Kaisa-Emilia Makkonen Interaction of Baculovirus with the Surface of Mammalian Cells and Intracellular Changes during Transduction Publications of the University of Eastern Finland Dissertations in Health Sciences KAISA-EMILIA MAKKONEN Interaction of Baculovirus with the Surface of Mammalian cells and Intracellular Changes during transduction To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Tietoteknia auditorium, Kuopio, on Friday, April 4th 2014, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences Number 225 Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland Kuopio 2014 Juvenes Print – Suomen Yliopistopaino Oy Tampere, 2014 Series Editors: Professor Veli-Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Olli Gröhn, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN (print): 978-952-61-1419-4 ISBN (pdf): 978-952-61-1420-0 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 III Author’s address: Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Supervisors: Professor Kari Airenne, Ph.D. Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Professor Seppo Ylä-Herttuala, M.D., Ph.D. Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland KUOPIO FINLAND Reviewers: Senior Lecturer David Mottershead, Ph.D. Research Centre for Reproductive Health Discipline of Obstetrics and Gynaecology Robinson Institute School of Paediatrics and Reproductive Health University of Adelaide ADELAIDE AUSTRALIA Docent Veijo Hukkanen, M.D., Ph.D. Department of Virology University of Turku TURKU FINLAND Opponent: Professor Markku Kulomaa, Ph.D. Institute of Biomedical Technology University of Tampere TAMPERE FINLAND IV V Makkonen, Kaisa-Emilia Interaction of Baculovirus with the Surface of Mammalian Cells and Intracellular Changes during Transduction University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 225. 2014. 71 p. ISBN (print): 978-952-61-1419-4 ISBN (pdf): 978-952-61-1420-0 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 ABSTRACT Baculoviruses are insect specific viruses generally encountered in nature. In the recent decades they have been widely utilized for different purposes of biotechnology. The discovery of the viruses’ capabilities to transduce mammalian cells has led to their wide use for gene transfer purposes in mammalian cells. Though the virus is able to transduce several types of mammalian cells, still most of the details on the baculoviral uptake into the traditionally nontarget cells are unclear. Also, the receptor that the virus utilizes has not been identified so far. The purpose of this thesis was to examine in detail the interaction of baculovirus virions with the surface of mammalian cells and identify the exact member of the heparan sulfate proteoglycan family to which the virus binds. According to our results, the baculovirus binds specifically to N- and 6-O-sulfated syndecan-1 at the surface of mammalian cells. This is the first time that the viral receptor in mammalian cells has been identified. Since the outcome of baculovirus transduction in vitro is not only dependent on the cell type but also on the conditions of transduction, we also wanted to examine how the use of different kinds of cell culture media affect the transduction efficiency. Our results show that the use of optimized cell culture medium RPMI 1640 leads to improved nuclear entry of baculoviruses and the medium effect is related to intermediate filament vimentin rearrangement. Since some mammalian cells are more permissive to baculoviral transduction compared to others, we also wanted to investigate what the possible crucial differences between the cells and their phenotypes were. According to our data, the poorly permissive cells showed differences in the expression of F-actin, vimentin, PKCα and PKCε compared to permissive cells. The use of RPMI 1640 lead to lower expression levels of syntenin and differences in the regulation of PKCα and ε along with a looser vimentin network. When intracellular kinase activation was further studied in these permissive and poorly permissive phenotypes, differences in kinase regulation were detected. In conclusion, this thesis reveals important aspects which influence baculoviral uptake and trafficking in mammalian cells and identifies for the first time a receptor that the virus uses to bind to and enter into mammalian cells, syndecan-1. National Library of Medicine Classification: QU 470, QU 475, QW 160, QW 162, QW 165 Medical Subject Headings: Baculoviridae; Receptors, Virus; Transduction, Genetic; Heparan Sulfate Proteoglycans; Syndecan-1; Vimentin; Protein Kinase C; Cells, Cultured; Culture Media VI VII Makkonen, Kaisa-Emilia Bakuloviruksen interaktio ihmissolujen pinnalla ja solun sisäiset muutokset transduktiossa Itä-Suomen yliopisto, terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero 225. 2014. 71 s. ISBN (print): 978-952-61-1419-4 ISBN (pdf): 978-952-61-1420-0 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706 TIIVISTELMÄ Bakulovirukset ovat luonnossa yleisesti tavattuja hyönteisspesifisiä viruksia. Viimeisten vuosikymmenten aikana niitä on laajasti hyödynnetty erilaisiin biotekniikan sovelluksiin. Kun virusten havaittiin ensimmäistä kertaa pystyvän siirtämään geenejä myös ihmissoluihin, on niitä sittemmin tutkittu paljon myös geeninsiirtotarkoituksia silmällä pitäen. Vaikka bakulovirus pystyykin siirtämään geenejä useisiin erilaisiin solutyyppeihin, viruksen liikkeistä näissä soluissa tiedetään vielä vähän. Myöskään reseptoria, jota virus hyödyntää ei ole aiemmin pystytty identifioimaan. Tämän tutkimuksen tarkoituksena oli tutkia yksityiskohtaisesti bakuloviruksen interaktiota ihmissolujen pinnan kanssa ja identifioida heparaanisulfaattiproteoglykaanien perheenjäsen, johon virus mahdollisesti ihmissoluissa sitoutuu. Tulostemme mukaan bakulovirus sitoutuu spesifisesti N- ja 6-O-sulfatoituun syndekaani-1 molekyyliin. Tämä viruksen ihmissoluissa käyttämä reseptori pystyttiin ensimmäistä kertaa selvittämään tässä tutkimuksessa. Koska bakulovirustransduktion lopputulos in vitro ei ainoastaan riipu käytetystä solutyypistä vaan myös transduktio-olosuhteista, halusimme tutkia kuinka erilaiset soluviljelymediumit vaikuttuvat virustransduktion tehokkuuteen. Tulostemme perusteella optimaalisen soluviljelymediumin (RPMI 1640) käyttö kasvatusmediumina johtaa viruksen lisääntyneeseen tumaan sisäänmenoon. Tämä lisääntynyt sisäänmeno perustuu solujen keskikokoisiin filamentteihin kuuluvan vimentiinin uudelleenjärjestymiseen. Koska jotkut ihmissolut ovat alttiimpia virusvälitteiselle