6KRSYxxM13 („x‟ represents any amino acid). Melen et al. (2003) demonstrated that importin 3 could also bind to NLS1 using a Glutathione S-transferase (GST)-pull down assay.   The NLS2 is a 19-amino acids sequence and is located between amino acids 198 and 216 of NP. This sequence was identified as a counterpart of an NLS of Thogoto virus NP, which accumulates in the nucleus but lacks the amino acid sequence of NLS1 of influenza A virus. NLS2 can function as an NLS to a limit range when it is fused to MxA (Weber et al., 1998), a large GTPase that accumulated in the cytoplasm (Aebi et al., 1989). Previous results about NLS2 are controversial. Cros et al. (2005) found that mutants of NLS1 using alanine substitution of basic amino acids impaired the nuclear import of NP, whereas mutation of basic amino acids at the C-terminus of NLS2 on NP did not affect the localization of NP. This observation indicates that NLS1 is the major NLS to mediate the nuclear import of NP. However, using vRNP isolated from influenza A virions, Wu et al. (2007a) performed both peptide competition and antibody blocking experiments and found that inhibiting either NLS1 or NLS2 resulted in the nuclear accumulation of vRNP in semi-permealized cells, which indicates that NLS1 and NLS2 act independently, as inhibition of either NLS could not abolish nuclear import of vRNP. To solve this controversy, in this thesis I further investigate the contribution of NLS2. In addition, Cros et al. (2005) only mutated the C-terminal basic amino acids of NLS2 to test nuclear import efficiency in their experiments. Thus, the necessity of other basic amino acids on the N-terminus of NLS2 still needs to be addressed. Finally, it still remains to be determined what are the binding partners of NLS2.    34 The obpNLS was discovered on NP of the WS/33L strain (Ketha and Atreya, 2008). It is 32-amino acids in length and is located between NLS1 and NLS2. There are only 9 influenza A virus strains containing an obpNLS (Table 1-4). With a change of amino acid at position 105 from methionine (M) to arginine (R) in the NP sequence of influenza A strains with this obpNLS, the 32 amino acids sequence was divided into two bipartite NLS: bpNLS-1 (90KKTGGPIYRRVDGKWRR106) and bpNLS-2 (105RRELILYDKEEIRRIWR121). This obpNLS has been demonstrated to be able to translocate a chimeric protein into the nucleus as a functional nuclear import sequence (Ketha and Atreya, 2008), but it remains to be shown whether it mediates the nuclear import of influenza A vRNPs.  Besides NP, the polymerase proteins PA, PB1, and PB2 all contain their own NLSs. During infection, after these proteins have been made in the cytoplasm, they are imported to the nucleus for vRNP assembling. PA has two independent NLSs: a NLS between amino acids 124 and 139 and an unclassified NLS between amino acids 186 and 247 (Nieto et al., 1992) (Figure 1-7). Both PB1 and PB2 have one NLS each: a 38-amino acids long classical bipartite NLS on PB1 (Nath and Nayak, 1990), and a 4-amino acid long classical NLS on PB2 (Figure 1-7). Another 47-amino acid long NLS has been found on PB2, and this NLS functions for perinuclear binding (Figure 1-7) (Mukaigawa and Nayak, 1991). It has been demonstrated that PB1-PA dimer would be formed in the cytoplasm and transported to the nucleus, whereas PB2 would be transported alone (Fodor and Smith, 2004). During nuclear import, PB1-PA dimer enters the nucleus via a non-classical transport pathway by binding to importin-5 (Deng et al., 2006a), while PB2 transports into the nucleus is through the classical nuclear import pathway by binding to importin 5 through its C-terminal NLS (Tarendeau et al., 2007).  35  Figure 1-7: Schematic representation of the NLS on the vRNP proteins NP, PA, PB1 and PB2. The protein sequences are based on influenza A virus, strain X:31. Numbers indicate amino acids positions. Amino acids in bold have been identified to be important for nuclear import. Amino acids underlined are basic amino acids that are essential for nuclear import. 36 Table 1- 4: Details of the 9 influenza A virus strains that contain a NP with obpNLS.  Accession Number Strain Host HN type Q1I2B5 A/WSN/1933 TS61 Human H1N1 Q1K9H2 A/WSN/1933 Human H1N1 Q9DLK6 A/WSN/1933 Mice H1N1 Q5Q142 A/swine/Korea/S109/2004 Swine H9N2 Q5Q165 A/swine/Korea/S190/2004 Swine H9N2 Q5Q173 A/swine/Korea/S175/2004 Swine H1N1 Q5Q179 A/swine/Korea/S83/2004 Swine H9N2 Q5Q187 A/swine/Korea/S81/2004 Swine H9N2 Q5Q195 A/swine/Korea/S10/2004 Swine H1N1  Abbrevation: HN type: hemaglutininin and neuraminidase classification type. Table adapted from Ketha and Atreya (2008).   37 1.2.7 Current antiviral treatments for influenza A virus infection  Influenza A virus is a major human pathogen that causes severe infection and affects millions of people worldwide. Many animal species can be infected by influenza A virus, including birds, swine, horses, and dogs. Therefore animals have become important hosts during influenza virus spreading. Influenza A virus is responsible for both epidemics and pandemics flu characterized by high morbidity and mortality. The current antiviral treatment for influenza A virus infection include vaccination and antiviral compounds, including M2 ion channel inhibitors (amantadine and rimantadine) and NA inhibitors (oseltamivir and zamivir). Since vaccines have to be reformulated each year depending on the genetic variability of the virus, an emerging influenza pandemic would be unlikely to be contained or treated by vaccination. Thus, it is important to gain more knowledge of the biology of this virus to be able to develop novel anti-influenza strategies. In this section I first describe the mechanism of action of the two drugs currently used to prevent or treat certain influenza A virus infection and then introduce recent studies directed towards the identification of novel anti-influenza A drugs.  Influenza M2 protein, which is embedded in the envelope of the influenza virus, acts as a proton channel (Betakova, 2007). During infection, after the virus enters its host cell through endocytosis, the lower pH inside the endosome lumen activates the M2 ion channel, which enables protons to flow from the endosome into the interior of the virion (Pinto and Lamb, 2006). This process is required for the dissociation of M1 and vRNPs inside the virion and the subsequent release of vRNPs in the cytoplasm. Amantadine and its methyl derivative rimantadine that inhibit this process were approved by USA Food and Drug Administration (FDA) in 1966 and 1994, respectively (Leonov et al., 2011; Wang et al., 1993). Amantadine and rimantadine are  38 all adamatane derivatives. Both compounds bind the N-terminal of M2, which blocks the flow of protons into the interior of the virion and prevents virus uncoating (Leonov et al., 2011). Unfortunately, the rapid emergence of drug resistant viruses has lead to a limited usage of these two drugs. Drug resistance to amantadine or rimantadine is associated with mutations at position 26, 27, 30, 31, 34, or 38 of M2 (Bright et al., 2005). Starting from 2005, more than 90% of seasonal H3N2 virus has Ser-to-Asn mutantion at position 31, which acquires adamatane resistance (Bright et al., 2005). In 2009, the novel A (H1N1) pdm09 virus replaced the seasonal H1N1 virus, however, it also contains the adamatane resistance mutation (CDC, 2009). The currently circulating high pathogenic avian influenza H5N1 was also found to be resistant to amantadine (Cheung et al., 2006; He et al., 2008). Therefore, the use of amantadine and rimantadine is no longer recommended.   Another target of anti-influenza drug is the viral neuraminidase (NA). During viral exit, the function of NA is to cleave off sialic acid from cell surface glycoproteins and to facilitate the release of viral particles from the host cell (Air and Laver, 1989; Gamblin and Skehel, 2010; Palese et al., 1974). Therefore, NA inhibitors should prevent the release of newly formed viral particles and the spreading of infection. The NA inhibitors were designed to be synthetic analogs of sialic acids, which compete with the natural substrate, NA, and block its enzyme active site. The NA inhibitors oseltamivir (Tamiflu®) and zanamivir (Relenza®) against both influenza A and B viruses were approved by the USA FDA in 1999 (Long et al., 2000). Although the NA active site is highly conserved, there are still a number of mutations in the NA protein at position 105, 119, 122, 136, 151, 152, 198, 224, 246, 274, 292, 294, or 371, which make the virus resistant to NA inhibitors (Samson et al., 2013). For example, a His-to-Tyr mutation at position 274 confers resistance to oseltamivir in influenza A viruses of the N1 subtype, including seasonal  39 A (H1N1) (Sheu et al., 2011), A pdm09 (H1N1) (Baz et al., 2009; Memoli et al., 2010), and highly pathogenic A (H5N1) (de Jong et al., 2005; Le et al., 2005). A few influenza A (H3N2) strains contain zanamivor resistant mutation at position 151 of NA (McKimm-Breschkin et al., 2003; Sheu et al., 2008). Fortunately, mutations associated with oseltamivir resistance do not confer zanamivor resistance. Although drug resistance towards NA inhibitors exists, the resistance among circulating influenza virus strains is currently low. In the 2013 to 2014 season, oseltamivir and zanamivir were still the drugs recommend by the center for disease control and prevention (CDC) in the USA (CDC, 2014).   Since antiviral resistance to available drugs has appeared rapidly, new antiviral targets and new drugs for treating influenza A virus infection are needed. Recently high-throughput screening studies have been conducted to identify compounds targeting other influenza A virus proteins. NP is the most abundant protein expressed during influenza infection. During vRNP assembly, NP monomers are able to oligomerize and to bind to RNA (Wu et al., 2007b; Ye et al., 2006). Though high throughput drug screening, Kao et al. (2010) discovered a compound, nucleozin, that can inhibit nuclear accumulation of NP and viral replication. Nucleozin was later found to be able to bind to NP, stabilize NP monomer interactions and prevent vRNP assembly (Gerritz et al., 2011). Besides NP, NS1 is also an attractive target for influenza antiviral drugs since it is able to inhibit interferon / response during influenza virus infection (Hale et al., 2008). In addition, NS1 interacts with mRNA export factor NXF1-NXT1 to prevent nuclear export of host mRNA (Satterly et al., 2007). Through a high-thoughput screening of 200,000 synthetic compounds, Mata et al. (2011) identified a compound, napthalimdies that could inhibit NS1 expression detecting by luciferase assay read-out. Further investigation showed that naphalimdies activates a  40 host factor, REDD1, that functions as an antagonist of NS1 to rescue interferon expression and inhibit influenza virus replication.   An alternative approach to inhibiting influenza infection is to interfere with the nuclear import of vRNPs, since this processes is necessary for viral replication. NLS1 and NLS2 from NP are known to mediate the nuclear import of vRNPs (Section 1.2.6). Sequence alignment showed that both NLSs on NP are highly conserved between different strains of influenza A virus (Kukol and Hughes, 2014), suggesting that these NLSs are ideal targets for novel antiviral research. However, experiments need to be performed to demonstrate the NLS1 and NLS2 are good anti-influenza targets.  1.3 Novel functions of vimentin intermediate filaments  Intermediate filaments (IFs) are the third major type of cytoskeleton elements besides microfilaments (MFs) and MTs. They are 10-12 nm in diameter, which is in between the diameter of MF (5-8 nm) and MT (25 nm) (reviewed by Fuchs and Weber, 1994). They are rod-shape filaments that provide the mechanical strength to cells. Unlike MF and MT, IFs have a non-polar structure and have no IF-specific associated motor proteins (Goldman et al., 2012; Herrmann et al., 2007).   Encoded by more than 65 different genes, IF proteins are subdivided into six major types (Table 1-3), depending on their distribution as well as their sequence homology (reviewed by Fuchs and Weber, 1994). Type I and II IF proteins are acidic and basic keratins, which associate in a 1:1 ratio to form heterodimers. These heterodimers then polymerize into IF. Type III IF proteins such  41 as vimentin, peripherin, desmin, and Glia fibrilary acid protein (GFAP) form both homo- and heteropolymeric IF filaments. Type IV IF proteins include three classical neurofilaments (NFs), NF-L, NF-M, and NF-H, which direct the elongation of axons. Type V IF proteins are nuclear lamins, which are exclusively distributed in the cell nucleus and form a meshwork of filaments to support the nuclear membrane.     42 Table 1-5: Identified IF family members and their tissue distribution. Type Protein Name Number of genes  Tissue distribution I Keratins (Acidic) >25 Soft epithelia, hard epithelia and hair II Keratins (Basic) >24 III Vimentin 1 Fibroblast, endothelium and Leukocytes Desmin 1 Muscle GFAP 1 Astrocytes Peripherin 1 Neurons Synemin 1 Muscle IV NF-L 1 Central nerve system neurons NF-M 1 NF-H 1 -Internexin 1 Nestin 1 Heterogenous Synemin 1 Muscle V Lamin A/C 1 Nucleus Lamin B1 1 Lamin B2 1 VI Phakinin 1 Eye lens Filensin 1  Table adapted from Coulombe and Wong (2004).   43 Vimentin, a 57 kDa protein, is one of the most widely expressed Type III IF. It is typically expressed in leukocytes, blood vessel endothelial cells, and mesenchymal cells such as fibroblasts (Azumi and Battifora, 1987). The secondary structure of vimentin consists of a central -helical rod domain, a non- helical N-terminal “head” domain, and a C-terminal “tail” domain. During assembling, vimentin molecules aggregate and polymerize through their central -helical rod domain to form highly stable short vimentin filaments, which then form long filaments (Herrmann et al., 2007). The network of vimentin constantly undergo highly dynamic assemble and disassemble process. The disassembling of vimentin is regulated by phosphorylation on the N-terminal domain by cellular phosphorylation kinases, which allows the filaments and vimentin monomers to move along MTs to the cell periphery (Prahlad et al., 1998; Yoon et al., 1998). Inside the cell, vimentin interconnects with MFs and MTs through a family of proteins classified as plectins. Each plectin protein contains binding sites for vimentin on one side and for MFs or MTs on the other side, promoting the association of these three cytoskeleton elements and controlling intracellular trafficking (Svitkina et al., 1998).   Apart from the well-known mechanical function inside the cell, many novel functions of vimentin, such as cell adhesion, cell migration, signaling, and membrane traffic, have recently been reported (reviewed by Dave and Bayless, 2014; Ivaska et al., 2007)). Interestingly, in recent years it has been shown that several viruses require the vimentin network for successful infection. These viruses include African swine fever virus (AFSV) (Stefanovic et al., 2005), HIV-1 (Thomas et al., 1996), Dengue virus (Teo and Chu, 2014), enterovirus (Du et al., 2014), Japanese encephalitis virus (Das et al., 2011), cytomegalovirus (Miller and Hertel, 2009) and parvovirus  44 (Fay and Pante, 2013). In the following sections I first introduce the novel functions of vimentin, and then I review our current knowledge of the role of vimentin in viral infection.   1.3.1 Overview of novel functions of vimentin The first novel vimentin function I will describe is the role of vimentin IF in the cellular position of organelles. Gao and Sztul (2001) found that vimentin could directly bind to formiminotransferase cyclodeaminse, a peripheral Golgi protein, to associate with the Golgi apparatus. Overexpression of formiminotransferase cyclodeaminse induces a dramatic rearrangement of vimentin IF, suggesting that Golgi compartments can regulate vimentin IF architecture.   The position of lysosomes inside the cell and their acidity can also be influenced by vimentin. Using vimentin null cells, Styers et al. (2004) showed that the lysosome distribution is altered from dispersed cytoplasmic localization in wild-type cells to a juxtanuclear position in the vimentin null cells (Styers et al., 2004). These authors used co-immunoprecipitation (Co-IP), and found that vimentin interacts with AP3, a heterotertrameric clathrin adaptor-like complex that carries vesicles between endosomes and lysosomes. Since the trafficking of endo-lysosomal chloride channel (CIC3) is controlled by AP3, the organelle pH is also modified in vimentin null cells, which was detected by the pH probe Lysosensor (Styers et al., 2004). These findings indicate a novel role of vimentin in organelle positioning, subcellular distribution of late endosome/lysosome membrane proteins, and luminal pH modification of endosome/lysosome through the interaction of vimentin with the adaptor complex AP3.    45 Another novel vimentin function that has been studied in some detail is the role of vimentin in cell adhesion through regulating of integrin function. In cell culture, focal adhesions are mediated by integrin interacting with the extracellular matrix (ECM). Integrins are heterodimer transmembrane proteins composed of two subunits, the  and  subunits. While the extracellular domain of integrin interact with ECM ligands, its cytoplasmic domain is connected to the actin cytoskeleton via an interaction with a protein complex containing vinculin, talin, and paxillin (Barczyk et al., 2010). It has been shown that vimentin and actin are associated with integrins via vinculin and plectin in a structure termed vimentin-associated matrix adhesions (Burgstaller et al., 2010; Gonzales et al., 2001). Vimentin plays a critical role in regulating the size and stability of vimentin-associated matrix adhesions. In addition, vimentin can also control integrin-ligand interactions through recognizing specific ECM components by integrin. For example, the extracellular domain of 21 integrin recognizes laminin and collagen in non-transformed epithelia cells, while it can only recognize collagen in transformed epithelial cells expressing vimentin (Maemura et al., 1995).   Besides organelle positioning and cell adhesion, vimentin is also involved in cell migration and invasion. During embryonic development, wound healing, and carcinoma biogenesis, epithelial cells lose their epithelial characteristics and gain mesenchymal properties, which is termed epithelial-to-mesenchymal transition. Increased vimentin expression is shown to associate with this process, leading to an alternation of cell morphology and an increase of cell motility (reviewed by Chernoivanenko et al., 2013). Studies have demonstrated that vimentin expression affects cell migration during wound healing. In vimentin knockout mice, wound healing is significantly delayed in adult animals, whereas wound healing in embryo is completely impaired  46 (Eckes et al., 2000). During cell migration, p21 induces phosphorylation of vimentin, which in turn causes vimentin IF disassembly and retraction from the cell surface where lamellipodia form (Helfand et al., 2011). This suggested that vimentin IFs at the cell periphery could inhibit the formation of lamellipodia during cell migration, while disassembly of vimentin IFs facilitates this process. Through analyzing the expression of vimentin in cancer cells, it has been shown that increased vimentin expression is highly correlated with increased cell motility, invasiveness, and tumor progression of various epithelial cancers including prostate cancer, breast cancer, malignant melanoma, lung cancer, central nerve system (CNS) tumors, and gastrointestinal tumors (reviewed by Satelli and Li, 2011). Since most studies indicate an association between over-expression of vimentin with increased invasive phenotypes, it has been suggested that vimentin may participate in carcinoma biogenesis events and thus serves as a potential target for cancer treatment ((Satelli and Li, 2011).     1.3.2 The role of vimentin during viral infection While the roles of actin filaments and MTs during viral infections have been extensively studied, the role of IFs has only recently started to be elucidated. This is in part due to the availability of commercial drugs to stabilize or depolymerize actin filaments and MTs, and the lack of drugs that stabilize or depolymerize IFs. However, recently, using acrylamide, a vimentin IF disruption reagent, or using cell lines from vimentin-/- mice, researchers have begun to reveal the involvement of vimentin during viral infection. With these experimental tools, it has been established that vimentin has roles in different steps of the infectious cycle of different viruses, including viral cell attachment and entry, viral assembly for viruses that replicate in the cytoplasm, viral replication, and viral trafficking.  47 Vimentin is distributed in the cytoplasm, around the perinuclear region, Golgi apparatus, and the endoplamic reticulum. Additionally, Mor-Vaknin et al. (2003) have demonstrated that vimentin is also expressed on the cell surface and exposed to the extracellular environment in activated macrophages. Cell surface vimentin was reported to help virus attach and internalize into these cells. For instance, during HIV-1 infection of macrophages and activated T lymphocytes, vimentin binds to the third hypervariable region (the V3 loop) of the gp120 viral envelope protein and induces the viral entry (Thomas et al., 1996). In the case of porcine respiratory and reproductive syndrome virus, the virus binds to vimentin expressed on the surface of African green monkey kidney MARC-145 cells through viral N protein and mediates virus entry into the cell (Kim et al., 2006; Wang et al., 2011). In addition, vimentin was also found to bind to enterovirus on the surface of human astrocytoma (U251) cells and Japanese encephalitis virus on the surface of porcine kidney cells to initiate viral infection (Das et al., 2011; Du et al., 2014).   Aggresomes are sites where misfolded proteins accumulate, which are localized at the MT organization center (MTOC). Vimentin has been found to form protective cages surrounding aggresomes to restrict the movement of the toxic misfolded proteins in the cytoplasm (Johnston et al., 1998). Some large DNA viruses hijack the aggresome pathway during infection. For example, AFSV is a large cytoplasmic DNA virus that assembles in the cytoplasm of infected cells. At the beginning of the infection, MT mediates rearrangement of vimentin to virus assembly sites close to the MTOC. DNA replication during infection activates calcium calmodulin-dependent protein kinase II (CaM kinase II) and induces phosphorylation of the N-terminal domain of vimentin, further facilitating the redistribution of vimentin into a cage surrounding the virus factory (Stefanovic et al., 2005). CaM kinese II has also been shown to induce vimentin reorganization and phosphorylation during dengue virus infection (Teo and Chu,  48 2014). During dengue virus infection, the nonstructural protein NS4A directly interacts with vimentin and induces vimentin reorganization. Together with CaM kinese II induced vimentin reorganization, vimentin redistributes to dengue virus replication complexes where viral replication occurs. The rearrangement of vimentin during either AFSV or dengue virus infection is required to prevent invasion of host factors into the virus assembly site and maintain successful replication of viral proteins.   Another function of vimentin is during replication of certain viruses. In the case of Dengue virus, a proteomic study identified vimentin interaction with heterogeneous nuclear ribonucleoproteins (hnRNPs), as well as vimentin interaction with dengue virus nonstructure protein 1 (NS1) (Kanlaya et al., 2010). Moreover, using acrylamide treatment to disrupt cellular vimentin, Kanlaya et al. (2010) showed that dengue virus NS1 expression was reduced and hnRNP nuclear expression decreased. This study indicates that the interaction between host vimentin, hnRNP, and virus NS1 protein facilitate dengue virus replication and release.   Finally, the use of acrylamide treatment during viral infection has implicated vimentin IFs in viral trafficking. For example, acrylamide treatment during bluetongue virus infection resulted in the virus accumulating inside infected cells and a significant decrease in viral release (Bhattacharya et al., 2007). Thus, the authors concluded that vimentin IFs is necessary for viral egress. Similar studies in cells infected with human cytomegalovirus (Miller and Hertel, 2009) or with parvovirus (Fay and Pante, 2013) showed that disruption of vimentin IFs by acrylamide treatment inhibits viral protein production and production of viral progeny. In these two studies, the authors also used cells from vimentin null mice and reported that the incoming viruses remained in the  49 cytoplasm these cells compared with wild type cells. This is an indication that vimentin IFs is involved in the cytoplasmic trafficking of incoming virus towards the nucleus.  As discussed in Section 1.1.4, the role of MFs and MTs during influenza A virus infection is very well characterized. However, the role of IFs, especially the role of vimentin, during influenza A virus infection remains to be investigated. Nevertheless, a proteomic study has recently identified vimentin as a cellular factor that co-precipitaded with vRNP (Mayer et al., 2007). Another paper published recently suggested that vimentin expression is upregulated during H5N1 infection (Liu et al., 2012). Since vimentn is involved in endosome/lysosome acidification (Section 1.3.1), my hypothesis is that vimentin is required during influenza infection since the virus needs a lower pH for uncoating. This is one of the hypotheses I address in this thesis (Chapter 5).  1.4 Research objectives Productive infection of influenza A virus requires the efficient use of the host endocytic and nuclear import mechanisms at the early phases of infection for delivering the viral genome into the host cell nucleus. Understanding the interaction between influenza A virus and the host cellular machinery will provide us with better insights on the infectious cycle of this virus, and at the same time, these studies will generate important data that could be used in the development of new approaches to inhibit influenza infection. NLSs on NP are highly conserved and serve to mediate the nuclear import of the influenza viral genome. It will be important to determine whether the NLSs of NP could serve as anti-viral targets. Therefore, understanding the contribution of the NLSs of NP to the nuclear import of the influenza genome is an important topic that needs to be studied. In particular, the role of NLS2 is still unclear and its cellular  50 binding proteins need to be identified. Moreover, understanding the role of vimentin during influenza A virus infection is yet another topic that needs to be explored. Thus, the specific aims for my PhD thesis are as follows:  1) To characterize the contribution of NLS2 to nuclear import and to identify its cellular binding partners. The questions for this aim are: What is the contribution of NLS2 of influenza A virus NP to nuclear import. Which amino acids of NLS2 are important for nuclear import? Which isoforms of importin α interact with NLS2 during nuclear import?  2) To explore whether the NLSs of influenza A virus NP are good candidates for novel antiviral approaches. 3) To study the role of vimentin during influenza A virus infection. The following are the rationales for these aims.  1.4.1 Aim 1: To characterize the NLS2 of influenza A virus nucleoprotein and to identify its cellular binding partners  In previous studies, the function of NLS2 from nucleoprotein is contradictory. Cros et al. (2005) showed that mutating basic amino acids on the C-terminus of NLS2 didn‟t have effect on NP localization. However, by performing an antibody inhibition assay, Wu et al. (2007a) revealed that NLS2 could function independently to mediate nuclear import of vRNP. Therefore, it is important to characterize the function of NLS2 during nuclea import. As NLS2 contains two basic clusters of basic amino acids that fits the protype cNLS category, I hypothesize that NLS2 is able to mediate nuclear import of a cytoplasmic reporter. In order to test this, NLS2 was annealed to a 5GFP cytoplasmic reporter to test its nuclear import efficiency. Since there are two basic clusters on each terminus of NLS2, I hypothesize that it could function as a putative classical bipartite NLS. To test this hypothesis, I performed site-directed mutagenesis of basic  51 amino acids of NLS2. As introduced in Section 1.2.5, NP binds to importin 1, 3, 5, and 7 and NLS1 can interact with importin 1, 3, and 5 in vitro (Gabriel et al., 2011; Sasaki et al., 2013; Wang et al., 1997). NLS2 was shown to function independently of NLS1 to mediate nuclear import of vRNP (Wu et al., 2007a). Therefore, I hypothesised that any of these importin  isoforms could bind to NLS2 to promote nuclear import. In order to identify which isoforms of importin  interact with NLS2 during nuclear import, RNAi depletion of certain importin  isoforms was used to investigate its involvement during NLS2 mediated nuclear import. In addition, both Co-IP and GST-pull down assays were also performed to characterize the interaction between NLS2 and importin . The results of these experiments are described in Chapter 3.   1.4.2 Aim 2: To explore whether the NLSs of influenza A virus NP are good candidates for novel antiviral approaches  Influenza A virus enters the nucleus for viral transcription and replication. NLS1 and NLS2 on NP have been shown to facilitate the nuclear import of vRNP (Wu et al., 2007a). Therefore, the function of NLS1 and NLS2 are critical during viral infection. I hypothesized that influenza A virus containing mutations on either NLS1 or NLS2 cannot enter the nucleus. To test this hypothesis, mutant viruses, with mutations on NLS1, NLS2 or both NLSs, were generated using the reverse genetic technique, and tested for infectivity. In addition, in order to determine whether the NLSs of NP are good antiviral targets, I tested whether inhibiting their function could restrain influenza A virus infection. In order to achieve this, a competition assay was developed, where exogenous NLS1 or NLS2 was introduced into influenza A virus-infected cells. The results of these experiments are described in Chapter 4.   52 1.4.3 Aim 3: To study the role of vimentin during influenza A virus infection As introduced in Section 1.3.1, vimentin interacts with AP-3 to regulate the endo-lysosomal machinery and endosome acidification. As influenza A virus hijacks the cellular endocytosis machinery to reach late endosomes where the vRNPs are released, I hypothesized that vimentin may function during the endocytic trafficking of influenza A virus and viral genome release. To test this hypothesis, I used vimentin deficient mouse embryonic fibroblast cells (vimentin-/-), as well as HeLa cells depleted of vimentin by RNAi. In addition, in order to investigate in which step of the influenza A virus infection vimentin is involved, I determined the location and distribution of several organelles of the endocytic pathway during infection A virus infection and compared the differences in cells with and without vimentin. The results of these experiments are described in Chapter 5.   53 Chapter 2 Materials and Methods 2.1 Cell culture HeLa cells, Madin-Darby canine kidney epithelial (MDCK) cells, human embryonic kidney (HEK) 293T cells (American Type Culture Collection, Manasas, VA, USA) and mouse embryonic fibroblast (MEF) vimentin+/+ and vimentin-/- (courtesy of Dr. Laura Hertel, Children‟s Hospital Oakland Research Institute, USA and Dr. Robert Evans, University of Colorado Health Sciences Center, USA) (Holwell et al., 1997) were maintained at 37 °C and 5% CO2 in Dulbecco‟s modified Eagel‟s medium (DMEM) (Sigma-Aldrich, St. Louise, MO, USA; Catalog number: D5671) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich; Catalog number: F1051), penicillin/streptomycin (Cellgro, Herdon, VA, USA; Catalog number: CO-02-CI), 2 mM L-glutamine (Cellgro, Catalog number: 25-005-CI) and 1 mM sodium pyruvate (Gibco by Life Technologies, Grand Island, NY, USA; Catalog number: 11360-070).  2.2 Reagents and antibodies Purified influenza A virus X: 31, A/Aich/68 (H3N2) (2 mg/ml) was purchased from Charles River (Charles River, North Franklin, CT, USA; Catalog number: 10100374). Importazole, an inhibitor of importin -mediated nuclear transport, was purchased from Sigma-Aldrich (Catalog number: SML0341). pHrodoTM red epidermal growth factor (EGF) conjugate (Catalog number: P35374) and Alexa Fluor 488 EGF complex (Catalog number: E-13345) were obtained from Molecular Probes by Life Technologies and used to track endosome movement and acidification  54 during EGF uptake. NucBlue Live ReadyProbe Reagent (Catalog number: R37605) was purchased from Molecular Probes by Life Technologies and used to visualize the cell nucleus during live cell fluorescence microscopy. The dextran uptake experiment was performed using Alexa Fluor 488 Dextran (10,000 MW, Anionic, Fixable) (Catalog number: D-22910). Site-directed mutagenesis was performed using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA; Catalog number: 200518). Co-IP was performed with the MACs anti-GFP starting Kit (Miltenyl Biotec, San Diego, CA, USA; Catalog number: 130-091-288).  All the antibodies used in this thesis are list in Table 2-1.      55 Table 2-1: List of antibodies used in this thesis.   Primary antibodies* Dilution used Names Company Catalog # IF WB Mouse anti-NP Acris AM01375PU 1:1000 1:1000 Mouse anti-M1 Acris SM1748P 1:1000 1:1000 Rabbit anti-importin 1 Abcam Ab70609 1:1000 1:5000 Mouse anti-importin 3 Abcam Ab53751 1:1000 1:1000 Mouse anti- importin 5 Invitrogen 37-0800 1:2000 1:2000 Mouse anti-vimentin Sigma Aldrich V6630 1:1000 1:100 Rabbit anti-vimentin Santa Cruz SC5565 1:100 N/A Rabbit anti-EEA1 Cell signaling 3288 1:100 N/A Rabbit anti-Rab7 Cell signaling 9367 1:100 N/A Rabbit anti-LAMP1 Abcam Ab24170 1:200 N/A Mouse anti-beta actin Abcam Ab8227 N/A 1:10000 Mouse anti-GAPDH Abcam Ab9485 N/A 1:10000 Mouse Anti-GFP Invitrogen A11121 N/A 1:1000 Mouse Anti-GST GenScript A00865 N/A 1:1000  Secondary antibodies Concentration used Names Company Catalog # IF WB Alexa Fluor® 488, goat Anti-Mouse IgG(H+L)  Invitrogen A-11001 1:2000 N/A    56 Table 2-1: List of antibodies used in this thesis.  Secondary antibodies Concentration used Names Company Catalog # IF WB Alexa Fluor® 405, goat Anti-Mouse IgG(H+L) Invitrogen A-31553 1:2000 N/A Alexa Fluor® 568, goat Anti-Rabbit IgG(H+L) Invitrogen A-11011 1:2000 N/A HRP goat anti-mouse IgG Sigma- Aldrich A4416 N/A 1:2000 HRP goat anti-rabbit IgG Sigma- Aldrich A0545 N/A 1:2000  *Abbreviations: immunofluorescence (IF), Western blot (WB), not applicable (N/A), early endosome antigen 1 (EEA1), lysosomal-associated membrane protein 1 (LAMP1), green fluorescence protein (GFP), Glutathione S-transferase (GST), and horse radish peroxidase (HRP).    57 2.3 Construction of recombinant plasmids  To generate 5GFP-NLS1, 5GFP-NLS2K, and 5GFP-NLS2R constructs, the respective DNA strands were synthesized, annealed, and ligated directly into the pEGFP-GFP5 vector (courtesy of Dr. Gergely L. Lukacs, McGill University). The synthetic primers containing adapters of the BamHI enzyme (New England Biolab, Ipswich, MA, USA, Catalog number: R0136T) at each end were annealed. The forward (F) and reverse (R) primers from the respective 5‟ and 3‟ sequences were as according to Table 2-2. The annealed DNA fragments were then ligated to the BamHI site of the pEGFP-GFP5 vector. All constructs were confirmed by sequencing and those with the right orientation of insert were chosen for the transfection experiment in HeLa cells.   QuikChange site-directed mutagenesis kit was used (Stratagene, La Jolla, CA, USA; Catalog number: 200518) to generate mutations within the 5GFP-NLS2K or 5GFP-NLS2R sequences. The 5GFP-NLS2K or NLS2R recombinant plasmid served as a template. The primers used to generate mutants are listed in Table 2-2. All constructs were confirmed by sequencing and those with right mutations were chosen for the transfection experiment in HeLa cells.  To generate constructs of Glutathion S-transferase- (GST) fusion importins, plasmids pANT7_cGST-tagged importin 1 (Catalog number: HsCD00078257), 3 (Catalog number: HsCD00076865), 5 (Catalog number: HsCD00077349), and 7 (Catalog number: HsCD00077921) were purchased from DNASU (Arizona State University, AZ, USA). Since their protein expression in bacteria cannot be efficiently induced by IPTG, these plasmids were used in this study as templates for PCR amplification of cDNA for different human importin  isoforms. To gain high-level expression of GST-tagged importin 1, 3, 5, and 7, PCR was performed  58 with specific GST-importin  primers listed in Table 2-2. The PCR products were purified, digested with BamHI (New England Biolabs, Catalog number: R0136T) and EcoRI (New England Biolabs, Catalog number: R0101T) enzymes, and subcloned into the pGEX-6P-2 vector (GE Healthcare, Mississauga, ON, Canada; Catalog number: 28-9546050). The sequence of recombinant constructs were sequenced the Nucleic Acid Protein Service at University of British Columbia.     59 Table 2-2: List of primers used to generate recombinant plasmids in this thesis. Primers were purchased from Integrated DNA Technologies.  Construct name Primer sequence*   5GFP-NLS1 F‟5‟-GATCCAATGGCGTCTCAAGGCACCAAACGATCATATGAACAATGCCG -3‟ R‟5‟-GATCCGGCATTTGTTCATACGATCGTTTGGTGCCTTGAGACGCCATTG -3‟ 5GFP-NLS2K F‟5‟-GATCCAAAACGTGGAATCAATGACCGAAATTTCTGGAGG GGTGAAAATGGACGAAAGACAAGGG -3‟ R‟5‟-GATCCCCTTGTCTTTCGTCCATTTTCACCCCTCCAGAAATTTCGGTCATTGATTCCACGTTTTG -3‟ 5GFP-NLS2R F‟5‟-GATCCAAAACGTGGAATCAATGACCGAAATTTCTGGAGG GGTGAAAATGGACGAAGGACAAGGG -3‟ R‟5‟-GATCCCCTTGTCCTTCGTCCATTTTCACCCCTCCAGAAATTCGGTCATTGATTCCACGTTTTG -3‟ 5GFP-NLS2K A1 F‟5‟-GATCCGCAGCAGGGATCAATGATCGGAACTTCTGGAGGGGTGAGAATGGAGAAAAACAAGACCGG -3‟ R‟5‟-GATCCTCTTGTTTTTCGTCCATTCTCACCCCTCCAGAAGTTCCGATCATTGATCCCTGCTGCTGGA -3‟ 5GFP-NLS2K A2 F‟5‟-GAGGGGTGAAAATGGAGCAAAGACAGCGCCGAATC -3‟ R‟ 5‟-GATCCGGCGCTGTCTTTGCTCCATTTTCACCCCTC-3‟ 5GFP-NLS2K A1+2 F‟5‟-GAGGGGTGAAAATGGAGCAAAGACAGCGCCGAATC -3‟ R‟ 5‟-GATCCGGCGCTGTCTTTGCTCCATTTTCACCCCTC-3 5GFP-NLS2R D1 F‟5‟-GATCCAAAACGTGGAATCAATGACCGAAATTTCTGGAGGGGTGAAAATGGAT-3‟ R‟5‟-GATCCTCCATTTTCACCCCTCCAGAAATTTCGGTCATTGATTCCACGTTTTG-3‟ 5GFP-NLS2R D2 F‟5-GATCCAAAACGTGGAATCAATGACCGAAATTTCTGGG-3‟ R‟5‟- GATCCCCAGAAATTTCGGTCATTGATTCCACGTTTTG-3‟ 5GFP-NLS2R D3 F‟5-GATCCAAAACGTGGAATCAATGACG-3‟ R‟5‟-GATCCGTCATTGATTCCACGTTTTG -3‟  60 Table 2-2: List of primers used to generate recombinant plasmids in this thesis. Primers were purchased from Integrated DNA Technologies.  Construct name Primer sequence 5GFP-NLS2K GF2K F‟5- GAAAATGGACGAAAGAAAAGGCCGGATCC -3‟ R‟5‟- GGATCCGGCCTTTTCTTTCGTCCATTTTC -3‟ 5GFP-NLS2K GF2R F‟5- GAAAATGGACGAAGGAAAAGGCCGGATCC -3‟ R‟5‟- GGATCCGGCCTTTTCCTTCGTCCATTTTC -3‟ 5GFP-NLS2K LF2K F‟5- GAAAATGGACGAAAGGCAAAGGCCGGATCC -3‟ R‟5‟- GGATCCGGCCTTGCCTTTCGTCCATTTTC -3‟ 5GFP-NLS2R LF2R F‟5‟- GAAAATGGACGAAGGGCAAGGCCGGATCC -3‟ R‟5‟- GGATCCGGCCTTGCCCTTCGTCCATTTTC -3‟ GST-importin  F‟5‟- CGCGGATCCTTGTACAAAAAAGTTGGC -3‟ R‟5‟- CCGGAATTCCTTTGTACAAGAAAGTGG -3‟  *Abbreviations: F, forward primer; R, reverse primer.    61 2.4 Transfection of recombinant DNA and small interference RNA (siRNA) 2.4.1 Transfection of recombinant DNA HeLa cells were grown in monolayers on glass microscope coverslips (Fisher Scienific, Loughborough, UK; Catalog number: 12-545-81) and the 5GFP-NLSs recombinant plasmids were transfected into these cells using Lipofectamine 2000 (Invitrogen by life technologies, Catalog number: 11668-019), according to the manufacturer‟s instruction. Twenty-four hours after transfection, the cells were fixed with 3% paraformaldehyede (PFA) in phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 2.9 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) for 15 minutes and washed with PBS three times. The coverslips were mounted onto microscope slides with prolong Gold antifade reagent containing DAPI (Invitrogen by life technologies, Catalog number: P-36931). Samples were visualized using an Olympus Fluoview FV1000 laser scanning microscope (Shinjuku, Tokyo, Japan) at the Life Science Institute imaging facility.   2.4.2 Small-interfering RNA (siRNA) transfection Sequences of siRNA targeting importin 1, 3, and 5 have been previously reported (Nitahara-Kasahara et al., 2007). These were:  CCAAGCUACUCAAGCUGCCAGGAAA for 1.  CAGUGAUCGAAAUCCACCAAUUGAU for 3.  CCGGAAUGCAGUAUGGGCUUUGUCU for 5.   To knockdown different importin  isoforms, HeLa cells were seeded 24 hours prior to siRNA  62 transfection. Cells were transfected with siRNA against importin 1, 3, or 5 at a final concentration of 25 nM. As control, ON-TARGETplus control siRNA (25 nM, Thermo Scientific, Waltham, MA, USA; Catalog number: D-0018-01-05) was used. To knockdown endogenous vimentin, SMARTpool:ON-TARGRTplus human vimentin siRNA (Thermo scientific, Catalog number: L-003551-00-005, 5 nmol) was transfected at a final concentration of 50 nM and the concentration of control siRNA was adjusted to 50 nM accordingly. Transfections were performed using Lipofectamine® RNAiMAX Reagent (Invitrogen by life technologies, Catalog number: 13778-150) according to the manufacturer‟s instructions. Expression of importin 1, 3, and 5 was assessed by Western blot and immunofluorescence microscopy 48 hours after transfection. Expression of vimentin was assessed by Western blot and immunofluorescence microscopy 72 hours after transfection.  2.5 Influenza A virus infection  HeLa cells, vimentin+/+, or vimentin-/- cells were seeded on glass microscope coverslips, and then mock infected or infected with purified influenza A virus X:31, A/Aich/68 (H3N2) at a multiplicity of infection (MOI) of 4 in DMEM supplemented with 0.2% FBS. Cells were incubated for 15 minutes at 4°C to allow the virus to bind to the cell surface. Cells were then moved to 37°C for 1 hour to allow virus internalization. After this incubation period, a mild acidic wash (PBS-HCl, pH 5.5 at 4°C) was performed to exclude the delayed uptake of attached, but not internalized virus particles. Subsequently cells were incubated at 37°C in DMEM supplemented with 2% FBS according to the times (beyond 1 hour) indicated in figure legends.  63 2.6 Fluorescence microscopy 2.6.1 Indirect immunofluorescence microscopy Cells on glass microscope coverslips were fixed with 3% PFA in PBS for 15 minutes, permeablized with 0.2% Triton X-100 (Sigma-Aldrich, Catalog number: T8787-50ML) in PBS for 5 minutes, and incubated with PBS containing 1% Bovine Serum Albumin (BSA) (Sigma-Aldrich, Catalog number: A2153) and 10% Goat serum (Sigma-Aldrich, Catalog number: G9023) for 1 hour at room temperature. Cells were then incubated with primary antibody for 1 hour at 37°C. Secondary antibodies containing fluorophores were diluted in blocking buffer. Cells were incubated with the secondary antibodies for 45 minutes at room temperature, following by mounting the coverslips with Prolong Gold antifade reagent containing DAPI (Invitrogen, Catalog number: P-36931). Samples were visualized using an Olympus Fluoview FV1000 laser scanning microscope (Shinjuku, Tokyo, Japan). The antibody dilutions are indicated in Table 2-1.   2.6.2 Epidermal growth factor (EGF) uptake Vimentin+/+ and vimentin-/- cells were seeded on 8-well glass bottom -Slide (Ibidi, Martinsried, Germany; Catalog number: 80827) the day before treatment. Five hours prior to the treatment, cells were gently washed twice with PBS and cultured in DMEM without FBS for starvation. Alexa Fluor 488 EGF complex (10 g/ml), pHrodoTM red EGF conjugate (3 g/ml), and NucBlue Live ReadyProbes Reagent (3 l) were prebound to the cells at 4°C for 15 minutes. Cells were then washed twice with PBS before adding live cell image solution (LCIS: 140 mM  64 NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 20 mM HEPES, pH 7.4) and transferring to 37°C tissue culture incubator. After 30 minutes incubation, cells were preceeded for live cell imaging in the 37°C chamber using Olympus Fluoview FV1000 laser scanning microscope (Shinjuku, Tokyo, Japan).   2.6.3 Dextran uptake  Vimentin+/+ and vimentin-/- cells were seeded on 8-well glass bottom -Slide (Ibidi, Martinsried, Germany; Catalog number: 80827) the day before treatment. Five hours prior to the treatment, cells were gently washed twice with PBS and cultured in DMEM without FBS for starvation. Alexa Fluor Dextran (10,000 MW, Anionic, Fixable) (0.5 mg/ml) and NucBlue Live ReadyProbes Reagent (3 l) were added to the cells and incubated at 37°C for 30 minutes. After this period of incubation, cells were washed four times with PBS before adding LCIS and preceded for live cell imaging in the 37°C chamber using Olympus Fluoview FV1000 laser scanning microscope (Shinjuku, Tokyo, Japan).  2.6.4 Quantification of fluorescence images  2.6.4.1 Quantification of nuclear import  The quantification procedure was previously described (Wu et al., 2007a). Briefly, the mean intensity of defined areas (20 pixels by 20 pixels) was measured in the nucleus (FN) and in the cytoplasm (FC) using imageJ software (National Institute of Health). The fluorescence of the  65 nuclear envelope as well as that of the nucleolus was not included in the quantification. The mean background intensity (MB) was also measured next to the quantified cell. The ratio of nucleus to cytoplasm fluorescence intensity was calculated by this equation:  Fn/c = [FN – (400*MB)]/[FC – (400*MB)] Where FN is mean intensity in the nucleus and FC is the mean intensity in the cytoplasm.  Data were obtained from a total of 85-100 cells per experiment from three independent experiments. Results were analyzed by two tailed unpaired Student‟s t-test or One-way ANOVA followed by Tukey‟s test using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). All data are represented as mean  standard error of the mean (S.E.M) and P<0.05 were considered significant.   2.6.4.2 Quantification of vesicle area  In order to analyze differences of endo/lysomal vesicle size between cells with and without vimentin, the vesicle area was quantified using image J. Confocal image were first opened with image J, converted to 8 bits and made into binary form. Using the analyze particle option, all the vesicles were marked and analyzed for their area. Statistical analysis was done according to Section 2.6.4.1.  2.6.4.3 Quantification of lysosome distribution To determine the re-distribution of lysosomes in vimentin-/- cells and vimentin RNAi knockdown cells, 100 cells per experiment were analyzed for their lysosome staining. The proportion of cells  66 showing accumulation of lysosomes at the perinuclear region as a percentage of total cells was counted. Statistical analysis was done according to Section 2.6.4.1.  2.6.4.4 Quantification of colocalization of NP and EEA1/Rab7 In order to gain a better insight about the NP localization during influenza A virus infection in vimentin+/+ and vimentin-/- cells, colocalization analysis was performed to quantify the amount of NP colocalized with EEA1 or Rab7 during each time point of infection using image J. Quantification of pixels in each channel was done with JACop plugin in image J software (Bolte and Cordelieres, 2006). Threshholds for each channel were determined on JACop using Costes automatic threshholding (Costes et al., 2004), and the amound of colocalization of pixels above threshold was determined. Statistical analysis was done according to Section 2.6.4.1.  2.6.4.5 Quantification of endosome acidification using phrodo EGF In order to compare the endosome acidification between vimentin+/+ and vimentin-/- cells, red dots representing acidified phrodo EGF and green dots represented Alexa Fluor 488 EGF, which also represents the EGF containing endosomes, were counted. Statistical analysis was done according to Section 2.6.4.1.  2.7 Western blot analysis Cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 0.5% NP-40, 10 mM phenylmethylsulfonyl fluoride (PMSF), 1  67 M pepstatin, 10% g/ml aprotinin, and 2 mg/ml leupeptin (Roche)) on ice for 30 minutes. Lyates were centrifuged at 15000 x g for 10 minutes at 4°C. The supernatant were diluted with 2X Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS and 0.01% Bromophenol blue, 5% -mecaptoethanol (Sigma Aldrich, Catalog number: M6250)) and boiled at a thermomixer (Eppendorf) at 98°C for 5 minutes. Equal amount of protein samples were loaded onto a SDS-PAGE. Proteins were transferred using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Biorad) to polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany; Catalog number: IPVH00010) as described by the instructions provided by the manufacturer. The protein expressions were detected by western blot using primary antibodies towards the targeting proteins, while using β-actin or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression as reference gene control. The antibody dilutions are listed in Table 2-1.  