geeninsiirrolle toisiin verrattuna, halusimme tutkia syitä, mitkä selittävät havaitut erot. Havaitsimme, että vähemmän permissiivisissä soluissa oli erilainen F-aktiinin, vimentiinin, PKCα ja PKCε ilmentyminen permessiivisimpiin soluihin verrattuna. Optimaalisen soluviljelymediumin (RPMI 1640) käyttö sai soluissa aikaan alentuneen synteniin ilmentymisen sekä erilaisen PKCα:n ja ε:n säätelyn. Tämän lisäksi niissä oli havaittavissa väljempi vimentiiniverkko. Solunsisäisiä kinaasiaktiivisuuksia tutkiessamme havaitsimme, että soluissa oli myös erilainen kinaasien säätely. Yhteenvetona voidaan todeta, että tämä työ tuo ilmi tärkeitä näkökulmia bakuloviruksen sitoutumiseen ja liikkeisiin ihmisoluissa vaikuttavista tekijöistä. Tämän lisäksi työssä onnistuttiin ensimmäistä kertaa tunnistamaan bakuloviruksen ihmisoluissa käyttämä pintareseptori, syndekaani-1. Luokitus: QU 470, QU 475, QW 160, QW 162, QW 165 Yleinen suomalainen asiasanasto: bakulovirukset; virusreseptorit; transduktio; syndekaani-1; vimentiini; soluviljely; elatusaineet VIII IX Acknowledgements Since the whole is greater than the sum of its parts, I want to thank all the people that have directly or indirectly contributed to my work. First and foremost, I would like to thank my supervisor Kari Airenne for the great guidance and support during my PhD studies. My second supervisor Seppo Ylä-Herttuala is also very much appreciated for the possibility to have free hands to do BV research as well as to work in such a unique and inspiring environment. I would also like to thank my pre-examiners Veijo Hukkanen and David Mottershead for doing such a big and thorough job in reviewing my thesis. David is also highly appreciated for the language revision. Collaborators from Jyväskylä, Paula Turkki and Varpu Marjomäki, are highly appreciated and acknowledged for the excellently shared projects. Especially Paula, I cannot highlight enough how happy I am of our collaboration during these years. I feel so lucky having to had the possibility to work and get to know you. Anssi Mähönen, I thank you for being such a patient and thorough supervisor when I first came to Ark to do my master’s thesis. You taught me the basis on everything there is to know about our area of interest. Thank you also for the well-functioning collaboration with the medium-studies. I also want to thank Johanna Laakkonen for always helping me in many ways along the way. Though I knew you were busy with multiple other things, you always had the time to give me a helping hand whenever or whatever I needed. I value your contribution highly. Marja Poikolainen, Jatta Pitkänen and Helena Pernu from the SYH-office are also much acknowledged for their endless help, guidance and advice. Tarja Taskinen, Anneli Miettinen and Joonas Malinen, you have always been most helpful. I highly value all the technical support you have given me during the years. All the lovely people in the ex-Ark research and in the SYH-group, I thank you for providing such a wonderful working environment. I am sure a nicer group of people to work with does not exist. Tiina, especially, I am happy to have shared so many cheerful moments both at and outside work. I am lucky to have such a wonderful friend as you are. Last but not least, I am extremely thankful for my dear fiancé and his family as well as my family, relatives and friends for all the support and precious moments in life outside work. I am privileged and deeply thankful to have you all in my life. Kuopio, March 2014 Emilia X XI List of the original publications This dissertation is based on the following original publications: I Mähönen AJ, Makkonen K-E, Laakkonen JP, Ihalainen TO, Kukkonen SP, Kaikkonen MU, Vihinen-Ranta M, Ylä-Herttuala S and Airenne KJ. Culture medium induced vimentin reorganization associates with enhanced baculovirusmediated gene delivery. Journal of Biotechnology 145: 111-119, 2010. II Turkki P*, Makkonen K-E*, Huttunen M, Laakkonen JP, Ylä-Herttuala S, Airenne KJ and Marjomäki V. Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization. Journal of Virology 87: 9822-9835, 2013. III Makkonen K-E*, Turkki P*, Laakkonen JP, Ylä-Herttuala S, Marjomäki V and Airenne KJ. 6-O sulfated and N-sulfated Syndecan-1 promotes baculovirus binding and entry into mammalian cells. Journal of Virology 87: 11148-11159, 2013. *Equal contribution The publications were adapted with the permission of the copyright owners. This dissertation also includes unpublished results. XII XIII Contents 1 Introduction ..................................................................................................................... 1 2 Review of the literature .................................................................................................. 3 2.1 BACULOVIRUSES...................................................................................................... 3 2.1.1 Baculovirus: biotechnology applications ..................................................................... 5 2.1.2 Baculoviruses as gene delivery vectors ........................................................................ 6 2.1.3 Factors affecting baculovirus transduction ................................................................ 8 2.1.4 In vivo applications of baculoviruses ............................................................................ 8 2.1.5 Production of baculoviruses ............................................................................................. 9 2.1.6 Entry and intracellular movements of baculovirus ............................................... 10 2.2 ECHOVIRUSES......................................................................................................... 11 2.3 CELL CYTOSKELETON .......................................................................................... 12 2.3.1 Actin ........................................................................................................................................ 13 2.3.2 Microtubules ........................................................................................................................ 13 2.3.3 Intermediate filaments ..................................................................................................... 13 2.3.4 Viral delivery and the cytoskeleton ............................................................................. 15 2.4 HEPARAN SULFATE PROTEOGLYCANS ........................................................... 15 2.4.1 Syndecans .............................................................................................................................. 17 2.4.2 Syndecan endocytosis and recycling .......................................................................... 18 2.4.3 Viral interactions with syndecans ................................................................................ 19 2.5 PROTEIN KINASE C................................................................................................ 19 2.5.1 PKCα ....................................................................................................................................... 20 2.5.2 PKCɛ ........................................................................................................................................ 21 3 Aims of the study .......................................................................................................... 23 4 Materials and methods ................................................................................................. 25 4.1 METHODS ................................................................................................................ 25 4.2 MATERIALS ............................................................................................................. 26 4.2.1 Cell lines................................................................................................................................. 26 4.2.2 Viral vectors.......................................................................................................................... 26 4.2.3 Antibodies ............................................................................................................................. 27 4.2.4 Constructs and siRNAs .................................................................................................... 27 4.2.5 Reagents and drugs ........................................................................................................... 28 4.2.6 Statistical analysis .............................................................................................................. 28 5 Results and discussion ................................................................................................. 29 5.1 BACULOVIRAL INTERACTION WITH HSPGS (II, III) ...................................... 29 5.1.1 Baculovirus interacts and colocalizes with SDC-1 ................................................. 29 5.1.2 SDC-1 expression regulates baculovirus transduction efficiency .................... 30 5.1.3 Baculovirus utilizes specific HSPG sulfation ........................................................... 30 5.1.4 SDC-1 and baculovirus virions form aggregates in poorly permissive cells 31 5.2 CELLULAR FACTORS AFFECTING EFFICIENT BACULOVIRUS TRANSDUCTION AND ECHOVIRUS-1 INFECTION (II) ........................................ 32 5.2.1 The expression levels of viral receptor does not explain the inefficient transduction/infection ................................................................................................................. 32 5.2.2 Cellular differences in poorly permissive and permissive cells ....................... 32 XIV 5.3 CELL CULTURE MEDIUM HAS AN EFFECT ON VIRUS TRANSDUCTION/INFECTION (I, II)...........................................................................34 5.3.1 Baculovirus transduction efficiency is culture medium dependent ................ 34 5.3.2 Cell culture medium also affects transduction/infection of other viruses .... 34 5.3.3 RPMI 1640 leads to improved nuclear entry of baculoviruses .......................... 35 5.3.4 RPMI 1640 leads to changes in vimentin network ................................................. 35 5.3.5 RPMI 1640 does not change SDC-1 expression or enhance EV-1 binding .... 36 5.3.6 NaHCO3 does not explain the RPMI 1640 effect ..................................................... 36 5.3.7. RPMI 1640 changes the properties of the cells ....................................................... 36 5.3.8 PKC activation with PMA leads to different changes in permissive and poorly permissive cells ............................................................................................................... 38 5.4 CELL SIGNALLING DURING BACULOVIRUS TRANSDUCTION (unpublished data) ..........................................................................................................39 5.4.1 Different kinases and pathways are active during baculovirus transduction in different media ......................................................................................................................... 39 6 Summary and conclusion..............................................................................................45 7 References .......................................................................................................................47 Appendices: Original publications I-III XV Abbreviations AcMNPV Autogapha californica IF Intermediate filaments multiple iPS Induced pluripotent cell nucleopolyhedrovirus MF Microfilaments AAV Adeno-assosiated virus MT Microtubules aPKC Atypical protein kinase C nPKC Novel protein kinase C ARF6 ADP-ribosylation factor 6 NPV Nucleopolyhedrovirus ATP Adenosine triphosphate ODV Occlusion-derived virion BV Budded virion PDZ Postsynaptic CAG Chicken β-actin promoter largeprotein/zonula CMV Cytomegalovirus occludens-1 cPKC Classical protein kinase C CS Chondroitin sulfate DAG Diacylglycerol PKA Protein kinase A ECM Extracellular matrix PKC Protein kinase C EV-1 Echovirus-1 PLC Phospholipase C F-actin Filamentous actin PMA Phorbol G-actin Globular actin GAG Glycosaminoglycan GlcNAc N-acetylglucosamine GlcNS N-sulphoglucosamine Polh Polyhedrin promoter GlcA Glucuronic acid PS Phosphatidylserine GPC Glypican PT Post transduction GV Granulovirus RhoA Ras homolog gene family HCV Hepatitis C virus HEV Hepatitis E virus RSV Rous sarcoma virus HIV-1 Human immune deficiency SDC Syndecan virus VSVG Vesicular stomatitis virus HSPG Heparate sulfate proteoglycan IdoA Iduronic acid PIP2 density-95/disc Phosphatidylinositol-4,5bisphosphate 12-myristate 13 acetate PML Promyelocytic leukemia nuclear bodies member A envelope G-protein XVI WPRE Woodchuck hepatitis post-transcriptional regulatory element 1 Introduction Gene therapy is a novel approach aiming to treat inherited or acquired diseases and pathologic conditions by means of replacing a deficient gene or introducing a new one to target cells, tissues or the affected organ with the help