The band intensity was quantified using image J as previously described (Davarinejad, 2014). Briefly, the rectangle tool was used to draw the same frame around each band. The intensity was then measured by analyzing the gray value inside the frame. In order to compare the protein band of interest, the relative densities was calculated by dividing by the band densities of the loading-control bands.   2.8 Generation of influenza A virus by reverse genetics The eight plasmids system was generously provided by Dr. Honglin Chen from The University of Hong Kong and Dr. Robert Webster from St Jude Children Research Hospital. The eight plasmids  68 contain the cDNA of the influenza A virus (strain A/PR8/1934/H1N1) (PHW2000-PA, PHW2000-PB1, PHW2000-PB2, PHW2000-HA, PHW2000-NA, PHW2000-M, PHW2000-NP, PHW2000-NS). In order to generate mutant virus containing mutation on NLS1, NLS2, or both NLSs site-directed mutagenesis to mutant basic amino acids on NLS1 or NLS2 in PHW2000-NP plasmid was used using primers according to Table 2-2. All constructs were confirmed by sequencing. Wild-type or mutant viruses were generated as previously described (Neumann et al., 2012). Briefly, a co-culture of MDCK (3×105) and HEK-293T (4×105) cells were seeded in a 6-well dish and cultured overnight. The next day, wild-type NP plasmid or mutant NP plasmid together with the other seven plasmids were transfected into co-cultured HEK293T/MDCK cells using Lipofectamine 2000 according to manufacture‟s instruction. The medium was changed into DMEM supplemented with 1% TPCK-trypsin (Sigma Aldrich, Catalog number: T1426-50MG), 2% FBS, penicillin/streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate after 24 h transfection. Cell culture supernatant containing wild-type or mutant viruses was obtained 72 h post transfection and subjected for electron microscopy or plaque assay.     2.9 Plaque assay Plaque assay was used to evaluate the virus progeny production after infection. Supernatant were obtained from infected cells and used to evaluate virus titers. MDCK cells were seeded in 6-well plates at a density of 7x105 cells/well 48 hours before plaque assay. Supernatants containing virus progeny were serially diluted using 10-fold dilutions. Two hundred µl of each dilution was added to each well in triplicates. The plates were incubated at room temperature for 1 hour on a shaker. After two times wash with PBS, 2 ml of nutrient agar overlay (1% agarose, 0.5% penicillin in  69 Minimum Essential Medium Eagle (MEM)) was added to each well and the cells were incubated at 37°C in 5% CO2 for 72 hours. Afterwards, cells were fixed with 4% PFA and stained with 1% crystal violet in 20% methanol. The plaques were counted and used for viral titer calculation. The virus titers were expressed as plaque forming units (PFU)/ml = [(numbers of plaques per well) X dilution]/(inoculum volume)  2.10 Electron microscopy To confirm whether influenza A viruses were successfully generated by reverse genetics, a 10-l drop of reverse genetic generated virus was placed on top of a parlodion/carbon coated copper EM grid that was previously glow discharged for 30 seconds. After 8 minutes, the grid was washed 4 times in drops of distilled water, and then negatively stained in 2% uranyl acetate for 1 minute. The grid was then visualized by a FEI Tecnai G2 spirit transmission electron microscopy (Life Science Institute Imaging Facility) operated at accelerated voltage of 120 kilovolts.   2.11 Co-IP  HeLa cells were transiently transfected with 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R constructs using Lipofectamine 2000 according to the manufacturer‟s instructions. As control, cells were transfected with the 5GFP vector. As mentioned in Section 1.1.2, the NLS bearing cargo binds to importin  in the cytoplasm, but this complex disassociates in the nucleus. In order to detect sufficient interaction between importin s and NLSs, we used Importazole, an inhibitor of importin  dependent nuclear import (Soderholm et al., 2011), which arrests the import cargo  70 bound to importin s in the cytoplasm. 40 mM Importazole was added to cells 24 hours post transfection for 4 hours as previously described (Kublun et al., 2014), and the cells were harvested in 1% NP40 buffer (50 m M Tris, 150 mM NaCl, 5% glycerol, 1% NP-40) with 1 protease inhibitor (Roche Applied Science, Indianapolis, IN, USA; Catalog number: 04693116001). The Co-IP was performed using MACs anti-GFP starting Kit (Miltenyl Biotec, Catalog number: 130-091-288) according to the manufacture instruction. Briefly, cell lysates were incubated with the anti-GFP MicroBeads at 4 °C for 30 min, followed by washing 4 times with 1% NP40 buffer and once with washing buffer (10 mM Tris-HCl, pH 6.8, 1 mM EDTA). Pre-heated 95C hot elution buffer provided by the MicroBeads kit was used to collect eluent as the immunopreciptate which was then subjected to SDS-PAGE analysis. Proteins were separated on 10% SDS-PAGE and transferred onto PVDF membranes. The western blots were stained with antibodies against GFP and against importin s, and proteins were visualized with the enhanced chemilluminescence system as recommended by the manufacturer.  2.12 Expression and purification of importin s pGEX-6P-2-GST-tagged importin  constructs were transformed into BL21 Escherichia coli (E.coli) cells and expressed in the cells under isopropyl -D-1-thiogalactopyranosid (IPTG) induction for 5 hours. Bacterial pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) containing 5 mg/ml lysozyme (Sigma-Aldrich, Catalog number: L7651)) and 1 protease inhibitor (Roche Applied Science, Catalog number: 04693116001) at room temperature for 30 min. The lysates were then sonicated and cleared by centrifugation at 13,000 × g for 10 min. Bacterial expression of the GST-importin  71  fusion proteins was confirmed by SDS-PAGE/western blot analysis using an anti-GST antibody. Cleared lysates containing the expressed GST-importin  fusion proteins were incubated with glutathione-sepharose 4 fast flow beads (GE Healthcare, Catalog number: 17-5732-01) at 4C overnight. GST-importin -bound sepharose beads were washed three times with PBS and an aliquot was eluted with elusion buffer (50 mM Tris-HCl, 10 mM reduced glutathione, 1mM DTT) and analyzed by 10% SDS-PAGE to quantitate the levels of purified GST-tagged importin  fusion proteins. The purified GST-tagged importin s immobilized on glutathione-sepharose beads were maintained at 4 °C for the GST-pull down assay.  2.13 GST-pull down assay To prepare the cell lysates for the GST-pull down assay, HeLa cells were transiently transfected with 1GFP-NLS1, 1GFP-NLS2K, or 1GFP-NLS2R constructs using Lipofectamine 2000 according to the manufacturer‟s instructions. Cells were harvested at 24 hours post transfection. And resuspend in 2% FBS buffer. GFP expression analysis was performed using flow cytometry. Fluorescence-actived cell sorting data were collected on a FACS Calibur (Becton Dickinson Immunocytometry Systems, San Jose, CA) at ubcFLOW cytometry facility and analyzed using FlowJo software (Tree Star). Cells were then harvested in 1% NP40 cell lysis buffer containing 1 protease inhibitor (Roche Applied Science, Catalog number: 04693116001). The lysates were cleared by overnight incubation at 4 °C with glutathione-sepharose 4 fast flow beads.  Equivalent amount of GST-tagged importin s immobilized on glutathione-sepharose beads were incubated with the pre-cleared HeLa cell lysates at 4°C overnight. The protein-bound beads were  72 washed with 1% NP40 lysis buffer and the bound proteins were eluted in 50 µl of elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The proteins were separated on 10% SDS-PAGE and the gels were stained with Coomassie Brilliant Blue or transferred onto PVDF membranes, followed by detection of proteins with primary and secondary antibodies (dilutions according to Table 2.1) and visualization of the proteins with the enhanced chemiluminescence system as recommended by the manufacturer.   2.14 RNA extraction and cDNA synthesis The cells were lysed, and total RNA was extracted using TRIzol reagent (Invitrogen by life Technologies, Catalog number: 15596-026) following the manufacturer‟s instructions. RNA concentrations were measured by absorbance at a 260-nm wavelength using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA). Reverse transcription was performed using the Quantitect Reverse Transcription Kit (Qiagen, Mississauga, ON; Catalog number: 205311), and 1 μg of total RNA was used according to the manufacturer's instructions.  2.15 Real-time qPCR Real-time qPCR was performed using the ABI Prism 7300 sequencing detection system (PerkinElmer Applied Biosystems, Foster City, CA) in a 96-well optical microplate. Influenza A virus M1 cDNA expression was detected. Primer set for M1 was:  Forward: 5‟- AGATGAGTCTTCTAACCGAGGTCG-3.  Reverse: 5‟-TGCAAAAACATCTTCAAGTCTCTG-3.  Probe: 5‟-/56-FAM/ TCAGGCCCCCGCAAAGCCGA /3BHQ_1/ -3‟.   73 GAPDH gene expression was used as reference control. 20 times the reaction mix of GAPDH primers and probes were obtained from Applied Biosystems by life technologies (Catalog number: Hs99999905_m1) as gene reference control. Each 20 l reaction contained 10 l 2X Taqman gene expression master mix (Applied Biosystems by Life Technologies, Catalog number: 436906), 300 nM of each primer and probe, and 25 ng of cDNA. The qPCR parameters were 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec, and 55°C for 1 min. The nucleotide sequences of the resultant PCR products were confirmed by sequencing. The amplification efficiency was determined by plotting log cDNA dilution against ΔCt (ΔCt = Ct.target - Ct.gapdh), the slope of which was close to zero, indicating maximal and similar efficiency of the target and reference genes. Three separate experiments were performed on different cultures, and each sample was assayed in triplicate. A mean value was used to determine the mRNA levels by the comparative Ct (2-ΔΔCt) method with GAPDH as a reference gene. 74 Chapter 3 Characterization of the NLS2 of Influenza A Virus Nucleoprotein and Identification of its Cellular Binding Partners  3.1 Introduction Two NLSs on NP are known to mediate the nuclear import of the influenza viral genome: NLS1 at the N-terminus of NP (Wang et al., 1997), and NLS2 spanning residues 198-216 (Weber et al., 1998). NLS1 only contains two basic amino acids and does not fit into the consensus of the ten types of NLSs (Table 1-1); therefore, it was defined as an unconventional NLS (Wang et al., 1997). Over the years, NLS1 has been studied in detail and is considered a major contributor for the nuclear import of influenza A virus NP protein and the vRNP (Cros et al., 2005; O'Neill et al., 1995). In contrast, NLS2 is less studied. NLS2 was first discovered through sequence alignment of Thogoto virus NP with influenza A virus NP (Weber et al., 1998). Through antibody inhibition and peptide competition experiments, Wu et al. (2007a) demonstrated that NLS2 alone could mediate nuclear import of vRNP, indicating that the function of NLS2 is important and should not be neglected. Therefore, it would be important to study the function of NLS2 as well as to identify the import machinery involved in NLS2-facilitated nuclear translocation, which could help us gain better knowledge of vRNP nuclear import machinery during influenza A virus infection.   NLS2 has two basic amino acid clusters at each terminus that are separated by a 13 amino acids linker and it has therefore long believed to be a putative classical bipartite NLS. However, there is no research to date characterizing this NLS. A classical bipartite NLS contains two clusters of basic amino acids whose integrity is essential for its function (Robbins et al., 1991). To confirm  75 that NLS2 is a classical bipartite NLS, it has to be demonstrated that each basic cluster function interdependently to mediate nuclear import. Furthermore, each basic amino acid has to be tested to validate its necessity for NLS2‟s nuclear import function.   Interestingly, through sequence alignment of NLS2 of several influenza A virus strains, I found that there are two subtypes of NLS2s: One with a lysine (K) residue at position 214 on NP (position 17 on the NLS), and the second with an arginine (R) at this position. I named these two subtypes of NLS2s, NLS2K and NLS2R, respectively. As NLS2 has not been studied in detail, it is unknown whether this single amino acid substitution confers any differences in the function of NLS2K and NLS2R.  Not only does NLS2 need to be characterized, but also which importin  isoform participates during NLS2-facilated nuclear import needs to be investigated. With at least seven isoforms in human cells, importin  functions as an adaptor to bridge the interaction between an NLS-containing cargo and importin  for the classical nuclear import pathway (Goldfarb et al., 2004). It has been well established that NP interacts with importin 1, 3, 5, and 7 in vitro (Wang et al., 1997). Previous studies also demonstrated that importin 1, 3, and 5 bind to the NLS1 of NP (Sasaki et al., 2013; Wang et al., 1997). However, the interaction of specific importin  isoforms with NLS2 during nuclear import still remains to be determined.   In this chapter, I systematically investigate the contribution of NLS2 to nuclear import using an in vitro cell model. Additionally, I identified different importin  isoforms that bind to NLS2. Based on my results, I propose that NLS2 is a weaker NLS than NLS1. With one basic amino  76 acid different, NLS2K contains two functional basic clusters on each end, while NLS2R only contains the N-terminal functional basic cluster. I also found out for the first time that NLS2 interacts with importin 1, 3, 5, and 7.  3.2 Results 3.2.1 Nuclear import function of NLSs from influenza A virus nucleoprotein Previously, Wang and colleagues have found that GFP or oligomers with four or less GFP molecules could freely diffuse into the nucleus, while a chimera protein containing five molecules of GFP in tandem (5GFP) remains in the cytoplasm (Wang and Brattain, 2007). Thus, 5GFP chimera protein becomes a useful tool to study the nuclear import process. To determine the function of NLS1 and NLS2 during nuclear import, the coding sequences of NLS1 and NLS2 of NP from influenza A/X-31 strain were inserted into a cytoplasmic reporter plasmid containing 5GFP. This strain of influenza contains a NLS2 with a lysine (K) at position 17, and was therefore termed NLS2K. Recombinant plasmids of 5GFP-NLS1 and 5GFP-NLS2, as well as the control vector 5GFP, were transfected into HeLa cells to visualize the subcellular localization of the encoded proteins 24 h post transfection using confocal laser scanning microscope. As expected, without NLS sequences, 5GFP was localized in the cytoplasm (Figure 3-1A). The fusion of NLS1 to 5GFP resulted in nuclear accumulation of this protein, while 5GFP-NLS2 was found both in the nucleus and the cytoplasm (Figure 3-1A). Quantification of the nuclear to cytoplasmic fluorescence ratio (Fn/c) of these cells suggests that NLS1 exhibits greater role towards nuclear import of the chimera protein compare to NLS2K (Figure 3-1B). 77  Figure 3-1: Fusion of either NLS1 or NLS2 to 5GFP induces nuclear import of the chimera protein. A) HeLa cells were transfected with a plasmid expressing 5GFP, 5GFP-NLS1, or 5GFP-NLS2. Cells were fixed 24 h post transfection and visualized by confocal microscopy. Scale bar, 10 µm. B) Quantification of the fluorescence ratio of nucleus to cytoplasm (Fn/c) from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, **P<0.01, Student‟s t-test). 78 3.2.2 One basic amino acid difference changes the nuclear import function of NLS2 In order to gain a clear understanding of the NLS2 conservation throughout different influenza A virus strains, putative amino acid sequences of NLS2 were analyzed using the ClustalW2 online software. The analysis indicated a single basic amino acid difference at position 17. While the H3N2 and H1N1 seasonal influenza A viruses have lysine (K) at this position, high pathological influenza A viruses, including 1918 Spanish flu, pandemic H1N1, avian H5N1and H7N7, and H7N9 (Figure 3-2A) contain arginine (R). Therefores, for this study these NLS2s were termed, NLS2K and NLS2R, respectively (Figure 3-2A). To evaluate whether this one basic amino acid difference between these two NLS2s has different contribution to nuclear import, 5GFP-NLS2K and 5GFP-NLS2R constructs were transfected into HeLa cells, and the localization of the expressed chimera proteins were visualized by confocal laser scanning microscopy 24 h after transfection. Both 5GFP-NLS2K and 5GFP-NLS2R localized throughout the nucleus and the cytoplasm of the transfected cells (Figure 3-2B). However, quantification of the Fn/c of these cells showed that 5GFP-NLS2K was transported into the nucleus to a slightly higher extent than 5GFP-NLS2R (Figure 3-2C), suggesting that the one basic amino acid difference between the influenza NLS2K and NLS2R could lead to distinct efficiency in nuclear import.   79   80 Figure 3-2: Fusion of either NLS2K or NLS2R to 5GFP induces nuclear import of the chimera proteins. A) Sequence alignment of NLS2 from nine strains of influenza A virus. The viral sequences and their GenBank accession numbers are as follows: AGZ60858.1, BAJ10044.1, AGQ48054, BAA99400.1, ACF54602.1, AHK10804.1, AAV48837.1, AAO52971.2, and ABI94585.1. Numbers indicate the amino acids position on NP. Asterisks indicate identical amino acids conserved in all sequences. A colon (:) represents amino acid homologous substitutions. Seasonal H3N2 and H1N1 influenza A viruses are A/Alaska/02/2013 (H3N2), A/PR8/1934 (H1N1), A/X-31 (H3N2). 1918 Spanish flu is A/1918 (H1N1). Pandemic H1N1 are A/pdm/2009 (H1N1), avian H5N1 and H7N7, and H7N9 are A/Avian/H5N1, A/Avian/ H7N7 and A/Shanghai/01/2014 (H7N9). The numbers 1 and 19 on 5GFP-NLS2K and 5GFP-NLS2R sequences correspond to 198 and 216 on the NP sequence. B) Confocal images of HeLa cells expressing 5GFP-NLS2K and 5GFP-NLS2R 24 h post transfection. Scale bar, 10 µm. C) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05, Student‟s t-test).     81 3.2.3 Both clusters of basic residues in NLS2K are important for efficient nuclear import Since there are two clusters of basic residues at each end of the NLS2K sequence, it was proposed that NLS2K is likely a classical bipartite NLS (Weber et al., 1998). To test the importance of these two clusters for the nuclear accumulation of 5GFP-NLS2K, I created two mutant plasmids containing alanine (Ala) substitutions of the basic residues in each cluster. The first mutant (A1 mutant) has Ala substitutions of Lys1 and Arg2, and the second mutant (A2 mutant) has Ala substitutions of Arg16, Lys17, and Arg19 (Figure 3-3A). In addition, I also created a third mutant plasmid containing Ala substitutions of the basic amino acids on both clusters of NLS2K (A1+2 mutant; Figure 3-3A). The mutants and wild-type (WT) 5GFP-NLS2K plasmids were transfected into HeLa cells, and the subcellular localization of the encoded proteins were observed 24 h post transfection using confocal laser scanning microscopy. As documented in Figure 3-3, B and C, the fusion protein bearing mutations in the first cluster of basic amino acid (A1 mutant), showed similar nuclear localization as the WT NLS2K. The fusion protein bearing mutations in the second cluster of basic amino acid (A2 mutant) showed a significant decrease in nuclear localization compared to the WT NLS2K. However, when both clusters of basic amino acids were mutated (mutant A1+2) the decrease of nuclear accumulation of the A2 mutant was amplified. This indicates that the two clusters of basic amino acids can independently mediate nuclear import of 5GFP.    82  Figure 3-3: Both the N-terminal and the C-terminal basic clusters of NLS2K mediate the nuclear import of 5GFP-NLS2K. A) Schematic representation of the wild type 5GFP-NLS2K and mutants. The two clusters of basic residues in NLS2K are shown in bold, and Ala substitutions are shown in red bold. B) Confocal images of HeLa cells expressing 5GFP-NLS2K and its mutants 24 h post transfection. Scale bar, 10 µm. C) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05, ***P<0.001, One-way ANOVA followed by Tukey‟s tests).  83 3.2.4 The N-terminal cluster of basic residues in NLS2R is the main contributor for nuclear import  I further carried out similar mutagenesis experiments on the basic clusters of NLS2R to test whether NLS2R is a classical bipartite NLS. Similar to the experiments described above, I created 3 mutant plasmids with alanine substitutions of the basic amino acid of the first cluster (A1 mutant), the second cluster (A2 mutant), and both clusters (A1+2 mutant)  (Figure 3-4A). The mutants and WT 5GFP-NLS2R plasmids were transfected into HeLa cells, and the subcellular localization of the encoded proteins were analyzed 24 h post transfection using confocal laser scanning microscopy. As documented in Figure 3-4, B and C, the mutation in the second cluster of basic amino acid (mutant A2) did not significantly decrease the nuclear accumulation of the fusion protein compared to the WT NLS2R. The mutation in the first cluster of basic amino acid (mutant A1) significantly decreased the nuclear accumulation of the fusion protein compared to the WT NLS2R. However, this defect was the same when both clusters of basic amino acids were mutated (mutant A1+2). These results indicate that NLS2R is not a bipartite NLS, and that the N-terminal cluster of NLS2R is the main contributor to the nuclear targeting role of NLS2R. These findings also explain the difference in the nuclear import of 5GFP-NLS2K and 5GFP-NLS2R (Figure 3-2, A and C), as NL2K contains two functional basic amino acids clusters, but NLS2R only has one cluster of basic amino acids that mediates nuclear import.    84  Figure 3-4: Mutations in the N-terminal basic cluster of basic amino acids of NLS2R reduce the nuclear accumulation of 5GFP-NLS2R. A) Schematic representation of the wild type 5GFP-NLS2R and mutations. The two clusters of basic residues in NLS2R are shown in bold, and Ala substitutions are shown in red bold. B) Confocal images of HeLa cells expressing 5GFP-NLS2R and its mutants 24 h post transfection. Scale bar, 10 µm. C) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, **P<0.01, One-way ANOVA followed by Tukey‟s tests).  85 3.2.5 The linker of NLS2K/R also contributes to the NLS2 function Since the C-terminal basic cluster on NLS2R does not contribute to the nuclear import of 5GFP (Figure 3-4), next I sought to identify whether this cluster is necessary for NLS2R‟s function by generating truncated NLS2R mutant without the C-terminal basic cluster. Not only the necessity of the C-terminal basic cluster needs to be investigated, the function of the linker within NLS2R/K also needs to be examined as we observe that the A1+2 mutants for both NLS2s could still induce nuclear import of 5GFP (Figure 3-4). Previous studies has suggested that the acidic residues, such as aspartic acid (D) and glutamic acid (E) in the linker region, as well as basic amino acids flanking the N- and C-termini can contribute to the interaction between bipartite cNLS and importin  (Marfori et al., 2011). Therefore, it is possible that the linker of NLS2/K/R might contribute to the function of NLS2K/R, in particular the two arginine residues in the NLS2R‟s linker region at positions 7 and 11 might be required. To test this, I first carried out alanine scaning of all residues of the linker if NLS2K/R and did not observe any significant reduction of nuclear import (Figure A1).   