of a gene transfer vector (Verma & Weitzman 2005). The vectors generally used for the delivery purposes are different types of viruses or DNA based plasmids. DNA based plasmids are normally delivered by direct injection or with the aid of liposomes or nanoparticles. The advantage of the plasmid based gene transfer system is that they are safe to use. However, the transfection efficiency is in most cases relatively low and transient thus limiting their use in cases where efficient long term expression is needed (Verma & Weitzman 2005). Today, the most efficient gene delivery vehicles are based on viruses which have a natural preference to enter and expand in mammalian cells. Within gene therapy applications, the most commonly used viruses today include retroviruses, herpesviruses, adenoviruses and adeno-associated viruses (Verma & Weitzman 2005). These are either categorized into RNA (e.g. retroviruses) or DNA based viruses (e.g. adeno and adeno-associated viruses, AAV). Where the adenoviruses lead to transient gene expression, retroviruses have the possibility to integrate within the host genome. AAVs and herpesviruses are generally thought to remain in the cells in episomal from. Baculoviruses are insect-specific viruses widespread in the nature. In recent decades, they have been widely utilized in protein production but have also proved to be useful as gene transfer vehicles in a wide variety of mammalian cells (Kost et al. 2005). The viruses have multiple important advantages such as their large transgene capacity, safety and the capability to transduce both dividing and non-dividing cells (Airenne et al. 2013). The transduction results in transient gene expression and is not cytotoxic to the target cells. Since baculoviruses are non-pathogenic for humans, vertebrates do not have a pre-existing immunity against them (Strauss et al. 2007). Many of these advantages are valued properties of viral vectors aimed to be used in different gene therapy applications. Though the baculovirus has been widely studied in the mammalian cell environment, much of the steps of the virus´s entry in non-target cells, starting from the initial binding and ending in transgene expression in the host cell nucleus, are mostly unknown. In this work, we focused on further determining and understanding the events leading to efficient transduction in mammalian cells. Understanding the changes and responses of the cells during transduction can not only help to engineer improved baculoviral vectors for future gene transfer purposes, but can also help in the selection of the most suitable targets for baculovirus-mediated gene delivery. 2 3 2 Review of the Literature 2.1 BACULOVIRUSES Baculoviruses are large (40–60 nm x 230–385 nm) enveloped insect-specific viruses with a double-stranded circular supercoiled DNA genome of approximately 80–180 kbp in size encoding for 90-180 genes (Summers & Anderson 1972; Rohrmann 2011; Okano et al. 2006). They naturally infect lepidoptera, hymenoptera and diptera hosts and today, over 600 members are known (Okano et al. 2006). Baculoviruses are very commonly encountered in nature and are also used as natural control agents in agriculture to protect human food crops from pests (Kost & Condreay 2002; Summers 2006). The viruses can be divided into two major groups based on their occlusion body morphology: nucleopolyhedroviruses (NPV) and granuloviruses (GV). GVs contain only one nucleocapsid per envelope whereas NPVs contain either single (SNPV) or multiple (MNPV) nucleocapsids per envelope (Figure 1). The occlusion bodies consist of a crystalline matrix composed of polyhedrin in NPVs and granulin in GVs. Both of these occlusion bodies are very stable and can resist most environmental conditions (Rohrmann 2011). According to the new classification, the viruses of Lepidoptera are further divided into Alpha- and Betabaculoviruses including NPVs and GVs, respectively, and those infecting Hymenoptera and Diptera are named Gamma- and Deltabaculoviruses (Jehle et al. 2006). Figure 1. Morphology of nucleopolyhedrovirus and granulovirus. Polyhedral occlusion bodies of nucleopolyhedroviruses contain multiple nucleocapsids and are further categorized as SNPV or MNPV. Granular occlusion bodies of granuloviruses contain only one nucleocapsid. In nature, baculoviruses (NPVs) have a biphasic infection cycle and the virions are present as two types; occlusion-derived virions (ODV) and budded virions (BV) (Blissard & Rohrmann 1990). They are similar in their nucleocapsid structure but differ in the origin and composition of their envelopes as well as their roles in the virus infection cycle. Where the budded virus spreads the infection within the host, the occlusion-derived virus spreads it between insect hosts and is also responsible for the primary infection (O´Reilly et al. 1994). The primary infection cycle of a baculovirus starts when the highly stable polyhedrin crystalline protein matrix coated ODVs enter an insect from a virus contaminated plant 4 (Herniou et al. 2003; Szewczyk et al. 2006) (Figure 2). The polyhedrin matrix dissolves in the alkaline environment of the insect midgut and releases the ODVs which then fuse with the columnar epithelial cells of the digestive tract (Granados & Lawler 1981). After cellular entry, the nucleocapsids are transported into the nucleus where transcription and virus replication take place. Baculovirus infection is divided into three phases and the viral genome replication stage is known as the early phase (0-6 h). At the later stage of infection, also known as the late phase, viral late genes are expressed and the machinery of the host cell is recruited to produce budded virus particles, which bud out of nucleus within vesicles. This phase extends from 6 h to 24 h post infection. The nucleocapsids are released from their vesicles on their way to the cell membrane and exit the cell from its basolateral side acquiring an envelope from the cell membrane. These viruses then enter other insect cells via adsorptive endocytosis (Volkman & Goldsmith 1985; Wang et al. 1997) and further spread the infection within the larva through the tracheal system and hemolymph (Keddie et al. 1989; Federici 1997). In the very late phase of infection (18-24 to 72 h) the production of budded viruses decreases and occluded virions are produced. The death of the larva results in the release of ODVs in the environment and facilitates the beginning of a new infection cycle. Figure 2. Infection cycle of baculovirus. The infection cycle begins when ODVs fuse with the columnar epithelial cells of the insect midgut. In the cells, the nucleocapsids are transported to the nucleus and the replication of the viral genome starts. At the later stage of the infection budded viruses are produced which spread the infection systemically in the larvae. Modified from (Ghosh et al. 2002). ODV and BV differ not only in their roles in the infection cycle but also in their structure (Figure 3). The polyhedrin coated matrix surrounding ODVs is missing from the BV virion. The different virions have also different lipid and protein profiles within their envelopes (Braunagel & Summers 1994). The structural difference is based on the origin of the envelopes. Where the ODV envelope is derived from the nuclear membrane of the insect cell, the BV envelope is acquired from the host cell membrane (Funk 1997). The budded virus contains, along with host cell proteins, viral coded proteins which are not present in 5 the ODV form (Braunagel & Summers 1994). One of these is the major envelope glycoprotein gp64 (Oomens et al. 1995) which forms spike-like peplomers in the rod end of the virus envelope focused on the side of the virus budding from the insect cells (Hefferon et al. 1999). Gp64 is crucial in the progress of the infection since it is responsible for the attachment (Hefferon et al. 1999) and entry (Blissard & Wenz 1992) into further host cells. Gp64 controls the pH-dependent membrane fusion which releases the nucleocapsid into the cell (Blissard & Wenz 1992). Gp64 is also necessary for the production of infective viruses and important in the virus egress from the cells (Oomens & Blissard 1999). Though ODVs and BVs are responsible for different stages of the infection they share some similar structural features. They both have similar DNA and nucleocapsid structures and both virions contain also the main nucleocapsid proteins vp39, p80 and p24 as well as the DNA binding protein p6.9 (Funk et al. 1997). P6.9 and vp39 together with vp1054 and vp91 are conserved structural proteins among all baculoviruses (Okano et al. 2006). Figure 3. Schematic of budded virus and occlusion derived virus. Budded virus has spike-like peplomers in the rod end of the virus which consist of gp64. Capsid protein vp39 is present in both forms of the virus. The prototype baculovirus is considered to be the -baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (Jehle et al. 2006). It is a large virus which has a rod shape appearance (Blissard & Rohrmann 1990). It is known to infect over 30 members of the Lepidoptera order (Carbonell et al. 1985). The virus has been widely studied and the sequenced genome of the virus is about 134 kbp in size encoding around 154 proteins (Ayres et al. 1994; Chen et al. 2013). The most important structural proteins of the budded form of the virus are the DNA binding protein p6.9, the main nucleocapsid protein vp39 and the envelope protein gp64 (Braunagel & Summers 1994). 2.1.1 Baculovirus: biotechnology applications The budded form of AcMNPV has been widely utilized in the baculovirus expression system and has become one of the most commonly used methods to produce recombinant proteins today (Kost et al. 2005). The insect cells commonly used in protein production are generally derived from Spodoptera furgiperda (Sf9 and Sf21AE) and Trichoplusia ni (BTI-Tn5B1-4) cells (Vaughn et al. 1977; Ikonomou et al. 2003). The advantage of the system is that nearly all necessary post-translational modifications take place excluding the Nglycosylation route, which is different between insect and mammalian cells. The production system is fast, easy and the scalability of the system allows the production of large quantities of the desired protein requiring that the genes are placed under the control of the strong AcMNPV polyhedrin (polh) or p10 promoter (Hu 2005; O´Reilly et al. 1994). Also promoters of the virus major capsid protein gene (vp39), the basic 6.9 kD protein gene, and 6 the viral ie1 gene can be utilized (O´Reilly et al. 1994; Miller 1993). In addition to production of proteins (Kost et al. 2005), the baculovirus expression system has been applied for drug screening (Kost et al. 2007), eukaryotic surface display (Oker-Blom et al. 2003), vaccination (Hu et al. 2008) and production of other viruses such as AAVs (Urabe et al. 2002) and lentiviruses (Lesch et al. 2008), as well as viral like particles (Noad & Roy 2003). Today, even the FDA and EMA accepted commercial baculovirus technology based vaccine products e.g. Cervarix®, Provenge® and FluBlok® (Hitchman et al. 2009; Lin et al. 2011; McPherson 2008) are available in the market. 2.1.2 Baculoviruses as gene delivery vectors Though baculoviruses are highly insect specific, it was proven especially in the 1980´s that AcMNPV is able to non-productively enter into vertebrate cells (Volkman & Goldsmith 1983). However, only in the 1990’s was it shown that AcMNPV can also be used for efficient gene delivery into vertebrate cells, especially hepatocytes, if containing an expression cassette having a target cell functional promoter (Hofmann et al. 1995; Boyce & Bucher 1996). Since then, the virus has been utilized in different types of in vitro and ex vivo gene transfer applications in many types of mammalian cells. These include a broad spectrum of cells of human, monkey, porcine, bovine, rabbit, rat, mouse, hamster, fish, sheep and avian origin (Table 1). In addition, baculoviral vectors have been successfully used in induced pluripotent stem cells (iPS) and other stem cell applications (Pan et al. 2013; Takata et al. 2011; Lei et al. 2011). Within all the studied cell types, the best targets have proven to be the ones from hepatic and kidney origin, whereas the poorest targets have been found to be from the hematopoietic origin (Airenne et al. 2013). Baculovirus has been shown to stimulate host antiviral immune responses in mammalian cell lines by promoting cytokine production (Abe et al. 2003; Beck et al. 2000; Gronowski et al. 1999; Abe et al. 2005; Abe et al. 2009; Boulaire et al. 2009; Chen et al. 2009; Han et al. 2009; Wilson et al. 2008). The virus exposure has been shown to result in the activation of tumor necrosis factor , interleukin 1 , and interleukin 1 expression as well as in the production of interferons (Beck et al. 2000; Gronowski et al. 1999). The involvement of toll-like receptors was suggested when AcMNPV was shown to induce the secretion of tumor necrosis factor and interleukin 6 as well as increased expression