Next, three truncated NLS2R constructs were generated and fused in frame to the C-terminus of the 5GFP construct as indicated in Figure 3-5A. The first mutant, 5GFP-NLS2R D1 (comprising amino acids: 1-15 of NLS2R), contains the N-terminal basic cluster of amino acids and the linker of NLS2, but not the C-terminal cluster of basic amino acids (Figure 3-5A). The second mutant, 5GFP-NLS2R D2 (comprising amino acids: 1-10 of NLS2R) was truncated before the arginine at position 11, and therefore contains only one of the basic residues of the linker. The third mutant, D3 (comprising amino acids: 1-6 of NLS2R) was truncated before the arginine at position 7, and therefore does not contain any of the basic residues of the linker. The mutants and WT plasmids  86 were then transfected into HeLa cells, and the subcellular localization of the expressed proteins were analyzed 24 h post transfection using confocal laser scanning microscopy (Figure 3-5B). I found that the truncated mutant 5GFP-NLS2R D1 lacking the C-terminal cluster of basic amino acids exhibited a slight decrease in nuclear accumulation of the chimera protein when compared with that of the full-length NLS2R (Figure 3-5B). Quantification of the fluorescence intensity confirmed this finding, with a mean Fn/c value of 0.57 for 5GFP-NLS2R, higher than that of 5GFP-NLS2R D1 (Fn/c of 0.425; Figure 3-5C). Interestingly, in the 5GFP-NLS2R site-directed mutagenesis experiment (Figure 3-4), the A2 mutant, which contains mutations in the C-terminal cluster, exhibited slight decrease in nuclear accumulation but it was not significant. This indicates that the C-terminal cluster is important for the function of NLS2, although its contribution is subtle. The D2 and D3 mutants were predominantly cytoplasmic (Figure 3-5B), with Fn/c of 0.31 and 0.28 respectively (Figure 3-5C). These ratios were similar for the control 5GFP (Figure 3-5C), which indicates that there was no nuclear import for the D2 and D3 mutants. These results imply that both the C-terminal basic cluster and the linker region of NLS2R are important to make NLS2R a functional NLS. 87  Figure 3-5: The linker of NLS2R also contributes to the NLS2R function. A) Schematic representation of the wild type 5GFP-NLS2R and its deletion mutants. Basic amino acids are shown in bold. B) Confocal images of HeLa cells expressing 5GFP-NLS2R and its deletion mutants 24 h post transfection. Scale bar, 10 µm. C) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05, **P<0.01, and ***P<0.001, One-way ANOVA followed by Tukey‟s tests). 88 3.2.6 Threonine at position 18 contributes to the function of NLS2K but not to the function of NLS2R The C-terminal cluster of basic amino acids of most classical bipartite NLSs only consists of K or R (Table 1-2). However, the C-terminal cluster of NLS2 contains a threonine (T) at position 18, instead of a K. To test whether changing this T to a basic amino acid could assist NLS2 in gaining nuclear import function, I mutated T18 to K18 in NLS2 and evaluated the nuclear import efficiency of this mutant. K18 was introduced into both 5GFP-NLS2K and 5GFP-NLS2R expressing plasmids (I termed these gain of function mutants GF2K and GF2R, Figure 3-6A). The mutants and WT plasmids were transfected into HeLa cells, and the subcellular localization of the expressed proteins was analyzed 24 h post transfection using confocal laser scanning microscopy (Figure 3-6B). The mutant chimera protein GF2K showed a reduction in nuclear accumulation compare to WT NLS2K. Quantification of the Fn/c ratio supported this observation: the mean Fn/c value for GF2K was 0.57 and that for WT NLS2K was 0.90 (Figure 3-6C). In contrast, there was not much difference between the Fn/c ratio of GF2R and WT NLS2R. Mutagenesis of T18 in NLS2R resulted in a chimera protein that was predominantly in the cytoplasm with a mean Fn/c of 0.52, while the WT NLS2R has a mean Fn/c of 0.58 (Figure 3-6C). These results imply that having basic amino acids in both termini does not make NLS2K or NLS2R a stronger NLS. Moreover, the decrease in nuclear accumulation of GF2K suggests that threonine plays a role in regulating the function of NLS2K but not that of NLS2R.  To investigate the role of T18 in NLS2 function, I then designed loss of function experiments by performing alanine substitution of T18 in both NLS2K and NLS2R (I termed these loss-of-function mutants LF2K and LF2R, Figure 3-7A). Mutants and WT plasmids were  89 transfected into HeLa cells, and the subcellular localization of the expressed proteins was analyzed 24 h post transfection using confocal laser scanning microscopy (Figure 3-7B). In this experiment, the fusion protein LF2K displayed a reduction of nuclear accumulation, indicating that T18 is essential for the function of the NLS2K‟s C-terminal cluster (Figure 3-7, B and C). However, the LF2R mutant fusion protein did not change its nuclear localization, which is consistent with the observation that the C-terminal cluster of basic amino acids is not important for nuclear import mediated by NLS2R (Figure 3-4). Taken together, these results imply that the threonine at position 18 can regulate nuclear import when it is next to lysine as in NLS2K but not when it is next to arginine as in NLS2R.  90  Figure 3-6: Substituting threonine with lysine at position 18 reduces the nuclear accumulation of 5GFP-NLS2K but not that of 5GFP-NLS2R. A) Schematic representation of the wild type 5GFP-NLS2K, 5GFP-NLS2R, and mutation plasmids. The two clusters of basic residues in NLS2K and NLS2R are shown in bold, and substitutions of T to K are shown in red bold. B) Confocal images of HeLa cells expressing 5GFP-NLS2K, 5GFP-NLS2R, and its mutants 24 h post transfection. Scale bar, 10 µm. C) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05, One-way ANOVA followed by Tukey‟s tests). 91  Figure 3-7: Mutation of threonine to alanine in the C-terminal cluster reduces the nuclear accumulation of 5GFP-NLS2K, but not that of 5GFP-NLS2R. A) Schematic representation of the wild type 5GFP-NLS2K, 5GFP-NLS2R, and mutation plasmids. The two clusters of basic residues in NLS2K and NLS2R are shown in bold, and substitutions of T to K are shown in red bold. B) Confocal images of HeLa cells expressing 5GFP-NLS2K, 5GFP-NLS2R, and its mutants 24 h post transfection. Scale bar, 10 µm. C) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05, One-way ANOVA followed by Tukey‟s tests). 92 3.2.7 Identification of the binding partners of NLS2 during nuclear import  3.2.7.1 Importin 3 is important for NLS2K-mediated nuclear import  Importin 1, 3, 5, and 7 are involved in the nuclear import of influenza A virus NP (Melen et al., 2003), which contains NLS1 and NLS2. Therefore, any of these importin  homologues may recognize NLS2. To identify whether any of these importin  is involved in the NLS2K nuclear import process, I first examined the involvement of these importin  isoforms in the nuclear import of 5GFP-NLS2 by transiently depleting the importin 1, 3, or 5 individually using siRNA and then transfecting the depleted cells with either 5GFP-NLS2K or 5GFP-NLS2R plasmids. As a control, cells were transfected with non-targeting siRNA and then with either 5GFP-NLS2K or 5GFP-NLS2R plasmids. Since the expression of 5GFP-NLS2K and 5GFP-NLS2R was low in importin 7 depleted cells (data not shown), I could not conclude whether importin 7 is important for the nuclear import of NLS2K or NLS2R containing cargo using this approach. Both western blot analysis and immunofluorescence microcopy showed that high efficiency knockdown of importin 1 (Figure 3-8, A and C), importin 3 (Figure 3-9C), and importin 5 (Figure 3-10, A and C) was achieved after transfection with 20 nM siRNA for 72 hours. 5GFP-NLS2K was then transfected into the importin -depleted cells to examine its nuclear import behavior. The subcellular localization of 5GFP-NLS2K was not altered in the importin 1- or 5-depleted cells (Figures 3-8A and 3-10A). In contrast, there was a significant inhibition of the nuclear accumulation of 5GFP-NLS2K in the 3-depleted cells (Figure 3-9, A and B). Quantification of the Fn/c ratio supported this observation, with a mean Fn/c value of 0.70 for the non-targeting siRNA-treated cells and 0.39 for importin 3 siRNA-treated cells.  93 These results indicate that importin 3 mediates the nuclear import of NLS2K-contaning proteins.   3.2.7.2 Importin 3 mediates nuclear import of NLS2R-containing proteins  Similar experiments were also performed to study the involvement of importin 1, 3, and 5 in the nuclear import of 5GFP-NLS2R. Using confocal laser scanning microscopy, my results showed that the subcellular localization of 5GFP-NLS2R did not change in the importin 3- or 5-depleted cells (Figures 3-12A and 3-13A). However, in importin 1-depleted cells, the nuclear import of 5GFP-NLS2R was significantly inhibited (Figure 3-11, A and B). Quantification of the fluorescence signal indicates that 5GFP-NLS2R was transported into the nucleus at a significantly lower efficiency (Fn/c of 0.29) in importin 1-depleted cells, compared to cells that were treated with non-targeting siRNA (Fn/c of 0.51) (Figure 3-11C). These results imply that importin 1 plays a critical role for NLS2R-mediated nuclear import.   A summary comparing the results of the RNAi experiments for 5GFP-NLS2L and 5GFP-NLS2R are presented in Table 3-1.    94  Figure 3-8: Depletion of importin α1 does not affect the nuclear accumulation of 5GFP-NLS2K. A) HeLa cells were treated with non-targeting siRNA or importin 3-siRNA for 48 h. After this time, the cells were then transfected with 5GFP-NLS2K plasmid for 24 h and prepared for immunofluorescence microscopy. Cells were probed with an anti-importin 1 antibody and DAPI to detect DNA, and visualized by confocal microscopy. Scale bar, 10 µm. B) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments. C) Western blot of cell lysates from non-targetting siRNA and importin 1-siRNA treated HeLa cells. The blot was probed with antibodies against importin 1 and -Actin. -Actin was used as a loading control. 95  Figure 3-9: Knockdown of importin α3 affects the 5GFP-NLS2K nuclear accumulation. A) HeLa cells were treated with non-targeting siRNA or importin 3-siRNA for 48 h. After this time, the cells were then transfected with 5GFP-NLS2K plasmid for 24 h and prepared for immunofluorescence microscopy. Anti-importin 3 antibody does not work for immunofluorescence staining. DAPI to detect DNA, and visualized by confocal microscopy. Scale bar, 10 µm. B) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05, Student‟s t-test). C) Western blot of cell lysates from Mock (non-targeting siRNA) and importin 3-siRNA treated HeLa cells. The blot was probed with antibodies against importin 3 and -Actin. -Actin was used here as a loading control.    96  Figure 3-10: Depletion of importin α5 does not affect the nuclear accumulation of 5GFP-NLS2K. A) HeLa cells were treated with non-targeting siRNA or importin 5-siRNA for 48 h. After this time, the cells were then transfected with 5GFP-NLS2K plasmid for 24 h and prepared for immunofluorescence microscopy. Cells were probed with an anti-importin 5 antibody and DAPI to detect DNA, and visualized by confocal microscopy Scale bar, 10 µm. B) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments. C) Western blot of cell lysates from Mock (non-targeting siRNA) and importin 3-siRNA treated HeLa cells. The blot was probed with antibodies against importin 5 and -Actin. The importin 5 antibody crossreacts with importin 7. The doublet represents importin 5 (upper band, indicated by *) and importin 7 (lower band). -Actin was used here as a loading control.     97  Figure 3-11: Knockdown of importin α1 reduces the nuclear accumulation of 5GFP-NLS2R. A) HeLa cells were treated with non-targeting siRNA or importin 1-siRNA for 48 h. After this time, the cells were then transfected with 5GFP-NLS2R plasmid for 24 h and prepared for immunofluorescence microscopy. Cells were probed with an anti-importin 1 antibody and DAPI to detect DNA, and visualized by confocal microscopy Scale bar, 10 µm. B) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, ***P<0.001, Student‟s t-test). C) Western blot of cell lysates from Mock (non-targeting siRNA) and importin 1-siRNA treated HeLa cells. The blot was probed with antibodies against importin 1 and -Actin. -Actin was used here as a loading control.     98  Figure 3-12: Knockdown of importin α3 does not affect the nuclear accumulation of 5GFP-NLS2R. A) HeLa cells were treated with non-targeting siRNA or importin 3-siRNA for 48 h. After this time, the cells were then transfected with 5GFP-NLS2R plasmid for 24 h and prepared for immunofluorescence microscopy. Anti-importin 3 antibody does not work for immunofluorescence staining. DAPI to detect DNA, and visualized by confocal microscopy. Scale bar, 10 µm. B) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments. C) Western blot of cell lysates from Mock (non-targeting siRNA) and importin 3-siRNA treated HeLa cells. The blot was probed with antibodies against importin 3 and -Actin. -Actin was used here as a loading control.    99  Figure 3-13: Depletion of importin α5 does not affect the 5GFP-NLS2R nuclear accumulation. A) HeLa cells were treated with non-targeting siRNA or importin 5-siRNA for 48 h. After this time, the cells were then transfected with 5GFP-NLS2R plasmid for 24 h and prepared for immunofluorescence microscopy. Cells were probed with an anti-importin 5 antibody and DAPI to detect DNA, and visualized by confocal microscopy Scale bar, 10 µm. B) Quantification of Fn/c from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments. C) Western blot of cell lysates from Mock (non-targeting siRNA) and importin 3-siRNA treated HeLa cells. The blot was probed with antibodies against importin 5 and -Actin. The importin 5 antibody crossreacts with importin 7. The doublet represents importin 5 (upper band, indicated by *) and importin 7 (lower band). -Actin was used here as a loading control.   100 Table 3- 1: Summary of importin  depletion experiments.   siRNA Nuclear import effect 5GFP-NLS2K 5GFP-NLS2R Imp 1 siRNA No effect Signicantly decreased Imp 3 siRNA Signicantly decreased No effect Imp 5 siRNA No effect No effect Imp 7 siRNA Unknown Unknown Abbreviation: Imp, importin. 101 3.2.7.3 Importin 1, 3, 5, and 7 bind to NLS2K and NLS2R  To investigate the interactions between NLS2K/R and four importin  isoforms, the binding specificity of each of importin  isoforms was determined. Recombinant GST-tagged importin 1, 3, 5, and 7 (immobilized with glutathione-sepharose beads) (Figure 3-14A) were incubated with HeLa cell lysates containing GFP-NLS2R or GFP-NLS2K. Since Sasaki et al. (2013) showed that NLS1 binds to importin 1, 3, and 5, GFP-NLS1 was used as positive control. GFP was used as negative control as it cannot interact with any importin  isoforms. Before cell lysis, GFP expression was detected by flow cytometry (data now shown) before lysis to insure there were equal amounts GFP-NLSs expressing in each condition. As expected, GFP did not interact with any of the importin  isoforms, and NLS1 interacted with importin 1, 3, and 5, but also with importin 7 (Fig 3-14A). Both GFP-NLS2R and GFP-NLS2K were also found interacting with importin 1, 3, 5, and 7 (Fig 3-14B).   To further analyze the binding between different isoforms of importin  and NLS2, the binding of 5GFP-NLS2K or 5GFP-NLS2R with importin 1, 3, 5, and 7 using Co-IP was next tested. For this approach, 5GFP-NLS1 and 5GFP were used as positive and negative controls, respectively. HeLa cells were transfected with 5GFP-NLS2K, 5GFP-NLS2R, 5GFP-NLS1, or 5GFP plasmids. In order to rescue more binding complex, importazole, an inhibitor of importin -mediated nuclear import through blocking the binding of RanGTP with importin  (Soderholm et al., 2011), was added to the transfected cells for 4 h after 24 h post transfection. Since NLS binds to importin  in the cytoplasm and dissociates from it in the nucleus, more binding complex of GFP-NLSs and importin  were obtained by using importazole. The lysates  102 containing protein complexes were then precipitated using anti-GFP magnetic beads. The immunoprecipitated proteins were eluted and analyzed by western blot to detect importin 1, 3, 5, and 7. The untreated cell lysate was also analyzed using western blot to test for endogenous importin 1, 3, 5, and 7 expressions. As expected, since 5GFP does not interact with any importin  isoforms, no importin  bands were observed in the immunoprecipitation from cell lysates of cells expressing 5GFP (Figure 3-15A). 5GFP-NLS1, as the positive control, interacted with importin 1, 3, 5, and 7 (Figure 3-15A). Both 5GFP-NLS2K and 5GFP-NLS2R were also found bound to importin 1, 3, 5, and 7 (Fig 3-15A). As documented in Figure 3-15B, quantification of the band intensity of the immmunprecipitates revealed that importin 1 was able to coimmuoprecipitated the highest amount of protein for 5GFP-NLS2R, whereas importin 3 precipitated approximately the same amount of NLS2K and NLS2R, which is in agreement with the previous importin  siRNA results (i.e., importin 1 reduceed nuclear import of 5GFP-NLS2R in importin 1-depleted cells Figure 3-9 and Table 3-1). Notably, NLS2R precipitated a larger amount of importin 5 and 7 than NLS2K (Figure 3-15B). However, I could not compare the binding levels between different importin  isoforms to one particular NLS, since different importin  antibodies present distinct antigen-binding capability and the endogenous levels of importin  isoforms varies.   Taken together, I demonstrate here that NLS2K and NLS2R are able to bind to importin 1, 3, 5, and 7 using GST-pull down and Co-IP assays.   103  Figure 3-14: Detection of the interactions between NLS2R and NLS2K with importin α1, α3, α5, and α7 using GST pull-down assay. A) GST-tagged importin 1, 3, 5 or 7 were expressed in E. coli and purified using glutathione-Sephorase beads. Purified proteins were subjected to 10% SDS-PAGE and stained by Coomassie brilliant blue. M, Precision Plus Dual Color standards. B) HeLa cells were transfected with GFP, GFP-NLS1, GFP-NLS2K, or GFP-NLS2R for 22 h and then harvested in lysis buffer containing 1% NP40. Cell lysates were incubated with GST-tagged importin 1 (Imp1), 3 (Imp3), 5 (Imp5), or 7 (Imp7) on immobilized glutathione-sepharose beads. The GFP-NLS1/2K/2R protein bound to GST-importin 1, 3, 5, and 7 were examined by western blot using an anti-GFP antibody. The amount of GST-importin 1 (Imp1), 3 (Imp3), 5 (Imp5), or 7 (Imp7) was detected by western blot using anti-GST antibody.   104  Figure 3-15: NLS2K and NLS2R bind importin 1, 3, 5, and 7 detected by Co-IP. A) HeLa cells were transfected with 5GFP, 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R for 22 h. Cells were then harvested in lysis buffer containing 1% NP40 and subjected to Co-IP using micro-GFP-magnetic beads. The amount of the specific importin  isoform that binds to NLSs was examined by western blot using anti-importin 1 (Imp1), 3 (Imp3), 5 (Imp5), and 7 (Imp7) antibodies. Cell extracts of transfected cells were used to detect endogenous importin 1, 3, 5, and 7 expressions. The amount of 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R was detected by western blot using an anti-GFP antibody. B) Quantification of the band intensity for the western blot shown in A. The amount of importin  immunoprecipitated was normalized by dividing the band intensity of 5GFP-NLS1/2R/2K by the band intensity of 5GFP-NLS.  105 3.3 Discussion In this study, I for the first time characterized the function of NLS2 of influenza A virus NP and identified the importin  isoforms that bind to this NLS. Using 5GFP fused to NLS2 I demonstrated that the function of NLS2 in promoting nuclear import is significant (Figure 3-1). Interestingly, two subtypes of NLS2s were discovered through sequence alignment (Figure 3-1A), NLS2K and NLS2R, which exhibited significant difference in nuclear import when fused to 5GFP. Using site-directed mutagenesis, it was shown that NLS2K behaves as a bipartite NLS, while NLS2R functions as a monopartite NLS (Figures 3-3 and 3-4). I further obtained deletion mutants of NLS2R to determine its exact length. The results indicate that both the C-terminal basic amino acid cluster and the linker are important for the nuclear import function of NLS2R (Figure 3-5), although the contribution of the C-terminal cluster appears to be weak (Figure 3-4, mutant A2). Functional analysis further revealed that substitution of threonine at position 18 efficiently inhibited localization of 5GFP-NLS2K, but not 5GFP-NLS2R to the nucleus. In addition, GST-pull down assays and Co-IP analysis revealed the importin  isoforms that interact with NLS2K and NLS2R. It was shown that four importin  isoforms, 1, 3, 5, and 7, were able to interact with both NLS2K and NLS2R in vitro. Using siRNA, I further illustrated that importin 3 is important to promote the nuclear import of 5GFP-NLS2K, whereas importin 1 is essential for the nuclear import function of NLS2R (Table 3-1).     Our laboratory had previously demonstrated that NLS2K alone could promote nuclear import of vRNP (Wu et al., 2007a). However, Cros et al. (2005) showed that inactivating NLS2K by mutating basic amino acids on its C-terminus had no effect on NP localization in transfected  106 HeLa cells. Ozawa et al. (2007) also reported that mutating C-terminus basic amino acids on NLS2R did not change the localization of NP compared to WT NP, which is contradictory to the results of Wu et al. (2007a). In this study, I showed that the two basic amino acid clusters on each end of NLS2K function interdependently in promoting nuclear import, which indicates that the function of NLS2K depends on basic amino acid clusters on its N-terminus and also its C-terminus. My study also revealed that the N-terminus of NLS2R is the main contributor to the nuclear import function of NLS2R instead of the C-terminal basic amino acids. My result explain the results reported by Cros et al. (2005) and Ozawa et al. (2007), in which these authors performed mutations only in their C-terminal cluster of NLS2 and neither NLS2K nor NLS2R was totally inactivated. Thus, I propose that NLS2 may exhibit more of a contribution in the nuclear import of NP than previously reported.    NLS2 was originally proposed to be a putative classical bipartite NLS, since it contains two stretches of basic amino acids on each end that are separated by a 13 amino acids-long linker (Weber et al., 1998). In my study, I conducted site-directed mutagenesis to test whether NLS2 is a classical bipartite NLS. My study showed that NLS2K presented stronger nuclear import promoting ability than NLS2R. By mutating the N-terminal basic cluster, the C-terminal basic cluster, or both clusters, the results indicate that NLS2K is a bipartite NLS, but NLS2R not. Thus, the difference in nuclear accumulation of the 5GFP chimera protein containing NLS2K or NLS2R could be explained by the phenomenon that NLS2K promotes nuclear import using both basic clusters, while NLS2R mainly uses its N-terminal basic amino acid cluster. A hallmark of a functional classical bipartite NLS is to have two interdependent clusters of basic amino acids at both ends (Robbins et al., 1991); with mutations on each cluster affecting nuclear import.  Therefore, based on our results, neither NLS2K nor NLS2R could be classified as a classical  107 bipartite NLS since it was shown that mutations on one of the basic clusters on NLS2K or NLS2R yielded similar nuclear accumulation of the chimeric protein compared with the WT 5GFP-NLS2K/R.   Since I found that NLS2R is not a classical bipartite NLS, I then studied the important functional regions of NLS2R by a series of truncated 5GFP-NLS2R constructs (Figure 3-5A). It was shown that each deletion mutant exhibited less and less nuclear import function, which indicates that both the C-terminal basic cluster and the linker might be necessary for NLS2R to interact with importin . The necessity of the C-terminal basic cluster in promoting nuclear import of 5GFP-NLS2R is in agreement with our site-directed mutagenesis data (Figure 3-4). I showed that mutating the C-terminal basic cluster (A2 mutant) slightly decreased the nuclear import of 5GFP (Figure 3-4), suggesting that the C-terminal may have a minor contribution to nuclear import. The result with the deletion mutant (5GFP-NLS2R D1) further implies that the C-terminal cluster is necessary for the function of NLS2R, which may possibly interact weakly with importin .     The linker region in a classical bipartite cNLS separates the two basic clusters, which interact with importin  through its major and minor binding sites. It has been thought that the linker does not make any specific contacts with importin  (Marfori et al., 2011). However, Kosugi et al. (2008) conducted activity-based profiling via systematically mutational analysis of classical bipartite NLSs and found that the acidic residues, such as aspartic acid (D) and glutamic acid (E) in the linker region have minor contribution to the nuclear import function of classical bipartite NLSs. In my study, I showed that the linker in NLS2R was important, as deletion of the linker region significantly reduced nuclear import of 5GFP (Figure 3-5). Since the linker of NLS2  108 contains D and E, it is possible that these acidic amino acids could contribute to the nuclear import function of NLS2.   