of activation ligands in murine macrophages (Abe et al. 2003). The role of toll-like receptor 9 and MyD88-dependent signaling pathway on activation of immune cells via baculoviral DNA has also been demonstrated (Abe et al. 2005). Viral DNA has also been shown to promote humoral and CD8+ T-cell adaptive responses and induce maturation of dendritic cells as well as the production of inflammatory cytokines (interferon αβ) against coadministered antigen (Hervas-Stubbs et al. 2007). However, other viral components and recognition pathways, e.g. toll-like receptor 3 (Chen et al. 2009) and toll-like receptorindependent routes (interferon regulatory factors 3 and 7) (Abe et al. 2003; Abe 2012) are also involved. The induction of antiviral effects in mammalian cells is shown to be cell type dependent (McCormick et al. 2004; Suzuki et al. 2010; Han et al. 2009). Several aspects support the use of baculoviruses as gene delivery vehicles. The most important one being the safety of these vectors, since the viruses have not been linked to any diseases outside the phylum Arthropoda (Burges et al. 1980). The viruses are not able to replicate in mammalian cells, thus offering a non-cytotoxic approach for gene transfer purposes in mammalian cells. The baculovirus is able to carry large amounts of foreign DNA (up to 38 kbp) (O´Reilly et al. 1994) and it is easy to manipulate (O´Reilly et al. 1994). The use of baculoviruses does not limit the investigator to dividing mammalian cells since the virus can also transduce nondividing cells (van Loo et al. 2001). The outcome of the virus transduction is transient, gene expression lasting from one to two weeks and does not pose an insertional mutagenesis risk. Transgene expression starts around 6 h after 7 transduction and the highest level of expression is normally achieved at 24-48 h after transduction (Boyce & Bucher 1996; Haeseleer et al. 2001). Table 1. Baculoviral transduced cell types from different origin. Species Cell lines References Primary cells References Human CHP212, DLD- (Airenne et al. 2009; Neural cells, (Sarkis et al. 2000; Ma et 1, ECV-304, Barsoum et al. 1997; pancreas β-cells, al. 2000; Condreay et al. FlC4, hCSMC, Boyce & Bucher 1996; bonemarrow 1999; HEK293, Hela, Kost & Condreay fibroblasts, 1996; Hofmann et al. 1995; HepG2, Huh7, 1999; Clay et al. keratinocytes, Ho et al. 2005; Gao et al. IMR32, KATO- 2003; Grassi et al. hepatocytes, 2002; Swift et al. 2013; III, MRC5, 2006; Ho et al. 2005; mesenchymal stem Gamble MG63, MT-2, Hofmann et al. 1995; cells, hepatic SAOS-2,SK-N- Ma et al. 2000; Sarkis stellate cells, MC, WI38, et al. 2000; Shoji et W12, 143TK al. 1997; Song & Boyce & & Bucher Barton 2012; Nicholson et al. 2005) prostate cells Boyce 2001; Song et al. 2003; Tani et al. 2001) Rabbit Monkey RAASMC (Airenne et al. 2009) COS-1, CV-1, (Aoki et al. 1999; Vero Condreay et al. 1999; Intervertebral disc (X. Liu et al. 2006; Lee et cells, chodrocytes al. 2009) Hepatocytes (Martyn et al. 2007) Tani et al. 2001; Yap et al. 1997) Rodent CHO, BHK, (Aoki et al. 1999; Rat hepatocytes, (Boyce & Bucher 1996; Hsu RGM- I, PC12, Boyce & Bucher 1996; rat chondrocytes, et al. 2004; Gao et al. N2a, L929 Ma et al. 2000; Sarkis rat liver stellate 2002; Liang et al. 2004; et al. 2000; Shoji et cells, mouse kidney Beck et al. 2000) al. 1997; Tani et al. cells 2001) Porcine CPK, FS-L3, (Aoki et al. 1999) PK-15 Coronary artery (Grassi et al. 2006) smooth muscle cells Bovine MDBK, BT (Aoki et al. 1999) Sheep FLL-YFT (Aoki et al. 1999) - - Fish EPC, TO-2, (Leisy et al. 2003; - - CHH-1, CHSE- Yokoo et al. 2013; 214, HINAE, Huang et al. 2011; MES1 Yan et al. 2009) Df-1, (Lu et al. 2007; Han Duck embryonic (Song et al. 2006; Ping et DT40,HD11 et al. 2010) cells, chicken al. 2006) Avian - - myoblast and embryonic fibroblast cells 8 2.1.3 Factors affecting baculovirus transduction The outcome of baculovirus transduction in vitro is generally cell dependent but can also be influenced by modifying cell culture conditions, optimizing the expression cassette within the virus and improving the virus entry by pseudotyping. Changes in the physical properties such as pH, transduction time, temperature and transduction volume have all been seen to have an effect on the outcome of the transduction (Hsu et al. 2004; Airenne 2009). Since baculoviruses are considered non-cytotoxic to mammalian cells, multiple readditions of the virus can be safely performed to pursue the enhanced expression levels (Hu et al. 2003; Wang et al. 2005). The use of chemical substances, such as the histone deacetylace inhibitors sodium butyrate and tricostatin A have also been shown to lead to improved transgene expression (Condreay et al. 1999), whereas the microtubule interfering substances nocodazole or vinblastine have been shown to aid baculovirus transport to the nucleus (van Loo et al. 2001; Salminen et al. 2005). Promoters active in mammalian cells, such as CMV (cytomegalovirus), RSV (Rous sarcoma virus) or CAG (chicken β-actin promoter), are generally used to drive the baculovirus mediated transgene expression since the virus´s own promoters are mostly inactive in non-target cells (Stanbridge et al. 2003). Genetic engineering by incorporation of tissues specific promoters into baculoviral vectors has been use to target transgene expression to certain tissues and cells (Park et al. 2001; Li et al. 2005; Wang & Wang 2006; Guo et al. 2011) whereas inducible promoters (Guo et al. 2011; McCormick et al. 2004) have been used to control the level of the expression. Incorporation of the woodchuck hepatitis post-transcriptional regulatory element (WPRE) into the baculovirus expression cassette has been shown to lead to enhanced transgene expression (Mähönen et al. 2007). Since baculovirus transduction is transient, can the incorporation of elements enabling long term expression from other viruses, such as from AAV (Palombo et al. 1998; Wang & Wang 2005), Epstein-Barr virus (Shan et al. 2006) and the transposon Sleeping beauty (Luo et al. 2012), be used to prolong the expression. Pseudotyping is based on the modification of viral envelope proteins in order to enhance virus tropism or improve transduction efficiency. Since gp64 is essential in the infection of insect cells and transduction of mammalian cells (Liang et al. 2005), addition of extra copies of gp64 on the viral envelope has been shown to lead to better transduction efficiency (Tani et al. 2003). Gp64 has been also modified to target different peptides or proteins, e.g. human immunodeficiency virus-1 (HIV-1) glycoprotein 120 (Oker-Blom et al. 2003; Boublik et al. 1995). Vesicular stomatitis virus envelope G-protein (VSVG) has been used not only to broaden virus tropism, but also to enhance transduction (Barsoum et al. 1997; Kaikkonen et al. 2006; Kitagawa et al. 2005; Pieroni et al. 2001; Tani et al. 2003). Transduction efficiency has also been successfully improved by directing the expression with ligands (Matilainen et al. 2006; Mäkelä et al. 2006; Ojala et al. 2001; Ojala et al. 2004; Oker-Blom et al. 2003; Riikonen et al. 2005; Räty et al. 2004). Other surface modifications that have aided in virus transduction are the incorporation of avidin (Räty et al. 2004), biotin (Kaikkonen et al. 2008), lymphatic homing peptide (Mäkelä et al. 2008) and polymer coating with polyethyl glycol (Kim et al. 2006; Kim et al. 2009; Kim et al. 2007; Kim et al. 2010) or polyethylenimine (Yang et al. 2009). 