In addition, threonine at position 18 of NLS2K was shown to contribute to the nuclear import function of NLS2K, as mutation of T18 on NLS2K decreased the nuclear accumulation of the chimera protein (Figures 3-6 and 3-7). In contrast, substitution of T18 on NLS2R did not change the nuclear localization of 5GFP (Figures 3-6 and 3-7). Besides the conserved basic residues, post-translational modifications such as phosphorylation could modulate nuclear import rates (Nardozzi et al., 2010b). For example, S111 adjacent to the NLS of SV40‟s large T-antigen has been shown to enhance its nuclear import (Hubner et al., 1997), while phosphorylation of T124 decreased nuclear accumulation of SV40 T-antigen (Jans et al., 1991). Since T is a potential phosphorylation site, it could be phosphorylated to promote the nuclear import function of NLS2K when it is adjacent to K (as in NLS2K), but not when it is next to R (as in NLS2R). As substitution of T18 on NLS2R did not result in changes in the nuclear localization of 5GFP.  We are the first group to study the role of different importin  isoforms in the NLS2-mediated nuclear import (Figures 3-8 to 3-13). I found that nuclear import of 5GFP-NLS2K was significantly decreased when importin 3 was depleted (Figure 3-9), and nuclear accumulation of 5GFP-NLS2R was reduced when importin 1 was depleted (Figure 3-11). I was not able to observe significant inhibition of 5GFP-NLS2K nuclear accumulations when I depleted importin 1 and 5. Similarly, siRNA depletions of importin 3 and 5 have no effect in the nuclear import function of NLS2R (Table 3-1). These results indicate that importin 3 and 1 are important for NLS2K- and NLSR-mediated nuclear import, respectively. However, other  109 importin  isoforms might still participate in NLS2K or NLS2R mediated nuclear import, because the seven importin  isoforms exhibit a broad functional redundancy (reviewed by Goldfarb et al., 2004)).     GST-pull down and Co-IP assays were then used to determine whether other importin  isoforms could also interact with NLS2K or NLS2R. The results of GST-pull down assays demonstrated that both NLS2K and NLS2R interact with importin 1, 3, 5, and 7. Using Co-IP, I was able to confirm that importin 1, 3, 5, and 7 interact with both NLS2K and NLS2R. In these experiments, importazole was added at 24 h after transfection to obtain higher amount of immunoprecipitated proteins in the cytoplasm. In the presence of importazole the newly expressed 5GFP-NLSs associated with importin  and did not enter the nucleus. I was able to show that NLS1 immunoprecipitated importin 1, 5, and 7 in cells treated with importazole (Figure 3-15), which is similar to the GST-pull down data (Figure 3-14). The interaction between importin 3 and NLS1 was difficult to interpret since the amount of immunoprecipitated protein was relatively low. This is either caused by the lower expression of endogenous importin 3 or the lower antigen binding of the importin 3 antibody used. NLS2R immunoprecipitated higher amount of importin 1, 5, and 7 than NLS2K (Figure 3-15). This was not expected, because NLS2K was a stronger NLS in mediating nuclear import than NLS2R (Figure 3-2). One possible explanation is that as discussed above T18 could serve as a potential phosphorylation site on NLS2K during nuclear import, and the binding of importin  to NLS2K might be enhanced by threonine phosphorylation. Loss of threonine phosphorylation during sample preparation for the co-IP experiments may have led to changes of importin α binding to NLS2K, as we did not add phosphatase inhibitors (e.g., sodium fluoride) into the lysis buffer. Thus, it could explain why  110 NLS2K exhibited better function in facilitating nuclear import in vivo, while we observed less amount of importin  binding to NLS2K than to NLS2R in the GST-pull down assays and Co-IP analysis.   In summary, I have characterized for the first time the contribution of NLS2 for the nuclear import of a chimeric protein, and found that it could exhibit different nuclear import efficiencies depending on only one basic amino acid difference. Furthermore, my results indicate that potential phosphorylation site(s) in NLS2K could enhance the binding affinity of this NLS to different importin α isoforms. Four importin  isoforms, importin 1, 3, 5, and 7, were found to bind to NLS2K/R in vitro. These findings increase our understanding about the role of NLS2 during nuclear import.  111 Chapter 4 NLSs of Influenza A Virus Nucleoprotein as Novel Antiviral Candidates 4.1 Introduction  The emergency of resistance by influenza A virus to current antiviral drugs has catalyzed an increasing number of investigations to help in the developing of new antiviral treatments to replace the existing drugs. NP is one of the most abundant viral proteins during influenza A virus infection. It plays a major role in the assembly of vRNPs with the single stranded influenza genomic RNA and three polymerase subunits, as NP monomers are able to oligomerize and bind to RNA (Prokudina-Kantorovich and Semenova, 1996; Wu et al., 2007b; Ye et al., 2006). NLSs on NP are also critical for its function as they are the sequences that mediate nuclear import not only of NP, but also of the vRNP (Cros et al., 2005; Wu et al., 2007a). Since NP has no cellular counterpart and is highly conserved throughout influenza A strains (Wu et al., 2007), NP may serve as a potential target for antiviral compounds.   Previous studies have conducted high-throughput screening to identify small molecules that interfere with the interaction between NP monomers or NP with vRNA (Gerritz et al., 2011; Kao et al., 2010; Lejal et al., 2013; Su et al., 2010). Nucleozin was one of the compounds discovered, which stabilizes NP monomers and prevents the interaction of NP with vRNA by inhibiting the formation of NP trimers (Kao et al., 2010). A second compound, Naproxen, was recently identified as the first inhibitor that directly blocks the interaction between NP and vRNA (Lejal et al., 2013). It targets the RNA-binding groove on NP and blocks the assembly of vRNP.    112 Despite the fact that nuclear import is an important function of NP, inhibition of nuclear import of NP has not been explored as a potential antiviral target. In order to establish a successful infection, influenza A virus must deliver its genome into the host cell nucleus for transcription and replication. NLSs on NP are critical for the delivery of the viral genome into the nucleus of the host cell. My hypothesis is that inhibiting the function of the NLSs of NP will inhibit the nuclear inport of vRNPs, and therefore virus production. Thus, I propose that the NLSs of NP are good targets for the development of anti-influenza drugs.   In Chapter 3, I described the nuclear import function of NLS2 and its import receptors. In this chapter, I further aim to verify the potential antiviral effect of targeting NLSs on NP. Using reverse genetics, I was able to generate viruses with mutations on the NLSs of NP and tested their infectivity. Wu et al. (2007a) performed a peptide competition assay in semi-permeabilized cells and successfully inhibited the nuclear import of purified vRNP in vitro by competing with the function of NLS1 and NLS2. Similar to their studies, here I developed a competition assay using intact cells transfected with constructs encoding the NLSs of NP and infected them with influenza A virus to investigate whether inhibiting the function of NLSs on NP during infection could impair influenza A virus production.      113 4.2 Results 4.2.1 NLS1 or NLS2 mutations on NP lead to the production of non-infectious influenza A viruses by reverse genetics  To demonstrate the roles of NLS1 and NLS2 during influenza A virus infection, we used an eight plasmids reverse genetic system (generously provided by Dr. Honglin Chen from The University of Hong Kong, and Dr. Robert Webster from St Jude Children Research Hospital) to test whether substituting WT NP for mutant NPs (with mutations on NLS1, NLS2, or both NLSs) could affect influenza viral infection. These eight plasmids contain full-length cDNAs coding for the viral proteins HA, NA, PB1, PB2, PA, M, NS, and NP of influenza A virus strain A/PR/8/34 (H1N1) ligated to the cloning vector pHW2000 as previously characterized by (Hoffmann et al., 2000). Here I performed site-directed mutagenesis of basic amino acids on NLS1 or NLS2 of the NP plasmid. As illustrated in Figure 4-1A, four mutants were generated:  1) NLS1 mutant (NLS1 MT) contains alanine substitution of K and R at position 7 and 8 on NLS1 of NP protein.  2) NLS2 A1 mutant contains alanine substitution of K and R at position 198 and 199 on the N terminus of NLS2 of NP.  3) NLS2 A2 mutant contains alanine substitution of K and R at position 213, 214, and 216 on the C terminus of NLS2 of NP.  4) NLS2 A1+2 mutant contains mutation of both NLS2 A1 mutant and NLS2 A2 mutant.  The last three mutants are similar to the 5GFP-NLS2K mutants studied in Section 3.2.3 (Figure 3-3A). To ensure that the NP gene was free of unwanted mutations, the wild-type (WT) NP plasmid and mutant NP plasmids were sequenced prior to performing the reverse genetics experiments. Next, DNA plasmids containing WT NP or NP mutants, and the plasmids for PA,  114 PB1, PB2, NS, HA, NA, and M were transfected into co-cultured MDCK and HEK293T cells to generate WT and mutanted viruses. Empty pHW2000 vector was used as a negative control. Supernatant of the transfected cells were harvested at 72 h post transfection. In order to confirm that viruses were successfully generated by reverse genetics, an aliquot of the supernatant was subjected to negative staining and visualized using an electron microscope. As shown in Figure 4-1B, we were able to generate both WT and mutanted virus containing mutants in either NLS1 or NLS2 of NP protein. All the viruses were enveloped and contained distinct spike projections on the surface (Fig. 4-1B).   In order to test the amount of virus generated, we performed western blots to detect the amount of NP and M1 expression in the supernatant. As expected, the supernatant of cells transfected with the empty pHW2000 vector, as a negative control, did not yield any bands in the western blots, and M1 and NP were detected in the supernatant of cells transfected with the 8 WT plasmids (Figure 4-2A). The expression of M1 was also detected in the supernatant of cells transfected with the 8 WT plasmids or with each of the NP mutants and the other 7 influenza plasmids, although the NLS1 mutant virus showed higher M1 expression than any of the NLS2 mutants (Figure 4-2A). The expressions of NP were similar in WT, NLS1 mutant, and NLS2 A1 mutant viruses (Figure 4-2A). However, there was a relatively weak expression of NP in the NLS2 A2 mutant virus. NP expression of the NLS2 A1+A2 mutant was too low to be detected using western blots.  Next, the viral titers were determined using plaque assays. I found that the supernatant from cells transfected with WT NP and the other 7 influenza plasmids contained virus as detected by plaque assays (Figure 4-1B), with a virus titer of 1.3x104 PFU/ml (Figure 4-1C). However, the  115 supernatant of cells transfected with any of the three NP mutants and the other 7 influenza plasmids were not able to generate any plaques (Figure 4-1, B and C). Our results indicate that viral infectivity was completely inhibited by NLS1 or NLS2 mutations in NP.   Taken together, reverse genetics offers a powerful tool to generate mutant viruses for specific purposes. In our experiments, we were able to obtain mutant viruses carrying NLS1 or NLS2 mutantions on NP, which were not infectious.   116  Figure 4-1: Wild type and NLS mutant viruses were successfully generated by reverse genetics. HEK293T and MDCK cells were transfected with the influenza A/PR/8/34 (PR8) 8-plasmids reverse genetics system. To generate NLS1 or NLS2 mutant viruses, the plasmid of WT NP was replaced for plasmids of NP containing mutations on NLS1, or NLS2. After 72 h of transfection, supernatants were harvested and analyzed by negative stain electron microscopy. A) Schematic representations of the WT NP and its mutants. Alanine substitutions of K or R are shown in red. B) Electron micrographs of WT or MT influenza A viruses negatively stained with uranyl acetate. Scale bar, 50 nm.  117  Figure 4-2: Mutations of NLS1 or NLS2 inhibit the infectivity of influenza A virus. A) Western blot analysis of NP and M1 expression in supernatants of cells that were transfected with the reverse influenza genetics system containing HA, NA, PB1, PB2, PA, M, NS, and NP (WT) plasmids or the first 7 plasmids and mutant NP plasmids NLS1 MT, NLS2K A1, NLS2K A2, or NLS2K A1 + A2 (as indicated in Figure 4-1A). As a negative control, pHW2000 empty vector was used for transfection. B) Results of plaque assays of MDCK monolayer cells infected with the supernatant of cells transfected with the reverse influenza genetic system as indicated in A. Cells were stained with crystal violet. The picture showed plaques forming of 10-2 dilution of supernatant containing wild type and mutant viruses. The results shown are representative of three independent experiments. C) Quantification of the viral titer in the supernatants estimated from the plaque assay results shown in (B). PFU = plaque forming units. Shown are the mean values and S.E.M measured from three independent experiments. 118 4.2.2 Competing with the NLS1 or NLS2’s function impair nuclear import of vRNP during influenza A virus infection In order to determine whether the NLSs of NP would serve as potential antiviral targets, here I developed a competition assay, which consists of transfecting HeLa cells with 5GFP-NLS1/NLS2 and then examining whether these cells could be infected with influenza A virus. My hypothesis is that 5GFP-NLS1/NLS2 will compete with the influenza A viral NLSs for binding to their nuclear import receptors, and this would inhibit the nuclear import of influenza A vRNPs and therefore the infection.  To determine the specificity of this competition assay, I first tested whether NLS1/NLS2 could interfere with cNLS-mediated nuclear import. Since 57% of the nuclear import that happens in cells are facilitated by cNLSs (Lange et al., 2007), it is important to eliminate the possibility that NLS1/NLS2 could compete with cNLS during nuclear import. The cNLS I used in this study is the NLS from SV40 large T-antigen, which is the prototype of monopartite cNLS. DsRed-cNLS was transfected into HeLa cells together with 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R and the subcellular localization of the encoded proteins was visualized 24 h post transfection using confocal laser scanning microscopy. As expected, 5GFP-NLS1 accumulated in the nucleus (Figure 4-3 A), whereas 5GFP-NLS2K- and 5GFP-NLS2R-transfected cells showed green fluorescence both in the cytoplasm and the nucleus (Figure 4-3, B and C). DsRed-cNLS was located in the nucleus and also in the nucleolus (Figure 4-3 A, B and C), which indicates that cNLS mediated nuclear import is not inhibited by competition with NLS1/NLS2.     119 In order to rule out that cNLSs in the cells do not compete with influenza A viral genomes for nuclear import, we transfected DsRed-cNLS into HeLa cells for 48 hours followed by infection with influenza A virus for 2 and 10 hours (Figure 4-4A). Immunofluorescent staining of NP was performed to determine the localization of influenza A vRNPs. As illustrated in Figure 4-5A, at 2h p.i., NP was located in the cytoplasm. At 10h p.i., NP was expressed and imported into the nucleus for vRNP assembly. Next, we performed infection after introducing DsRed-cNLS into HeLa cells (Figure 4-4B). Similar to mock infected cells, NP resided in the cytoplasm at 2 h p.i., and mostly accumulated in the nucleus at 10 h p.i., indicating the inability of a cNLS to compete with the nuclear import of influenza A vRNPs.   To determine whether exogenous NLS1 or NLS2 could inhibit nuclear import of the vRNP, I infected HeLa cells expressing 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R with influenza A virus. Immunofluorescent staining of NP was performed to determine the localization of influenza A vRNPs. Same as we observed Figure 4-5A, NP was primarily located in the cytoplasm at 2 h p.i., and accumulated in the nucleus by 10 h p.i. (Figure 4-5A). Next, I expressed 5GFP-NLSs plasmids before performing the infection. As illustrated in Figure 4-5B, C and D, NP was found throughout the cytoplasm at 2h p.i. However, at 10 h p.i., NLS1-, NLS2K-, and NLS2R-expressing cells revealed delayed nuclear import of NP, as most of NP was predominantly in the cytoplasm. Moreover, NLS2R-expressing cells showed weaker inhibition in the nuclear import of NP (Figure 4-5D). Quantification of Fn/c of NP staining showed that NP accumulation is significantly higher at 10 h p.i. than at 2 h p.i. (Figure 4-5E). This is because the influenza A virus strain used in this experiment contains NLS2K instead of NLS2R. These results showed that impairing NLS1 or NLS2‟s function by competition with proteins containing these  120 NLSs will inhibit the nuclear import of influenza A viral genome, which suggests that NLS1 and NLS2 are good targets for anti-influenza drugs development.  Taken together, I showed that introducing NLSs-containing proteins into cells will inhibit nuclear import of the influenza A viral genomes during infection, suggesting that developing antiviral drugs containing peptides corresponding to the NLSs of influenza NP might be a good approach for anti-influenza therapeutic.  121  Figure 4-3: 5GFP-NLS1, NLS2K, and NLS2R do not affect the nuclear import of DsRed-cNLS. HeLa cells were co-transfected with DsRed-NLS and 5GFP-NLS1 (A), 5GFP-NLS2K (B), or 5GFP-NLS2R (C). The cells were visualized by confocal microscopy 24 hours post transfection. DNA was detected by staining with DAPI. Scale bar, 10 µm. DAPI, blue; 5GFP-NLSs, green; DsRed-cNLS, red. 122  Figure 4-4: DsRed-cNLS does not inhibit the nuclear import of influenza A virus NP.A) Mock transfected HeLa cells were infected with influenza A virus (strain A/X-31 H3N2) for 2 and 10 hours. B) HeLa cells were transfected with DsRed-NLS for 48 h and then infected with influenza A virus (strain A/X-31 H3N2) for 2 and 10 h. Cells were labeled with a monoclonal NP antibody. DNA was detected by DAPI staining. Scale bar, 10 µm. DAPI, blue; DsRed-cNLS, red; NP, grey. C) Quantification of Fn/c of NP and DsRed-cNLS staining from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, ***P<0.001, One-way ANOVA followed by Tukey‟s tests).  123  124   125 Figure 4-5: Transfection of 5GFP-NLS1, 5GFP-NLS2K, or 5GFP-NLS2R plasmids inhibits nuclear import of influenza A virus. A) Mock transfected HeLa cells were infected with influenza virus (strain A/X-31 H3N2) for 2 and 10 h. B) to D) HeLa cells were transfected with 5GFP-NLS1 (B), 5GFP-NLS2K (C), or 5GFP-NLS2R (D) for 48 h and then infected with influenza A virus (strain A/X-31 H3N2) for 2 and 10 h. Cells were labeled with a monoclonal NP antibody. DNA was detected by DAPI staining. Scale bar, 10 µm. DAPI, blue; 5GFP-NLSs, green; NP, red. E) Quantification of Fn/c of NP staining from confocal images using Image J Software. Fluorescence ratios of at least 85 different cells were quantified in each condition and the mean of these ratios was generated as the value of one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.05 and ***P<0.001, One-way ANOVA followed by Tukey‟s tests).    126 4.3 Discussion In this chapter, I first generated mutant viruses containing mutated NLS1 or NLS2 using reverse genetics to examine the role of these highly conserved sequences during influenza A virus infection. Using EM, I showed that viruses containing NP mutants with substitution of basic amino acids for alanine on NLS1 or NLS2 were generated using reverse genetics (Figure 4-1). However, I found that these mutant viruses exhibited no infectivity (Figure 4-2). This result indicates that NLS1 and NLS2 on NP play a critical role during influenza A viral infection. Further, I developed a competition assay by infecting cells expressing exogenous 5GFP-NLS1 or 5GFP-NLS2. Using this assay, I demonstrated that both the exogenous NLS1- and NLS2-expressed proteins inhibited the nuclear import of influenza A virus, and were therefore able to impair influenza viral infection.   In addition to analyzing the mutant viruses obtained by reverse genetics, I further conducted western blots to detect both NP and M1 in the supernatant of cells transfected with the WT and mutant NP plasmids (Figure 4-2A). I was able to observe M1 expression in all the viruses with slight differences, which could be due to different efficiency in generating viruses in each condition. Surprisingly, NP expression was relatively low in NLS2 A2 mutant, and for the NLS2 A1+2 mutant I was not able to detect a NP band. This suggests that these two mutant viruses contain less NP than other types. This may due to improper packaging of vRNP. Previous studies showed that R199 on NLS2, which is also the mutated site in NLS2 A2 and NLS2 A1+2, lies in the RNA-binding groove on NP and was shown to be involved in the incorporation of vRNA into vRNP (Li et al., 2009). This could explain my results: as NLS2 A2 and NLS2 A1+A2 mutants contain the R199 mutation, their NP protein may not successfully assemble into vRNP and  127 package into the virion; therefore, NP expression is relatively low in these two mutants.      NP, the major protein component of influenza A virus is a multi-function protein during the life cycle of the virus. It binds to the vRNA and the viral polymerases to assemble the vRNPs (Noda et al., 2006). The vRNP complex is essential for vRNA transcription, replication and virus packaging. NLSs on NP are not only important for nuclear import of vRNP, but also essential for mRNA synthesis, vRNA transcription, nucleolus accumulation, and viral replication (Ozawa et al., 2007). By generating viruses that contain either mutated NLS1 or NLS2 using a reverse genetic approach, I have demonstrated that virus containing these mutations were produced but not infectious (Figures 4-1 and 4-2), suggesting that both NLS1 and NLS2 are important for a successful infection of influenza A virus. My results are in agreement with previous studies that showed that alanine substitution of K7 and R8 on NLS1 not only disrupt nuclear localization of NP (Cros et al., 2005; Ozawa et al., 2007; Sasaki et al., 2013), but also lead to a reduction of viral mRNA synthesis (Ozawa et al., 2007).   Similar to my results with the reverse genetics system, Sasaki et al. (2013) also found that mutant virus was not generated after alanine substitution of R8 on NLS1 in the NP plasmid and transfection of this together with the other 7 influenza plasmids. Thus my results, together with those of Ozawa et al. (2007) and Sasaki et al. (2013) indicate that NLS1 plays a critical role during influenza A virus infection.   I also found that although viruses were generated by the reverse genetics approach they were not infectious when the NP plasmid contained mutations on NLS2 (Figures 4-1 and 4-2). In Chapter 3, I showed that NLS2, a weaker NLS than NLS1, could significantly promote nuclear import of  128 the 5GFP chimera protein (Figure 3-1). Besides the nuclear import function, other studies have revealed that NLS2 also plays vital roles in nucleolus accumulation, RNA binding, vRNA transcription, and, therefore, viral replication (Li et al., 2009; Ozawa et al., 2007). For example, the R199 residue on the N-terminal basic cluster of NLS2 has been shown to be involved in the binding of NP with RNA (Li et al., 2009). Ozawa et al. (2007) also demonstrated that multiple basic amino acid changes in NLS2 (R213, K214, and R216) completely impaired vRNA transcription, NP nucleolus accumulation, and viral replication. Therefore, my results together with previous studies suggest that NLS2 has an essential role during influenza A virus infection. Since both NLS1 and NLS2 are highly conserved throughout several influenza A virus strains and are critical for viral infection (Wu et al., 2007b), I propose that both NLS1 and NLS2 could serve as potential antiviral targets.   My results with the competition assay further supports the idea that the NLSs of NP are good targets for the developing of anti-influenza drugs. I showed that introducing NLS1 or NLS2 into cells interrupted the nuclear import of the influenza A viral genome during infection (Figure 4-5 B, C and D). These results suggest that NLS1 or NLS2 competed with influenza A virus vRNP for nuclear import and blocked vRNP nuclear import independently. It also clearly indicates that not only NLS1, but also NLS2 is important for the nuclear import of vRNP during influenza A virus infection, as either NLS1 or NLS2 inhibited nuclear import of vRNP.   In Chapter 3, I have demonstrated that similar to NP (Sasaki et al., 2013), NLS1 and NLS2 interacted with importin 1, 3, 5, and 7. Thus, since NLS1 and NLS2 facilitated nuclear import using the same nuclear import receptors as NP, adding either exogenous NLS1 or NLS2  129 during infection would bind to 1, 3, 5, and 7, making these import receptors unavailable to mediate the nuclear import of vRNPs, and therefore inhibiting influenza A virus infection.   In order to confirm that exogenous NLS1 or NLS2 did not compete with cellular cNLSs during my competition assays, and that the nuclear import of vRNPs was not affected by cellular cNLSs, I also performed competition assays between NLS1 or NLS2 with the monopartite cNLS from SV40 Tag (PKKKRKV), and determined whether this cNLS inhibited the nuclear import of influenza A vRNPs during infection. My results showed that the SV40 Tag cNLS neither inhibited NLS1- or NLS2-facilitated nuclear import nor impaired the entry of the vRNP into the nucleus (Figures 4-3 and 4-4). Previous studies have shown that similar to NLS1, NLS2, and NP, SV40 Tag cNLS interacts with importin 1, 3, and 5 (Melen et al., 2003; Miyamoto et al., 1997; Nadler et al., 1997; Sekimoto et al., 1997). Interestingly, Melen et al. (2003) showed that although SV40 Tag cNLS and NP bind to the same importin  isoforms, interaction with importin  was through different sites. Specifically, SV40 Tag cNLS was shown to interact with importin  through its major site (ARM 2-4), whereas NP exhibited binding to importin  through its minor site (ARM 7 and 8) (Melen et al., 2003). Since SV40 Tag cNLS did not interfere with the NLS1- nor NLS2-mediate nuclear import of 5GFP-NLS1/2 or vRNP, my results confirm that NLS1 and NLS2 could be good targets for anti-influenza drugs, because cellular NLSs might not interfere with NLS1/2 function.  Competition assays to test the function of NLS1 or NLS2 for nuclear import has been previously published Cros et al. (2005); Wu et al. (2007a) are in agreement with my competition results. Cros et al. (2005) showed that NLS1 peptide competed with bacterialy-expressed GFP-NP for  130 import receptors in digitonin-permeablized cells, and thereby inhibited NP nuclear import (Cros et al., 2005); Wu et al. (2007a) documented that adding NLS1 or NLS2 peptides to digitonin-permeablized cells reduced the nuclear accumulation of chemically purified and fluorescently-labeled vRNPs. As these competition assays were performed using bacterialy expressed NP or isolated vRNP digitonin-permeablized cells, it is uncertain whether these may contain conformational changes compare to vRNP or NP inside virions. Nevertheless, I obtained similar results as those published (Cros et al., 2005); Wu et al. (2007a) by introducing exogenous NLS1 or NLS2 to compete with vRNP during infection in intact cell, confirming the role of NLS1 and NLS2 during a real influenza virus infection.   In summary, my findings indicate that NLS1 and NLS2 are important for nuclear import and viral production during influenza A virus infection and could serve as potential targets for antiviral drug development. This study also confirms the important role of NLS2 that I found in Chapter 3, and indicate that NLS2 should not be neglected.  131 Chapter 5 Characterization of the Role of Vimentin During Influenza A Virus Infection  5.1 Introduction  Influenza A virus hijacks the endocytic pathway during the early stages of infection. This pathway is very dynamic, and distinct endosome populations are involved. Early endosomes (EEs) are the main sorting station for incoming ligands, and they subsequently mature to late endosomes (LEs) (Huotari and Helenius, 2011). During endosome maturation, the Rab7 GTPase is recruited to LEs to replace Rab5 through a Rab conversion process (Rink et al., 2005). Subsequently, LEs acquire lysosomal components (e.g., lysosome (LY)-associated membrane proteins (LAMPs)), experience a drop in their luminal pH (from ~6.5 to ~5.0), and move to the perinuclear region (Huotari and Helenius, 2011). The lower pH in LEs is critical during influenza A virus infection, as it triggers the conformational change of HA, which in turn mediates the membrane fusion between endosomal and viral membranes. At the same time, the lower pH also activates the M2 ion channel to pump protons into the interior of the virion, triggering the dissociation between vRNP and M1 (Section 1.2.3). These two processes are necessary for the release of vRNPs in the cytoplasm and their subsequent import into the nucleus.  During influenza A virus infection, actin filaments and microtubules facilitate viral internalization and endosomes travelling towards the perinuclear region (Section 1.2.4) (Lakadamyali et al., 2003; Sun and Whittaker, 2007). However, the involvement of vimentin intermediate filaments during influenza viral infection has not being studied in detail. Although, Arcangeletti et al. (1997) showed that vimentin is rearranged during influenza A virus infection, and that acrylamide  132 treatment, which disrupts the vimentin network, impaired virus production. More recently, Mayer et al. (2007) identified vimentin as a cellular protein that immunoprecipitated with vRNP using mass spectrometry analysis. Thus, to completely understanding the infectious cycle of influenza A virus it is necessary to determine the role of vimentin IF during influenza infection.   Previous research showed that vimentin plays important roles in vesicular membrane traffic (Styers et al., 2005). Vimentin filaments are required for cellular transport of endosomes, lysosomes, and organelles, and for positioning of organelles of the endocytic pathway. The membrane adaptor complex AP3 interacts with vimentin and assists in vesicle formation, and vesicle budding and uncoating. In vimentin-deficient fibroblasts, LE and lysosome reporter molecules were redistributed (Styers et al., 2005). In addition, an increased pH of endocytic organelles was detected with lysosensor (Styers et al., 2004). As an acidic environment (pH 5.5) in LEs is critical for influenza A virus membrane fusion with the endosomal membrane to release the vRNPs in the cytoplasm, I hypothesize that vimentin could be involved in the release of vPNPs from LEs, and has thereby an important role during influenza A virus infection.   In this study, I first determined whether vimentin is involved during influenza A virus infection by detecting differences in viral production between cells with and without vimentin. I found a significant inhibition of influenza A viral production in vimentin null cells and cells transiently depleted of vimentin by RNAi. To investigate in which step of the influenza A virus infection vimentin is involved, I dissected the endocytic pathway during infection and compared the distribution and pH of EEs and LEs in cells with and without vimentin. My results suggest that vimentin is required during the escape of influenza A virus from late endosomes.  133 5.2 Results  5.2.1 Vimentin is required for efficient influenza A virus infection  In a proteomic study, vimentin emerged as one of the proteins that could interact with vRNP (Mayer et al., 2007). To validate the involvement of vimentin during influenza A virus infection, I first examined infectivity and viral production using a vimentin-/- mouse embryonic fibroblast (MEF) cell line (Colucci-Guyon et al., 1994). A wild type MEF cell line (termed here vimentin+/+) was used as a control. Cells were infected with influemza A virus, and the production of M1 mRNA and expression of viral M1 were analyzed at different time points after infection by qRT-PCR and western blot, respectively. I found a significant decrease of viral M1 mRNA and M1 expression in vimentin-/- cells compared to vimentin+/+ cells (Figures 5-1 and 5-2). In vimentin+/+ cells, I observed a rapid elevation of viral M1 mRNA and M1 starting at 2 h p.i., which indicates that the virus had successfully entered the cell and started transcription as early as 2 h p.i. However, in vimentin-/- cells, the expression of M1 mRNA showed subtle changes in the first 5 h p.i., and reached the relative maximum level by 10 h p.i., which was still 20 times lower compared to M1 mRNA expression in vimentin+/+ cells at same time (Figure 5-1). Moreover, both NP and M1 protein expression also exhibited significant differences between vimentin+/+ and vimentin-/- cells (Figure 5-2). There were subtle changes in both NP and M1 protein expression during the first 5 h p.i. in vimentin-/- cells (Figure 5-2B). NP and M1 protein levels reached their relative maximum by 10 h p.i. in vimentin-/- cells (Figure 5-2, A and B), which were also significantly lower compared to vimentin+/+ cells at same time p.i.   To examine whether the presence of vimentin affects the production of infectious progeny virions in cells, we infected vimentin+/+ and vimentin-/- cells with influenza A virus for 17 h and harvested  134 the supernatant for plaque assays. As documented in Figure 5-3, significantly lower amounts of progeny virions were produced in vimentin-/- cells compared to that in vimentin+/+ cells (2.18x104 pfu/ml versus 6.04x104 pfu/ml). This result indicates that the production of virus was inhibited in vimentin deficient cells.   Taken together, our results suggest that vimentin is involved in influenza A virus infection as viral infectivity and production was dramatically inhibited in vimentin-/- cells. Because defects in M1 mRNA expression were observed at early time points (2 h p.i and 5 h p.i), vimentin may play a role during the early stages of influenza A virus infection.  135  Figure 5-1: Viral M1 mRNA expression decreased in vimintin null cells. Vimentin+/+ and vimentin-/- cells were incubated with influenza A virus (MOI of 1) for 15 min on ice to allow the virus to bind to the cell surface. Cells were then rinsed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37 C in a CO2 incubator for different time periods. To analyze the production of M1 mRNA, RNA was extracted and cDNA was synthesized. Real-time PCR was performed using primers directed against M1. Mouse gapdh was used as a reference gene. Shown are the mean values and S.E.M measured for three independent experiments (n=3).     136  Figure 5-2: The viral M1 and NP protein expression was attenuated in vimentin null cells. Vimentin+/+ and vimentin-/- cells were incubated with influenza A virus (MOI of 1) for 15 min on ice to allow the virus to bind to the cell surface. Cells were then washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37 C in a CO2 incubator for different time periods. The cells were then harvested and the whole cell lysates were subjected to western blot. A) M1, NP, GAPDH, and vimentin protein expressions were detected using anti-M1, anti-NP, anti-GAPDH, and anti-vimentin antibodies. B) Quantification of the relative density of M1 and NP bands from the western blots. The intensity of M1 and NP bands were measured using ImageJ and normalized with the relative intensity of GAPDH bands. Shown are the mean values and S.E.M measured for three independent experiments (n=3).   137   Figure 5-3: Influenza viral progeny production was significantly decreased in vimentin-/- cells. Vimentin+/+ and vimentin-/- cells were incubated with influenza A virus (MOI of 1) for 15 min on ice to allow the virus to bind to the cell surface. The cells were rinsed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37 C in a CO2 incubator for 17 hours. Supernatant was harvested and viral titers were determined using plaque assays. A) Plaques on MDCK cell monolayers were stained with crystal violet. The picture showed three replicates of the plaque assay using 10-2 dilution of the supernatant from infected vimentin+/+ and vimentin-/- cells. B) The infectious viral titer in the supernatant was calculated by plaque assay results. PFU = plaque forming units. Shown are the mean values and S.E.M measured for three independent experiments (n=3, ***P<0.001, Student‟s t-test).       138 5.2.2 The distribution and morphology of late endosomes and lysosomes are altered in both vimentin-/- cells and vimentin depleted HeLa cells. Since endosomal organelles are mislocalized in vimentin null cells (Styers et al., 2005), and influenza A virus uses the endocytic pathway during infection I first investigated whether these defects contributed to the decrease of influenza A virus infection that were observed in the vimentin null cells. For this, I first determined the cellular position of EEs, LEs, and LYs by immunolabeling of these organelles in both vimentin+/+ and vimentin-/- cells. EEs were immunostained with an EE antigen 1 (EEA1) antibody, LEs were detected using an antibody against the small GTPase Rab7 antibody, and the lysosomal marker LAMP1 was used to localize lysosomes. As illustrated in Figure 5-4A, EEA1 showed the characteristic punctuated cytoplasmic staining in both vimentin-/- cells and vimentin+/+ cells, and the distribution of EEA1 was identical in both cell types. However, the sizes of EEA1-stained vesicles were significantly larger in vimentin-/- cells compare to vimentin+/+ cells (Figure 5-4D). The average EE vesicle area was 0.120  0.004 m2 for vimentin+/+ cells and 0.160  0.002 m2 for vimentin-/-cells.   In contrast to the unchanged cellular position of EEA1, Rab7 was remarkably mislocalized in vimentin-/- cells. As illustrated in Figure 5-4B, Rab7-stained organnels were found all around the nucleus in vimentin+/+ cells, but were accumulated on one side of the nucleus in vimentin-/- cells. Quantification of the percentage of cells yielding asymmetric perinuclear accumulation of Rab7 showed that there was a significant increase of cells showing this pattern in vimentin-/- cells (Figure 5-4E). The percentage of cells showing perinuclear accumulation of Rab7 at one side of the nucleus was 56.83%  3.65% in vimentin-/- cells compared to 9%  4.93% in vimentin+/+ cells (Figure 5-4E). Moreover, the size of LEs was significantly increased in vimentin-/-cells compared  139 with vimentin+/+ cells, as revealed by quantification of their area (Figure 5-4D). The average LE vesicle area in vimentin+/+ cells and vimentin-/- cells was 0.15  0.01 m2 and 0.25  0.02 m2, respectively (Figure 5-4D).   Immunolabeling with the anti-LAMP1 antibody also revealed differences between the vimentin+/+ cells and vimentin-/- cells. As documented in Figure 5-4, C and D, the lysosomal antigen LAMP1 showed a punctate cytoplasmic staining in vimentin+/+ cells, while they were compactly localized at one side of the perinuclear region in vimentin-/- cells. Indeed, quantification of the percentage of cells yielding asymmetric perinuclear accumulation of LAMP1 showed that there was a significant increase in the number of cells that show this pattern in vimentin-/- cells (Figure 5-4, C and D). The percentage of cells showing perinuclear accumulation of LAMPI at one side of the nucleus was 66%  8% in vimentin-/- cells compared to 17%  3% in vimentin+/+ cells (Figure 5-4E).  140   141      142 Figure 5-4: The distribution of late endosomes and lysosomes, but not early endosomes is modified in vimentin null cells. Vimentin+/+ and vimentin-/- cells were seeded on coverslips 24 h prior to fixing. The coverslips were subjected to fixation and processed for immunofluorescence confocal microscopy. Cells were stained for EEA1 (A), Rab7 (B), and LAMPI (C). DNA was detected by staining with DAPI. Alexa Fluor 647 phalloidin was used for actin filaments staining. Scale bar, 10 µm. DAPI, blue; endosomal and lysosomal markers, green; Alexa Fluor 647 phalloidin, grey. D) Quantification of EEA1 and Rab7 labeled endosome vesicle area in vimentin+/+ and vimentin-/- cells using Image J software. E) Quantification of the proportion of cells showing accumulation of Rab7 and LAMP1 immunostaining at the perinuclear region as a percentage of total cells. For the quantification in (D) and (E), at least 85 cells were scored for each condition and the mean value was generated for one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, **P<0.01, Student‟s t-test). 143 In order to confirm that the changes of endosomes and lysosomes are caused by vimentin, a transient knockdown study using vimentin specific siRNA was carried out in HeLa cells. As documented in Figure 5-5 A, the expression level of vimentin was substantially reduced in cells treated with 50 nM siRNA. Next, the siRNA vimentin-depleted cells and cells treated with non-targeting siRNA (as control) were imunostaining for EEA1, RAb7, and LAMP1. EEs exhibited a punctate staining pattern in vimentin siRNA treated-cells, similar to the pattern observed in non-targeting siRNA treated-cells (Figure 5-5B). Similar to vimentin-/- cells, I also observed enlargement of EEs in the vimentin depleted-cells. The average EE vesicle area was 0.11  0.01 m2 and 0.16 0.01 m2 in control cells and vimentin depleted cells respectively (Figure 5-5E).   The vimentin siRNA transfected-cells also showed the same defects in Rab7 and LAMP1 localization as the vimentin-/- cells. The staining of Rab7 accumulated on one side of the nucleus after vimentin RNAi depletion (Figure 5-5C). The percentage of cells showing perinuclear accumulation of Rab7 at one side of the nucleus was 61.08%  4.46% in vimentin depeleted cells compared to 15.67%  2.24% in non-targetting siRNA treated cells (Figure 5-5F). In addition, similar to vimentin-/- cells, the size of LEs was also increased in vimentin siRNA transfected-cells compared with non-targetting siRNA treated-cells (Figure 5-5E). The average LE vesicle area was 0.110  0.004 m2 in control cells and 0.18  0.01 m2 in siRNA vimentin-depleted cells (Figure 5-5E). After siRNA depletion of cellular vimentin, LAMP1 was also shown redistributed and more accumulated at one side of juxtanuclear region (Fig. 5-5D), which is similar to vimentin-/- cells. The percentage of cells with LAMP1 accumulation on one side of the nucleus  144 was 10%  1% for the control cells and 78%  3% for the vimentin siRNA transfected-cells (Figure 5-5F).   Taken together, our results show that morphological as well as positional alterations of both LEs and LYs occur with vimentin deficiency in our cell models. These results indicate that vimentin is important for endosomal trafficking and processing of molecular cargo in the endo-lysosomal maturation pathway. 145   146  147 Figure 5-5: Knockdown of vimentin modifies late endosomal and lysosomal distribution, but not early endosomal distribution. HeLa cells were transfected with 50 nM of non-targeting siRNA or vimentin siRNA for 72 h. A) Western blot of cell lysates from siRNA tranfected HeLa cells. GAPDH was used as loading control. B) Confocal images of siRNA transfected HeLa cells immunolabeled with an anti-vimentin antibody and EEA1 (B), Rab7 (C) or LAMP1 (D). DNA was detected by DAPI staining. Scale bar, 10 µm. DAPI, blue; endosomal and lysosomal markers, green; vimentin, red. E) Quantification of EEA1 and Rab7 labeled endosome vesicle area using Image J software. F) Quantification of the proportion of cells showing accumulation of Rab7 and LAMP1 immunostaining at the perinuclear region on one side of the nucleus as a percentage of total vimentin-depleted cells. For the quantification in (E) and (F), at least 85 different cells were quantified in each condition and a mean value was generated for one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, **P<0.01, Student‟s t-test).  148 5.2.3 Vimentin is required for the escape of the influenza genome from late endosomes  Influenza virus utilizes the cellular endocytosis pathway to reach LE, and endosomal trafficking and maturation play an important role during the release of the influenza genome from endosomes.  As we described above, influenza A virus replication is significantly reduced in vimentin-/-cells, and these cells have defects on LE and LY organization. Based on these results, we sought to examine whether the defect on LE organization in vimentin-/-cells is the cause of the reduced influenza A virus replication in vimentin-/-cells. For this purpose, I first performed an immunofluorescence microscopy colocalization study of EE and LE markers with influenza NP protein in influenza A virus infected vimentin+/+ and vimentin-/-cells. The NP antibody used labeled not only soluble NP, but also NP on vRNPs. The detailed staining of NP with EEA1 or Rab7 for all time points were shown in Figures A2-A5 in the Appendix, in Figures 5-6 and 5-7 I selected to show only the time points that illustrate the sequential events happening during influenza A virus infection. Quantification was also performed to compare the colocation of NP with EEA1 or Rab7 in vimentin+/+ and vimentin-/- cells (Figure 5-8). As illustrated in Figure 5-6, in influenza infected vimentin+/+ cells, NP was shown at the cell surface at 15 min p.i. At 30 min p.i., NP was highly colocalized with EEA1, an early endosome marker (Figure 5-8A). At 1 h p.i some NP was colocalized with Rab7, a marker for LE (Figure 5-8B). However, at 2 and 5 h p.i., NP was located in the nucleus, indicating that vRNPs were released from LEs and entered the nucleus for viral transcription and replication, and NP was translated in the cytoplasm and transported back into the nucleus for vRNP assembling. Finally, by 7 h p.i, NP was mostly located in the cytoplasm, indicating that most vRNPs were exported to the cytoplasm.  The results of the immunofluorescence microscopy colocalization study of EE and LE markers  149 with influenza NP for vimentin-/-cells are documented in Figure 5-7. Similar to the vimentin+/+cells, NP was located at the cell surface in vimentin-/- cells at 15 min. However, at 30 min p.i., NP was still mostly localized at the cell surface in vimentin-/- cells (Figure 5-7). Only 8% colocalization of NP and EEA1 was observed (Figure 5-8A). This is in contrast to the vimentin+/+ cells, in which NP was highly colocalized with EEs at 30 min p.i. (Figure 5-8A), and indicates a delay in influenza A virus uptake in vimentin-/- cells compare to in vimentin+/+ cells. By 1 h p.i., NP was found highly colocalized with EEA1 in the vimentin-/- cells (Figures 5-7 and 5-8A). In contrast to vimentin+/+ cells, vimentin-/- cells exhibited enlarged early endosomal staining at 1 h p.i.. Most vRNPs were found in LEs by 2 h p.i., as was identified by co-immunolabeling of Rab7 and NP protein (Figure 5-8B). At 5 h p.i., while NP have already been translated and progeny NP was located in the nucleus in vimentin+/+ cells (Figure 5-6), vRNPs still remained in LEs in vimentin-/- cells (Figures 5-7 and 5-8B). In addition, we observed enlarged late endosomal compartments distributed throughout the cytoplasm at both 2 and 5 h p.i. in the vimentin-/- cells. By 7 h p.i., the vimentin-/- cells had most of NP label at the perinuclear region in what appeared to be virus-containing vesicles, some of which were positive for Rab7 (Figures 5-7 and 5-8B).  These results suggest that the lack of vimentin leads to accumulation of influenza A virus within Rab7 positive LEs. The morphology changes of EE and LE during influenza A virus infection also indicate that there could be a defect in the endosome maturation process in vimentin-/- cells.  150   151 Figure 5-6: Sequential subcellular events of NP during influenza A virus infection in vimentin+/+ MEFs. Vimentin+/+ cells were seeded on glass coverslips 24 h prior to infection. Cells were incubated with influenza A virus (MOI = 2) for 15 min on ice to allow virus binding to the cell surface. The cells were washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37C in a CO2 incubator for different time periods. The cells were probed with anti-NP antibody and anti-EEA1 or anti-Rab7 antibody. DNA was detected with DAPI staining. Scale bar, 10 µm. Zoom images showed high magnification of areas in squares of merge images. Scale bar, 2 µm. DAPI, blue; EEA1 or Rab7, green; influenza NP, red. All experiments were repeated three times and representative confocal microscopy images are shown. 