2.1.4 In vivo applications of baculoviruses The first in vivo gene transfer attempts with baculoviruses were performed into the livers of rats and mice in the late 1990´s (Sandig et al., 1996). Subsequently several different types of animal models (Table 2), including ones of mouse, rat and rabbit origin, have been used to study gene transfer capabilities of the virus in the tissue environment. Though baculoviruses don´t suffer from pre-existing immunity in humans or other vertebrates (Strauss et al. 2007), the virus is quickly inactivated by the components of the serum complement (Hofmann et al. 1995). The inactivation results when the classical (Hofmann & 9 Strauss 1998; Georgopoulos et al. 2009) and the alternative (Hoare et al. 2005) pathways are activated. As a result of the complement susceptibility of the baculoviral virion, the best in vivo targets have been found to be the immunopriviledged tissues such as the eye (Kinnunen et al. 2009) or the brain (Wu et al. 2009). Accordingly, studies have revealed that the central nervous system and testis are also good targets for the virus (Airenne et al. 2010). Due to the neutralizing capabilities of complement, approaches to overcome its effect have included the use of complement inactivators, e.g. soluble complement receptor type 1 (Hoare et al. 2005), cobra venom factor (Sarkis et al. 2000) and compstatin (Georgopoulos et al. 2009) which have all proven their usefulness in protecting the virus from inactivation. Shielding the viruses with complement interfering factors, such as decay acceleration factor (Huser et al. 2001), factor H like protein, C4b-binding protein and membrane cofactor have also aided in the protection of the virus from the immune system (Kaikkonen et al. 2010). Table 2. In vivo gene transfer studies performed with baculovirus. Species Tissue Reference Mouse Brain, testis, eye, abdomen, muscle, liver, lung, systemic administration, nasopharyngeal carcinoma, tumors (Wu et al. 2009; Sarkis et al. 2000; Tani et al. 2003; Park et al. 2009; Haeseleer et al. 2001; Huang et al. 2008; Strauss et al. 2007; Pieroni et al. 2001; Hofmann & Strauss 1998; Kircheis et al. 2001; Kaikkonen et al. 2010; Nishibe et al. 2008; Kim et al. 2006; Hoare et al. 2005; Yang et al. 2009; Pan et al. 2010; Mäkelä et al. 2008; Wang et al. 2008; Balani et al. 2009; Kitajima et al. 2008; Luo et al. 2013; Molinari et al. 2011; Luo et al. 2012) Rat Brain, liver, eye, dorsal root ganglia, biodistribution analysis, heart (Laitinen et al. 2005; Kaikkonen et al. 2006; Räty et al. 2006; Li et al. 2004; Lehtolainen et al. 2002; Sarkis et al. 2000; Wang & Wang 2006; Wang & Wang 2005; B. H. Liu et al. 2006; Wang et al. 2006; Ong et al. 2005; Li et al. 2005; Paul et al. 2012; Lesch et al. 2011) Rabbit Artery, muscle, intervertebral disc, eye (Airenne et al. 2009; Kaikkonen et al. 2006; X. Liu et al. 2006; Kinnunen et al. 2009; Heikura et al. 2012) Honeybee Several tissues (Ando et al. 2007) Fish Embryo (Wagle & Jesuthasan 2003; Wagle et al. 2004) Dog Femoral artery (Paul et al. 2013) Butterfly Wings (Dhungel et al. 2013) Chicken Intravenous inoculation (Luo et al. 2013) 2.1.5 Production of baculoviruses Baculoviruses are produced in insect cells, today most commonly in the Sf-9 cell line, originally derived from fall armyworm (Spodoptera frugiperda) ovarian tissue (Vaughn et al. 1977). The most generally used method to generate baculoviruses today relies on the Bac-To-Bac transposon based system (Invitrogen). The system utilizes a site-specific transposition, Tn7 to insert foreign genes into a bacmid DNA (baculovirus genome) propagated in E. coli cells (Luckow et al. 1993). In practice, the transgene is inserted into a 10 donor plasmid which is transferred into a baculovirus shuttle vector by site-specific transposition in E. coli. The recombinant bacmid containing colonies are selected by bluewhite color screening with LacZ, the bacmid is isolated and used to transfect insect cells. Pure recombinant primary viruses are achieved in 7 to 10 days and the production normally results in high titers of the virus (O´Reilly et al. 1994). The system has been further modified to reduce background and is known as the BVBoost system (Airenne et al. 2003). The virus production protocol is simple and can be performed at biosafety level 1 (Airenne et al. 2011; O´Reilly et al. 1994; Burges et al. 1980). Recent development in membrane-based technology purification also enables virus production at high-scale with good manufacturing based practice (Vicente et al. 2009). 2.1.6 Entry and intracellular movements of baculovirus Though the binding and entry of baculoviruses into mammalian cells have been studied in many types of cells, much of the molecular details remain still unknown (Airenne et al. 2013). Due to the wide tropism of the virus it has been previously speculated that baculoviruses utilize non-specific electrostatic interactions and attach to the surface of mammalian cells via a general cell surface molecule, such as a phospholipid or a heparan sulfate proteoglycan (HSPG) (Wu & Wang 2012; O’Flynn et al. 2013; Duisit et al. 1999; Tani et al. 2001). It was previously shown that the removal of HSPGs with heparinase treatments leads to a reduction in baculovirus binding (Duisit et al. 1999). Recently it was also shown that baculovirus has a heparin binding motif within gp64 (Wu & Wang 2012). Both of these findings support the possible role of HSPGs in cell surface binding. However, gp64 has also been shown to interact with cell surface phospholipids (Tani et al. 2001; Kamiya et al. 2010; Kataoka et al. 2012; O’Flynn