152  153 Figure 5-7: Sequential subcellular events of NP during influenza A virus infection in vimentin-/- MEFs. Vimentin-/- cells were seeded on glass coverslips 24 hours prior to infection. Cells were incubated with influenza A virus (MOI = 2) for 15 min on ice to allow virus binding to cell surface. The cells were washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37C in a CO2 incubator for different time periods. The cells were probed with anti-NP antibody and anti-EEA1 or anti- Rab7 antibody. DNA was detected with DAPI staining. Scale bar, 10 µm. Zoom images showed high magnification of areas in squares of merge images. Scale bar, 2 µm. DAPI, blue; EEA1 or Rab7, green; influenza NP, red. All experiments were repeated three times and represented confocal microscopy images are shown. 154  Figure 5-8: Colocalization analysis of NP and EEA1 or Rab7 during influenza A virus infection. Quantification of the percentage of NP colocalized with EEA1 (A) or Rab7 (B) in vimentin+/+ and vimentin-/- cells for the experiments shown in Figures 5-6 and 5-7. Colocalization was measured using Image J JACop plugin. At least 85 cells were quantified in each condition and the mean value was generated for one experiment. Shown are the mean and standard error of S.E.M analyzed from the values of three independent experiments.    155 5.2.4 Lack of vimentin affects the endosomal pH In Section 5.2.3, I found out that vRNPs were trapped in LEs in vimentin-/- cells. As influenza A virus has a strict low pH requirement for their vRNPs to escape from LEs, I hypothesize that vimentin-/- cells could have modification in the pH of the lumen of LEs. In order to test this hypothesis, we followed the traffic of a pH-sensitive fluorescence conjugated EGF (pHrodo-EGF). Similar to influenza A virus, EGF follows the clathrin-mediated endocytosis pathway similar to influenza A virus, as it is directed to LEs and reached LYs for degradation (Lamaze et al., 1993). Previous studies demonstrated that pHrodo-EGF is weakly fluorescent outside of cells at neutral pH, but after internalized through endocytosis, it becomes brightly fluorescent in the acidic endosomes (Suprynowicz et al., 2010). Therefore, using pHrodo-EGF could indicate whether there are differences in the pH of organelles of the clathrin-mediated endocytic pathway between vimentin+/+ and vimentin-/- cells.   In order to monitor the traffic of EGF, we use Alexa Fluor 488-conjugated EGF to track the movement of EGF. Concurrently, pHrodo EGF was added together with Alexa Fluor 488 EGF to visualize the acidification inside the EGF-containing endosomes. For this analysis, both EGF were bound to serum-starved vimentin+/+ and vimentin-/- cells at 4 C, followed by washing off the unbound EGF and transferred to 37 C. After 30 min, a punctate pattern of intracellular green and red fluorescence was observed in vimentin+/+ cells, which is consistent with endocytosis of Alexa Fluor 488 EGF and acidification of EGF-containing endosomes (Figure 5-9A). The complete merging of red and green fluorescence was visualized and quantified at 60 and 90 min, which indicates that both pHrodo EGF and Alexa Fluor 488 EGF were localized to the same endosomal compartment, which properly acidified (Figure 5-9A). In contrast, in vimentin-/- cells,  156 the prebound pHrodo EGF did not become fluorescent after warming the cells to 37 C for 30 min, indicating that endosome acidification did not occur at this time point. At 60 and 90 min, the number of endosomes exhibiting fluorescence in vimentin-/- cells was significantly lower than that in vimentin+/+ cells (Figure 5-9B). This result suggests that the acidification in endosomes of vimentin-null cells was significantly impaired, which could cause influenza A virus accumulation inside the LEs.   Since influenza A virus could enter cells through clathrin-mediated endocytosis and macropinocytosis, we also studied dextran uptake, which enters the cell via macropinocytosis (Rossman et al., 2012) in vimentin+/+ and vimentin-/- cells. Since macropinocytosis is mediated by actin that ruffles the plasma membrane (Lim and Gleeson, 2011), we would not expect an dextran uptake deficiency in vimentin-null cells. As illustrated in Figure 5-10A, at 40 min after dextran incubation, Alexa Fluor 488 dextran showed a punctate pattern in both vimentin+/+ and vimentin-/- cells. As expected, I did not observe difference in uptake efficiency between these two cell lines (Figure 5-10B). However, there was a general increased in dextran-positive vesicles in vimentin-/- cells compare to vimentin+/+ cells (Figure 5-10A). The average vesicle size in vimentin-/- cells was 0.18  0.02 m2, whereas this number was 0.12  0.01 m2 in vimentin+/+ cells (Figure 5-10B). Since dextran need to reach LEs /LYs for degradation, the enlarged vesicle might represent LEs and LYs.         157  158  Figure 5-9: The acidification of EGF-containing endosome is inhibited in vimentin-/- fibroblast cells. A) Trafficking and acidification of endosome in live vimentin+/+ and vimentin-/- cells after cellular uptake of Alexa Fluor 488 EGF and pHrodo EGF were examined at different time points (30 min, 60 min, and 90 min). Scale bar, 10 μm. NucBlue live ready probe reagent, blue; Alexa Fluor 488 EGF complex, green; pHrodo-red-conjugated EGF, red. B) Quantification of the percentage of acidified endosomes in vimentin+/+ and vimentin-/- cells using Image J software. At least 85 cells were quantified in each condition and the mean value was generated for one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments.   159  Figure 5-10: The enlargement of endo/lysosomal compartment during dextran uptake in vimentin-/- fibroblast cells. Vimentin+/+ and vimentin-/- cells were treated with 0.5 mg/ml Alexa Fluor 488 dextran for 30 mins at 37C. A) Confocal image of dextran uptake in live vimentin+/+ and vimentin-/- cells. NucBlue live ready probe reagent, blue; Alexa Fluor 488 dextran (10 kDa), green. B) Quantification of dextran uptake efficiency and vesicle size of dextran-containing endosomes or LYs using Image J software. At least 85 cells were quantified in each condition and the mean value was generated for one experiment. Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, *P<0.1, Student‟s t-test). 160 5.3 Discussion Here, we systematically studied the role of vimentin during influenza A virus infection. We observed that distribution and morphology of EE, LE, and LY compartments changed in vimentin null cells. The improper acidification in endocytic compartments that happens in cells lacking vimentin further resulted in the trapping of influenza vRNPs in LE compartments, which resulted in reductions in influenza A virus genome expression, and viral progeny production. These data indicate that vimentin plays important roles in endocytic trafficking, maturation, and acidification, which further regulate influenza A virus infection.   During influenza A virus infection, (Arcangeletti et al., 1997) reported that the vimentin network is rearranged around the nucleus at 8 h p.i. and completely collaped around the nucleus by 16 h p.i.. By immunolabeling NP and organelles of the endocytic pathway I found that there was a defect in the endocytic trafficking of influenza A virus in vimentin-null cells (Figure 5-7). Through immunofluorescence staining, I also observed an alteration in LEs and LYs distribution (Figures 5-3 and 5-4). Since vimentin interacts with the clathrin adaptor-like AP-3, which regulates sorting of membrane proteins into vesicles and carries vesicles between endosomal-lysosomal compartments (Styers et al., 2004), it is not surprised that the LEs and LYs were mislocalized in vimentin-null cells. This also explains why the distributions of virus-containing endosomes were different during influenza A virus infection.   Besides changes in the localization of endo/lysosomal compartments, I also observed the enlargement of EEs, LEs, and lysosomes in cells lacking vimentin (Figures 5-4 and 5-5). During  161 endocytic trafficking, EEs undergo fusion and fission to mature into LEs, which then fuse with lysosomes for cargo degradation (Huotari and Helenius, 2011). There is a constant influx and efflux of membrane happening during the fusion and fission process. While the fusion process causes enlargement of endosome vesicles, the fission process recovers endosome vesicles to its normal sizes (Gruenberg and Maxfield, 1995). Previous studied showed that MTs are critical for this fusion and fission process. For example, nocodazole, a MT depolymerizing drug, induces the enlargement of EEs due to an increased fusion rate and an impaired tubular fission rate (Skjeldal et al., 2012). Similarly, treating cells with the actin filament depolymerizing drugs, Latruncluin B or Jasplakinolide, induced enlarged EEs (Ohashi et al., 2011) and LEs (Drengk et al., 2003), further blocking endosomal transport and movement. Since the vimentin network is dynamically integrated with MTs and actin filaments through several motor proteins and plectins (Section 1.4), it is highly possible that the fission process is inhibited in cells lacking vimentin, which would lead to the enlargement of EEs and LEs.   During influenza A virus infection, I observed that the virus particles accumulated in Rab7-positive vesicles in vimentin-null cells. However, uncoating of the virion and transport of vRNPs to the nucleus did not occur (Figure 5-7), and, as a consequence, the viral protein expression and viral progeny production were significantly impaired (Figures 5-1, 5-2, and 5-3). I hypothesized that the accumulation of viral particles in Rab7-positive LEs may be due to improper acidification inside the virus-containing endosomes. Using a pH-sensitive EGF conjugate, pHrodo-EGF, together with Alexa Fluor 488 EGF, we were able to monitor the endosome trafficking and acidification. As a result, we observed an inhibition in endosome acidification in vimentin-null cells (Figure 5-8). These data show that the luminal pH dynamics of endocytic compartments is impaired in vimentin-null cells.  162 The specific low pH in LEs is also critical for the infection of many other viruses. For example, adenovirus enters cells by clathrin-mediated endocytosis, and the pH inside the endosome plays an important role in inducing conformational changes in the viral particle, which is critical for efficient infection (Scherer and Vallee, 2011). Dengue virus uses the anionic lipid membrane of LE for low pH-dependent fusion during infection (Zaitseva et al., 2010). Parvoviruses use receptor-mediated endocytosis for host cell entry and is internalized in endosomes, where the low pH triggers their release in the cytoplasm (Vihinen-Ranta et al., 2004). Interestingly, Fay and Pante (2013) also observed reduced parvovirus replication in vimentin null cells, which also suggested that vimentin could play a role in MVM escaping from endosomes during infection. Taken together, my results and previous studies suggest that vimentin could be involved in general in the infection cycle of viruses that require low endosomal pH during infection.   In summary, we have documented a previously unsuspected role of vimentin during influenza A virus infection. Our results showed that influenza A virus could not efficiently replicate in vimentin-null cells. Further investigations suggested endosome positioning and acidification were defective in these cells, which led to the accumulation of influenza A virus in LE vesicles.  163 Chapter 6 General Discussion and Future Perspectives  The early stages of influenza A viral infection before viral replication includes several critical steps. These are: 1) virus binding to the cell surface, 2) cellular uptake of incoming virus by endocytosis, 3) actin filament- and microtubule-mediated trafficking of virus-containing endosomes towards the nucleus, 4) release of vRNPs from LEs through fusion of the viral and endosomal membranes triggered by the acidity of LEs, and 4) nuclear import of vRNPs. Although the molecular mechanisms of some of these steps have been very well characterized, in particular the viral binding to their target cells and the escape of vRNPs from LEs, other steps, such as the details of the nuclear import of the vRNPs and whether the intermediate filaments are involved in the infectious cycle of the virus are less characterized. In this thesis, I investigated the role of vimentin intermediate filaments during influenza A virus infection, and characterized the contribution of the NLS2 of NP to nuclear import. My research objectives as listed in Chapter 1 include characterizing the function of NLS2 (Chapter 3), identifying the cellular binding partners of NLS2 during nuclear import (Chapter 3), determining whether NLSs on influenza A virus NP could be antiviral targets (Chapter 4), and identifying the role of vimentin during influenza infection (Chapter 5). In the following pages I discuss my results for each of these objectives.   6.1 Towards dissecting the function of NLS2 Nuclear import of influenza vRNPs is a critical step in the influenza A viral infections cycle, and this study is the first one to reveal the role of NLS2 of NP in promoting nuclear import and to identify the amino acid on this NLS necessary for this process. As shown in Chapter 2, NLS2 can  164 significantly facilitate nuclear import of 5GFP, although it is less efficient than NLS1. In addition, the competition studies described in Chapter 3 indicated that NLS2 is important in the infection cycle of influenza A virus as 5GFP-NLS2 is able to compete with the NLSs of influenza A virus NP for importin s, and inhibit influenza infection.   NLSs on NP facilitate nuclear import twice during infection. First, when incoming vRNPs are released from endosomes into the cytoplasm, they utilize the NLSs of NP to gain access to the nucleus for viral replication. Second, after NP is synthesized in the cytoplasm during infection, NP is transported into the nucleus using its NLSs in order to assemble progeny vRNPs. After that, the progeny vRNPs are exported into the cytoplasm, where the NLSs become hidden (Wu and Pante, 2009). Therefore, progeny vRNPs are unable to return to the nucleus. NLS1 and NLS2 on NP can facilitate nuclear import of vRNP independently (Wu et al., 2007a). NLS1 is located on the N-terminus of NP and is highly exposed on vRNP. NLS2, on the other hand, is less exposed (Wu et al., 2007b). The third obpNLS, which is located between NLS1 and NLS2 of only 9 influenza A virus strains, was shown to efficiently mediate nuclear import of GFP (Ketha and Atreya, 2008). Having multiple NLSs on its RNPs is important for an effective influenza A virus infection. For example, if the N-terminal NLS1 is cleaved off by host proteases during infection, the other NLSs could still functionally promote nuclear import of vRNPs.  Sequence alignments between different strains of influenza A virus revealed that two subtypes of NLS2 exist, which have a basic amino acid difference at position 17: NLS2K has a lysine, while NLS2R has an arginine. I found that the nuclear import efficiency was different between NLS2K and NLS2R (Chapter 3). Interestingly, all the highly pathogenic influenza A virus strains I  165 selected for sequence alignments contain NLS2R on NP, while most of the seasonal flu strains have NLS2K on NP. This is surprising since NLS2R is significantly less efficient in promoting nuclear import of 5GFP than NLS2K. Thus, having NLS2R on NP would not increase the efficiency of nuclear transport of vRNPs of high pathogenic influenza strains. Besides its nuclear import function, NLS2 is also important for vRNA transcription, for incorporating of NP onto vRNP, and for the nucleolus accumulation of NP (Li et al., 2009; Ozawa et al., 2007). Therefore, it will be interesting to determine whether having NLS2R instead of NLS2K will confer any differences in post-nuclear import processes, which could change the infectivity for highly pathogenic strains.        The efficiency of a NLS in promoting nuclear import of a cytoplasmic protein lies on its binding affinity to importin  and the expression level of importin  in the cells (Hodel et al., 2006; Hu and Jans, 1999; Yang et al., 2010). The classical bipartite NLS interacts with importin  through a major and a minor binding site on importin  (Section 1.1.4, Figure 1-3). The P1‟-P2‟ residues in a classical bipartite NLS are critical for the binding of the NLS to the importin  minor site while the P2-P5 residues are important for the binding of the NLS to the importin  major sites (Fontes et al., 2003; Marfori et al., 2011). For NLS2K, 1KR2 correspond to P1‟-P2‟, while 16RKTR19 correspond to P2-P5. Studies have shown that P2 is the most critical residue in the binding of a classical bipartite NLS to the major binding site of importin  (Marfori et al., 2011). Structural analysis suggested that amino acid K suits best at P2 in a NLS for the binding with threonine T155 and asparagine N192 on importin  (numbers indicating the position of amino acid on importin ). The K to R mutation at P2 would result in a decrease in binding affinity between the NLS and importin , as the R side chain is longer than the K side chain and could not  166 maintain the hydrogen bond with negatively charged residues inside the importin  major binding pocket (Colledge et al., 1986; Marfori et al., 2011). Sequence alignment of cNLSs and cNLS-like sequences suggested that K at P2 is strictly conserved among classical bipartite NLSs (Fontes et al., 2003). However, the NLS2 from influenza NP contains R instead of K at P2. Therefore, this explains why NLS2 is not very efficient in promoting nuclear import.   Post-transcriptional modifications such as phosphorylation, acetylation, ubiquitination, and small ubiquitin-like modifier modification (sumoylation) have been known to regulate nuclear-cytoplasmic trafficking of protein (reviewed by Gill, 2004; Nardozzi et al., 2010b; Sadoul et al., 2011). Phosphorylation usually occurs on serine (S), threonine (T), tyrosine (Y), or histidine (H) residues (Ciesla et al., 2011). Previous studies have shown that phosphorylation within an NLS could enhance the binding affinity of it to importin  (Alvisi et al., 2008; Nardozzi et al., 2010b). Epstrain-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is a protein that functions as a transactivator for genome transcription inside the nucleus. It was shown that phosphorylation of S385 on EBNA-1 NLS is essential for binding to importin 5 (Kitamura et al., 2006). Substitution of S385 by A or N decreased both the nuclear accumulation of EBNA-1 and the binding affinity of EBNA-1 to importin 5 (Kitamura et al., 2006; Nardozzi et al., 2010a). In my study, I showed that alanine substitution of T18 significantly reduced the nuclear accumulation of 5GFP-NLS2K, but not that of 5GFP-NLS2R. Thus, a possible explanation of this phenomenon is that T18 at the C-terminal of NLS2K could be phosphorylated, leading to its nuclear accumulation while the phosphorylation may be inhibited in NLS2R due to the large side chain of neighboring amino acid R17.    167 Besides phosphorylation, other post-transcriptional modifications could also regulate nuclear import. Acetylation has been shown to regulate the cellular localization of proteins through nuclear import and export (Choudhary et al., 2009). During acetylation, the acetyltransferase transfers the acetyl group from acetyl coenzyme A to epsilon amino group on lysine residue. This action has also been suggested to regulate nuclear import by interfering with the interaction between NLS and importin /. Previous studies on the nuclear import of SRY, a Y chromosome-encoded DNA-binding protein, suggested that the acetyltransferase p300 induces the acetylation of a single lysine residue on SRY, as it is important for its nuclear import function. While acetylation promotes nuclear import of SRY by increasing its interaction with importin , deacetylation can induce cytoplasmic retention of SRY (Thevenet et al., 2004). Interestingly, acetylation of K22 on importin  has been demonstrated to promote its interaction with importin , further regulating nuclear import (Bannister et al., 2000). Other studies have also suggested that ubiquitination and sumoylation could modify lysine residue and regulate nuclear transport of cellular proteins (reviewed by Gill, 2004). Nuclear import of PTEN, a tumor suppressor, dependents on monoubiquitination of lysine residues. Amino acid substitutions at two lysine residues (K13 and K289) result in cytoplasmic accumulation of PTEN, which lead to Cowden syndrome (Trotman et al., 2007). The nuclear localization of another tumor suppressor, PML, is also modified by sumoylation and mutation of the SUMO receptor lysine in PML will alter the distribution of PML (Best et al., 2002). Recently, Han et al. (2014) showed that K7 on NLS1 of influenza A virus NP is sumoylated. This modification is important for intracellular trafficking of NP and also viral growth. In this paper, the influenza A virus strain used was A/WSN/1933 which contains NLS2R instead of NLS2K. Therefore, it is still unknown whether K17 on NLS2K is sumoylated or not. In Chapter 3, I have demonstrated that with one basic amino acid difference,  168 NLS2K showed better function in promoting nuclear import of 5GFP than NLS2R. Post-transcriptional modification of K at position 17 on NLS2K may be one of the mechanisms that lead to the higher nuclear import efficiency of NLS2K compared to NLS2R.   6.2 Role of importin  in mediating the nuclear import of NLS2-containing proteins  During the nuclear import of influenza vRNPs, importin  binds to NLSs on NP to facilitate the vRNP transport through the NPC. Previously, it was reported that NP binds to importin 1, 3, and 5 using a yeast two-hybrid and GST-pulldown assays (Melen et al., 2003; Wang et al., 1997). More recently, it has been demonstrated that NLS1 interacts with importin 1, 3, and 5, and that importin α3 is the major binding partner of NLS1 (Sasaki et al., 2013). Moreover, these authors also found that S9 on NLS1 is highly involved in the interaction between importin 3 and NLS1. My study showed that NLS2, which is located at positions 198-216 of NP, binds to importin 1, 3, 5, and 7 (Chapter 3). The siRNA depletion experiments further suggested that importin 3 is essential to promote the nuclear import of NLS2K-containing proteins, and that importin 1 is critical for facilitating the nuclear import of NLS2R-containing proteins.   Importin  specificity is important for viral and cellular protein nuclear transport. A previous study has been shown that avian influenza A virus changes importin  specificities upon infecting mammalian cells to develop a successful infection (Resa-Infante and Gabriel, 2013). For efficient viral replication, an avian virus was shown to depend on importin 3, while mammalian viruses  169 depend on importin 7 (Gabriel et al., 2011). Upon infecting mammalian cells, avian influenza virus gains mutations on PB2 and NP, leading to a switch from importin 3 to importin 7 (Gabriel et al., 2011). In addition, it has also been reported that PB2 from influenza A/Thai/KAN-1/04 (H5N1) binds poorly to importin 7, while PB2 from influenza A/Victoria/3/75 (H3N2) exhibits strong binding to importin 5 (Resa-Infante et al., 2008). Consistent with the findings described by others, I was also able to show that influenza A virus changed binding specificity for importin  isoforms. Importin 3 is important for nuclear entry mediated by NLS2K from seasonal flu strains, whereas importin 1 is vital for nuclear import facilitated by NLS2R from high pathogenic strains (Table 3-1). Since importin  isoforms were shown not only to promote nuclear entry but also viral transcription (Resa-Infante et al., 2008; Sasaki et al., 2013), importin  specificity may also affect viral transcription between viruses containing NLS2K and NLS2R. Therefore, further experiments should be performed to determine whether the importin  specificity could influence viral transcription for viruses containing NLS2K or NLS2R.   The competition between import substrates for their importin  could also regulate nuclear import efficiency. When two substrates are added together to a nuclear import assay, they would compete with each other for their own preferred importin  (Quensel et al., 2004). Previous studies and my study showed that NLSs on NP and NP protein itself could all bind to importin 1, 3, 5, and 7 (Gabriel et al., 2011; Melen et al., 2003; Sasaki et al., 2013; Wang et al., 1997). Therefore, exogenous NLSs added during influenza A virus infection could compete with influenza A virus vRNPs for binding to importin 1, 3, 5, and 7. My results showed that vRNP nuclear import was inhibited in the presence of 5GFP-NLS1 or 5GFP-NLS2, suggesting  170 that NLS1 or NLS2 are able to compete with vRNPs for importin  during nuclear import.    6.3 NLSs of NP as potential antiviral targets  At present, the major strategies used to control influenza A virus infection include vaccination and antiviral drugs. The use of vaccination requires timely production. Therefore, vaccination is of limited used in preventing emerging viral strains or in the case of a pandemic. Current drugs for influenza A virus treatment are targeted to either M2 ion channel or NA. Unfortunately, the rapid emergence of drug resistant viruses has lead to a limit use of these two types of drugs. Therefore, there is a need to identify new antiviral targets of influenza A virus that are conserved for developing antiviral treatments. Given that NLSs on NP are highly conserved, investigation into compounds that inhibit the nuclear import function of NLS1 and NLS2 may lead to a new era of influenza A virus drug developments.   So far, two compounds on the market have been reported to inhibit nuclear transport. Leptomycin B is a specific inhibitor for CRM1-mediated nuclear export (Nishi et al., 1994). Importazole, a recent discovered cell permeable compound, was found to inhibit the function of importin  by altering its interaction with RanGTP (Soderholm et al., 2011). Both of these compounds inhibit classical nuclear export and import. In Chapter 3, importazole was used to treat 5GFP-NLS1-, 5GFP-NLS2K-, or 5GFP-NLS2R-transfected cells to obtain more importin -NLSs binding complexes. After treatment of the cells with importazole, I visualized less nuclear accumulation of 5GFP in transfected cells (data not shown), indicating that importazole successfully inhibits nuclear import mediated by NLSs from influenza NP protein. However, since importazole  171 impairs all the translocations of importin  from the cytoplasm to the nucleus, it can cause strong cytotoxicity and cannot be used to treat influenza A virus infection. To obtain a better compound that could not interfere with the nuclear import of other cellular protein, further work could apply high-throughout drug screening to identify compounds that affect NLS1- and NLS2-facilitated nuclear import but not other nuclear import pathways in the host cells.  Besides small molecule compound inhibitors, peptide inhibitors can also be investigated to restrain nuclear import mediated by NLS1 and NLS2. Peptide inhibitors in general have stronger interaction with targets than small molecules compounds via protein-protein interactions. One strategy for developing peptide inhibitors for nuclear import is to use peptide sequences that have high specificity and affinity to the cargo or cargo binding protein. Previous studies have applied this strategy for designing peptide inhibitors to block nuclear import mediated by the importin / pathway. Kosugi et al. (2008) generated two peptide inhibitors, bimax1 and bimax2, with high affinities for importin  to inhibit the importin / pathway. These two inhibitors allow importin  binding to importin  in the cytoplasm and prevent importin  releasing of the cargo when it enters the nucleus. Therefore, these high-affinity peptides antagonize cargo releasing, preventing the recycling of importin  from the nucleus to the cytoplasm. Currently, many peptide inhibitors have been developed which are cell permeable. Lin et al. (1995) has generated a peptide inhibitor SN50 containing the NLS of NF-B P50 subunit conjugated with the hydrophobic domain of Kaposi fibroblast growth factor. This hydrophobic domain is known to interact with lipid bilayers and facilitate the import of synthetic peptides into cells (von Heijne, 1990). Using this peptide inhibitor, Torgerson et al. (1998) demonstrated that the nuclear import of NF-B, AP-1, NFAT, and STAT1 was attenuated. Competition of the NLS on SN50 and the  172 NLSs on NF-B, AP-1, NFAT, and STAT1 for importin  is the mechanism of this inhibition phenomenon, which could also be explored to develop peptide inhibitors for influenza A virus infection.    In my study, I demonstrated that transfection of NLS1 or NLS2 plasmids into cells could inhibit nuclear import of vRNPs during infection (Section 4.2.2). Wu et al. (2007a) also showed that NLS1 and NLS2 peptides together could hinder nuclear import of vRNP in digitonon-permeablized cells. Additionally, NLS1 and NLS2 did not inhibit the classical nuclear import pathway mediated by importin /. This suggested that a peptide inhibitor containing NLS1 and NLS2 could be developed for inhibition of influenza infection since they have high specificity in competition with the nuclear import of vRNPs but not for the nuclear import of cellular proteins. Nowadays, peptide inhibitors have been developed for cancer treatment, hormone maintaining, and viral infection (McGregor, 2008). Peptides have high specificity and affinity for targets and they are easy to synthesis. Owning to their small size, they are easy to penetrate deeper into the tissue and generally have no immunogenic effect (McGregor, 2008). Recent studies have also shown that by binding serum albumin onto peptide inhibitors, the peptides could resist to serum or tissue protease, which largely increased the stability of therapeutic peptides (Dennis et al., 2002; Subramanian et al., 2007). Besides that, lots of cell penetrating peptides has been developed and conjugated with potential therapeutic peptides to delivery into cells (reviewed by Copolovici et al., 2014). A cell penetrating peptide typically either contains high abundant numbers of positive charged amino acids in side the sequence, like TAT peptide (GRKKRRQRRRPPQ) derived from HIV1 TAT protein, or has alternative polar and non-polar amino acids in the sequence, like this model amphipathic peptide  173 (KLALKLALKALKAALKLA) (Stalmans et al., 2013). Therefore, development of a cell-penetrating NLS1 and NLS2-containing peptide could not only serve as an effective inhibitor for nuclear import of viral genome but also offer a promising future for influenza treatment.   6.4 Role of vimentin during influenza A virus infection In Chapter 5, I investigated the role of vimentin during influenza A virus infection. Taking the advantage of vimentin null MEF cells, I showed that the endosomal trafficking and acidification were compromised in these cells, which resulted in the accumulation of influenza vRNPs in the late endosomal compartments during infection. The involvement of vimentin in the endosome sorting machinery was first described during the study of vimentin-AP-3 interaction by Styers et al. (2004). These authors showed that vimentin interacts with the adaptor complex AP-3 through its 3 subunit. AP-3 is responsible for trafficking of lysosome-associated membrane proteins from EE-associate tubules to LEs and then to LYs (Peden et al., 2004) and carries vesicles between endosomal-lysosomal compartments (Robinson, 2004). In vimentin-/- cells, Styers et al. (2004) showed that AP-3 was localized at the juxtanuclear region together with LAMP1. Our results also demonstrated that both Rab7 and LAMP1 were redistributed to the perinuclear region in both vimentin-/- cells and in vimentin-depleted cells (Figure 5-4 and 5-5). These results indicate that vimentin is able to regulate the sorting function of AP-3 and to control the positioning of AP-3 as well as endo-lysosomal compartments. Interestingly, Peden et al. (2004) revealed that loss of AP-3 leads to an increased incorporation of LAMP1 and CD63 in the recycling pathway, resulting in an increased LAMP1 and CD63 over the plasma membrane. In contrast, both Styers et al. (2004) and I were not able to observe these changes in LAMP1 in  174 vimentin-null cells, which suggests that vimentin may possess the role of regulating endo-lysosomal compartments positioning independent of AP-3.   In my study, I demonstrated that the endosome acidification process was impaired in vimentin null cells. Using pHrodo-EGF, I showed that the acidification of EGF-containing endosomes was not proper. This result is in agreement with our infection result that showed that influenza vRNPs were trapped in LEs in vimentin null cells. Vimentin could interfere with endosome acidification by inhibiting the endo-lysosomal chloride channel, CIC-3 (Styers et al., 2004). Previous studies demonstrated that the V type H+-ATPase (V-ATPase) and chloride channels regulate endosomal acidification (Marshansky et al., 2002). During acidification, the V-ATPase translocates the protons from the cytoplasm into the endosomal lumen, which reduces the net pH inside the endosome. This process is accompanied with inward movement of Cl-, which is responsible for shunting the positive potential created by active H+ entry (Grabe and Oster, 2001; Nilius and Droogmans, 2003; Sonawane and Verkman, 2003). The CIC type Cl- channels are responsible for endosomal Cl- conductance in cells. CIC-3 presented in LEs and LYs is responsible for maintaining the pH of these organelles (Jentsch et al., 2002). Hara-Chikuma et al. (2005) found that endosomal acidification and Cl- accumulation were significantly compromised in CIC-3 deficient mice, suggesting that CIC-3 Cl- channels are responsible to maintain endosomal acidification. Intriguingly, the subcellular distribution of CIC-3 is regulated by AP-3. CIC-3 is packed in AP-3-derived vesicles to travel to LEs and LYs, which in turn favors acidification inside those compartments (Salazar et al., 2004). Since vimentin is able to regulate the sorting function of AP-3, it could further control the targeting of CIC-3 to endo/lysosomal compartments. Therefore, in vimentin null cells, the deficiency of endosomal acidification may be due to impaired bound between LEs/LYs membrane and CIC-3, which may be due to the disrupted  175 function of AP-3.  Besides endocytic trafficking, the roles of vimentin during other infection processes also need to be investigated. During influenza virus infection, Arcangeletti et al. (1997) observed that vimentin was rearranged to the perinuclear region and it eventually collapsed around the nucleus at 16 h p.i.. The rearrangement of vimentin is regulated by phosphorylation of its N-terminal domain (Ando et al., 1989; Chou et al., 1991; Inagaki et al., 1987). During ASFV infection, it has been demonstrated that the DNA replication of ASFV leads to phosphorylation of vimentin by calcium calmodulin-dependent protein kinese II, inducing vimentin rearrangement into a cage to prevent movement of viral components into the cytoplasm (Stefanovic et al., 2005). Phosphorylation of vimentin by Rho-associated coiled coil-containing kinase (ROCK) during DENV2 infection also induces vimentin rearrangement to the perinuclear region (Lei et al., 2013). Furthermore, the perinuclear distributed vimentin co-localizes with DENV NS1 during infection, indicating that perinuclear localized vimentin could serve as a replication site for DENV (Lei et al., 2013). Is vimentin phosphorylated during influenza infection? Wheeler et al. (1990) observed an increase in phosphorylation of vimentin following the incubation of polymorphonuclear leukocytes with influenza A virus, suggesting that influenza infection may lead to the rearrangement of vimentin. However, the specific mechanism of vimentin rearrangement after viral infection still remains to be elucidated. In my study, I have demonstrated that vimentin could regulate endocytic organelle positioning. Since influenza A virus travels along the endocytic pathway to the perinuclear region for viral genome releasing, the rearranged of vimentin during viral infection may serve as a mean for the vRNP releasing into cytoplasm. Besides that, it is still unclear whether the vRNP uses the cytoskeleton for trafficking in the cytoplasm after uncoating and before nuclear import. Martin and Helenius (1991b) have found  176 that actin and MT-depolymerizing drugs did not block the nuclear import of vRNPs, suggesting that these two types of cytoskeleton elements are not critical during vRNP transport in the cytoplasm. Since vimentin is a putative binding partner of vRNP (Mayer et al., 2007), it will be interesting to test whether the vimentin network could support vRNP transport towarts the NPC after viral uncoating.   Furthermore, whether vimentin is involved in influenza viral transcription needs to be determined. By proteomic analysis, it was found that vimentin interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 and K (Kanlaya et al., 2010). The majority of hnRNPs are localized inside the nucleus and some of them can shuttle between nucleus and cytoplasm (Pinol-Roma and Dreyfuss, 1993). The hnRNPs play essential roles in mRNA biogenesis, transcriptional regulation, and pre-mRNA splicing (Chaudhury et al., 2010). As they shuttle between nucleus and cytoplasm, they also participate in mRNA translation (Dreyfuss et al., 2002). Using acrylamide treatment to disrupt the vimentin network, Kanlaya et al. (2010) detected reduced nuclear hnRNP expression. Thus, in vimentin null cells, reduced vimentin expression may decrease the expression of nuclear hnRNP, which may lead to defects in transcription and translation. Interestingly, hnRNP K participates in influenza A virus RNA splicing together with NS1 binding protein (NS1-BP) (Tsai et al., 2013), which is a host protein that interacts with influenza NS1 during infection (Wolff et al., 1998). The interaction of hnRNP K with NS1-BP was found to promote splicing of M1 mRNA, which yields the M2 RNA segment (Tsai et al., 2013). Therefore, as an interaction partner of hnRNP K, vimentin may control viral transcription and replication through the interaction between hnRNP K and NS1-BP during influenza A virus infection.   177 6.5 Proposed model of influenza A virus early infection steps.  From the results presented in this thesis and previous published studies, I would like to propose the model depicted in Figure 6-1 for the early steps of infection of influenza A virus. To start a productive infection, influenza A virus must be transported from the cell periphetry to the nuclear compartment. Influenza A virus is internalized and transported to EEs in an actin-dependent way (Figure 6-1, Step 1). The virus-containing EEs travel to the perinuclear region via dynein-directed movement on MTs and mature into mature endosomes (MEs) (Figure 6-1, Step 2). MEs further move along MTs and mature into LEs (Lakadamyali et al., 2003). Vimentin IFs are required at this step for maintain the position of LEs (Figure 6-1, Step 3). In the next step, AP3 together with vimentin IFs control the targeting of CIC-3 to LEs and LYs compartment (Styers et al., 2004), which inturn reduces the pH in LEs. This pH drop triggers membrane fusion between LEs and the viral envelope and further enables vRNPs release into the cytoplasm (Figure 6-1, Step 4). The presence of vimentin IFs are critical for this step of infection. This model is supported by my result in vimentin null cells, which show that the luminal pH of endocytic compartments is impaired in these cells (Figure 5-7) and resulted in the accumulation of influenza vRNPs in the late endosomal compartments (Figure 5-6). Upon releasing into the cytoplasm, vRNPs are then actively transported in to the nucleus. NLS1 and NLS2 on vRNP can interact with four importin  isoforms, importin 1, 3, 5 and 7, to facilate the nuclear import process of vRNPs (Figure 6-1, Step 5). If NLS1 and NLS2 are mutated or their functions are inhibited by competition, nuclear import of vRNPs is inhibited, which further impair viral replication and production (Chapter 4).      178   Figure 6-1: Proposed scheme of the early steps of infection of influenza A virus. Five steps are illustrated and labled 1-5. (1) Influenza A virus enters a host cell and transports to EEs in an actin-dependent manner. (2) EEs travel along MTs and mature into MEs. (3) MEs move along MTs further mature into LEs. Vimentin is involved in LEs positioning. (4) Vimentin together with AP-3 mediate acidification of LEs and release of vRNPs into the cytoplasm. (5) Importin 1, 3, 5 or 7 binds to NLS1 and NLS2 on vRNPs and facilates the nuclear import of vRNPs into the nucleus.      179 6.6 Future directions  6.6.1 Does any post-transcriptional modifications affect the function of NLS2K?  As described in Chapter 3, I have demonstrated that K17 and T18 on NLS2K play an important role for the nuclear import function of NLS2K. Previous studies have pointed out that K is a potential site for post-transcriptional modifications, such as phosphorylation and acetylation, and T is a potential site for post-transcriptional modifications, such as ubiquitination and sumoylation. These modifications interfere with nuclear import efficiency (Gill, 2004; Nardozzi et al., 2010b; Sadoul et al., 2011). Therefore, it will be interesting to determine whether NLS2 contains any of these post-transcriptional modifications and whether these are responsible for an increasing of its binding affinity to importin . In order to determine whether phosphorylation on NLS2 is involved in the function of this NLS, various phosphorylation inhibitors including protein kinase A (PKA) inhibitor, PKB inhibitor, PKC inhibitor, PKG inhibitor, extracellular signal-regulated kinase 1/2 inhibitor and calmodulin-dependent protein kinase II inhibitors could be added to 5GFP-NLS2K transfected cells to determine if there is any changes in the subcellular localization of 5GFP. In order to determine whether acetylation, ubiquitination, or sumoylation are involved in the NLS2K function, cell lysates can be obtained after 5GFP-NLS2K transfection and subjected to western blot. Acetylation-lysine antibody (Cell signaling, Catalog number: 9441) can be used to detect acetylation on NLS2K. Ubiquitination detection kit (R&D system, Catalog number: K415) and sumoylation detection kit (R&D system, Catalog number: K425) can be used to detect ubiquitination and sumoylation on NLS2K.       180 6.6.2 Characterization of the importin  interaction with NLS2K and NLS2R   In Chapter 3, I have demonstrated that NLS2K and NLS2R can interact with importin 1, 3, 5, and 7 using GST-pull down assays and Co-IP analysis. However, the exact contact sites on both NLS2 and importin  are unknown. This is, it is unknown which importin  binding pocket interacts with NLS2 and which amino acid residues on this NLS are responsible for such a binidng. In order to determine the binding site on importin  for NLS2, X-ray crystallography can be used to determine the crystal structure of IBB-importin  in complex with NLS2K/R. IBB-importin  does not include the IBB domain of importin , which keeps the cNLS binding site open to interact with NLSs. In order to establish amino acids on NLS2K/R that are necessary to bind to importin , NLS2K/R mutants containing mutations on basic amino acid clusters or alanine substitution on single basic amino acid can be generated by site-directed mutagenesis and subject to GST-pull down and Co-IP assays using the same protocols I developed in Chapter 3. Characterization of the interaction between importin  and NLS2 and the amino acids on NLS2 that are involved in this interaction can provide us with more information of the host-viral interaction during influenza A virus infection.   6.6.3 Drug screening for potential compounds targeting NLS1 and NLS2 from influenza A virus NP  In Chapter 4, I have shown that inhibiting the nuclear import function of NLS1 or NLS2 by a competition assay inhibited the nuclear import of vRNPs and impaired viral infection. Since NLS1 and NLS2 from NP are highly conserved, they could serve as potential drug targets. Therefore, a high throughput drug screen could be used to identify potential compounds that  181 inhibit NLS1‟s and NLS2‟s nuclear import function. Specifically, 5GFP-NLS1 or 5GFP-NLS2 could be transfected into cells and compounds could be added after transfection. As a control, 5GFP-cNLSs, containing the cNLS from SV40 Tag or nucleoplasmin can be used. Potential antiviral compounds should decrease the nuclear accumulation of 5GFP-NLS1 and 5GFP-NLS2, while not changing the nuclear accumulation of 5GFP-cNLSs.    6.7 Concluding remarks  Prior to this study, the role of NLS2 was poorly understood, and the role of vimentin in influenza A virus infection was yet to be defined. We are the first group that systematically studied the function of NLS2 and identified its binding partners. I have demonstrated that NLS2 is able to mediate the nuclear import of a cytoplasmic reporter protein through interactions with four importin  isoforms. My site-directed mutagenesis results indicated that NLS2K mediates the nuclear import through two basic amino acid clusters at its N- and C-terminus, while NLS2R only utilizes the N-terminal basic cluster. Moreover, I showed that both NLS1 and NLS2 on NP are critical for influenza A virus infection, since either mutating these NLSs or inhibiting them can impair viral infection. I also addressed the role of vimentin during influenza A virus infection for the first time, and my findings suggest that vimentin plays a role in endosome positioning and acidification, which is critical for influenza A virus trafficking and viral genome release into the cytoplasm.  Influenza A virus causes serious infectious, and is a life-threatening pathogen, especially in children, seniors, and immunocompromised patients. 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Shown are the mean and S.E.M analyzed from the values of three independent experiments (n=3, one-way ANOVA followed by Tukey‟s tests were performed and the result indicated that there was not significant decrease on the Fn/c).  208     209 Figure A-2: NP and EEA1 staining during influenza A virus infection in vimentin+/+ MEFs. Vimentin+/+ cells were seeded on glass coverslips 24 h prior to infection. Cells were incubated with influenza A virus (MOI = 2) for 15 min on ice to allow virus bind to the cell surface. The cells were washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37C in a CO2 incubator for different time periods. The cells were probed with anti-NP antibody and anti-EEA1 antibody. DNA was detected with DAPI staining. Scale bar, 10 µm. Zoom images showed high magnification of areas in squares of merge images. Scale bar, 2 µm. DAPI, blue; EEA1, green; influenza NP, red. All experiments were repeated three times and represented confocal microscopy images are shown.  210  211 Figure A-3: NP and EEA1 staining during influenza A virus infection in vimentin-/- MEFs. Vimentin-/- cells were seeded on glass coverslips 24 h prior to infection. Cells were incubated with influenza A virus (MOI = 2) for 15 min on ice to allow virus bind to the cell surface. The cells were washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37C in a CO2 incubator for different time periods. The cells were probed with anti-NP antibody and anti-EEA1 antibody. DNA was detected with DAPI staining. Scale bar, 10 µm. Zoom images showed high magnification of areas in squares of merge images. Scale bar, 2 µm. DAPI, blue; EEA1, green; influenza NP, red. All experiments were repeated three times and represented confocal microscopy images are shown. 212     213 Figure A-4: NP and Rab7 staining during influenza A virus infection in vimentin+/+ MEFs. Vimentin+/+ cells were seeded on glass coverslips 24 h prior to infection. Cells were incubated with influenza A virus (MOI = 2) for 15 min on ice to allow virus bind to the cell surface. The cells were washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37C in a CO2 incubator for different time periods. The cells were probed with anti-NP antibody and anti-Rab7 antibody. DNA was detected with DAPI staining. Scale bar, 10 µm. Zoom images showed high magnification of areas in squares of merge images. Scale bar, 2 µm. DAPI, blue; Rab7, green; influenza NP, red. All experiments were repeated three times and represented confocal microscopy images are shown. 214      215 Figure A-5: NP and Rab7 staining during influenza A virus infection in vimentin-/- MEFs. Vimentin-/- cells were seeded on glass coverslips 24 h prior to infection. Cells were incubated with influenza A virus (MOI = 2) for 15 min on ice to allow virus bind to the cell surface. The cells were washed with ice-cold infection medium to remove the unbound virus particles. The bound viruses were allowed to internalize at 37C in a CO2 incubator for different time periods. The cells were probed with anti-NP antibody and anti-Rab7 antibody. DNA was detected with DAPI staining. Scale bar, 10 µm. Zoom images showed high magnification of areas in squares of merge images. Scale bar, 2 µm. DAPI, blue; Rab7, green; influenza NP, red. All experiments were repeated three times and represented confocal microscopy images are shown.  

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