et al. 2013) suggesting that the baculovirus entry is a multistep process requiring several cell surface factors. The actual internalization of the baculovirus has been shown to be mediated by lipid rafts (Kataoka et al. 2012; O’Flynn et al. 2013) and is associated within cholesterol rich areas (Laakkonen et al. 2009; Kataoka et al. 2012; O’Flynn et al. 2013). Internalization has also been shown to lead to extensive membrane ruffling of the cell surface (Laakkonen et al. 2009). The entry mechanism is thought to be endosytosis, either pinocytosis or phagocytosis (Laakkonen et al. 2009; Matilainen et al. 2005; Kataoka et al. 2012) since none of the clathrin, caveolae-, flotillin-, GPI-anchored protein-enriched- or IL-2R-mediated endocytosis routes were seen to be involved (Laakkonen et al. 2009). Other mechanisms, such as clathrin mediated endocytosis as well as macropinocytosis have also been suggested (van Loo et al. 2001; Kataoka et al. 2012; Matilainen et al. 2005) highlighting the fact that several different entry routes can exist and entry can be cell type dependent. Virus uptake has been shown to rely on actin and the trafficking is regulated by Ras homolog gene family member A (RhoA), ADP-ribosylation factor 6 (Arf6) and dynamin (Laakkonen et al. 2009). Following cell entry, the virus is transported within vesicles until the pH-dependent fusion of the viral envelope with the endosome releases the capsid into the cytoplasm (Figure 4) (Laakkonen et al. 2009; Blissard 1996; Matilainen et al. 2005). The escaped nucleocapsid is transported in the cell with the aid of actin filaments and the nucleocapsid enters the nucleus via nuclear pores (Goley et al. 2006; Ohkawa et al. 2010; Au et al. 2012). When the virus is in the nucleus, the nucleocapsid disassembles and the viral DNA is released (Kukkonen et al. 2003; van Loo et al. 2001; Laakkonen et al. 2008; Au et al. 2012). In the nucleus, baculovirus virions localize into discrete foci in the nuclei and induce accumulation of promyelocytic leukemia (PML) nuclear bodies (Laakkonen et al. 2008). 11 Figure 4. Baculovirus entry mechanism in insect and in mammalian cells. Baculovirus binds to the surface of the cells and is then internalized. In the cell, the virus escapes from the endosome and is transported to the nucleus. Modified form (Stanbridge et al. 2003). 2.2 ECHOVIRUSES Echovirus-1 (EV-1) is a small nonenveloped single-stranded RNA virus which belongs to Enterovirus genus of Picornaviridae family. The virus is 24-30 nm in size and the genome is approximately 7.5 kilobases long. 32 different serotypes of enteroviruses are known. They comprise a large group of common human pathogens which cause a wide range of different illnesses from minor fever to aseptic meningitis, encephalitis, paralysis and myocarditis (Dahllund et al. 1995). EV-1 infects a wide variety of α2β1 integrin expressing cells as the virus uses the integrin as its receptor (Bergelson et al. 1992). The virus life cycle begins with attachment of the virus to the cell surface receptor, entry of the virus into the cell and 12 finally into the release of the single-strand, positive-sense RNA genome from the capsid into the cytoplasm, where the translation and replication of the genome takes place. EV-1 causes the clustering of its receptor at the cell surface which then leads to virus internalization primarily via macropinocytosis. The internalization is dependent on Rac1, Pak1, PLC, and protein kinase Cα activation. This is followed by gradual accumulation of the viruses in perinuclear endosomes (Marjomäki et al. 2002; Upla et al. 2004; Karjalainen et al. 2008). 2.3 CELL CYTOSKELETON The cell cytoskeleton is a dynamic network which adapts to the environmental and internal changes of the cell. The components of the cytoskeleton are responsible for connecting the cell to the environment as well as organizing the contents of the cell and enabling the cell to move and change shape (Wickstead & Gull 2011). The cytoskeleton consists of three main types of filaments; actin filaments, microtubules and intermediate filaments (Figure 5). The most important differences between these filaments are their physical properties such as stiffness, polarity, assembly and the type of molecular motors which they associate with (Fletcher & Mullins 2010). These cytoskeletal polymers are controlled by several regulatory proteins which are responsible for different tasks. These include filament formation, growth determination and promotion, depolymerization and disassembly, crosslinking, stabilization and organization into high-order network structures (Fletcher & Mullins 2010). Figure 5. Cytoskeletal elements of the cell. Blue represents microtubules, green actin and red intermediate filaments. Modified from (Godsel et al. 2008). 13 2.3.1 Actin Actin, also called microfilament (MF), has many important roles within the cells. Though actin is present throughout the cytosol, it is predominantly found near the plasma membrane (Fletcher & Mullins 2010). Actin can be found in cells either as free monomeric actin or linear filament. Monomeric subunits of actin are called globular actin (G-actin). When G-actins polymerize they form a fiber which is called filamentous actin (F-actin). All actin monomers have the same head-to-tail assembly in the actin fiber which thus shows polarity. Actin filaments have a fast growing plus-end and a slow growing minus-end. In cells, filamentous actin has a double-stranded helix appearance and the fibers are approximately 7-9 nm in diameter and several micrometers in length (Fletcher & Mullins 2010). Actin filaments are found in cells in bundles consisting of aligned long filaments and three-dimensional networks of branched actin filaments (Vignaud et al. 2012). Actin polymerization requires adenosine triphosphate and is reversible. The assembly and disassembly of actin filaments is regulated by actin-binding proteins such as Arp2/3 (Cooper & Schafer 2000). Actin interacts with motor protein myosins which move towards the extremities of the filaments (Vignaud et al. 2012). The polarity of actin has an important role not only in the actin assembly but also in myosin movement (Cooper 2000). 2.3.2 Microtubules Microtubules (MTs) are dynamic hollow rods (25 nm in diameter) in which 13 protofilaments are assembled around a hollow core in parallel orientation. Microtubules consist of tubulin which is formed of globular α/β tubulin dimers. Tubulin heterodimers assemble into the same orientation thus leading to the polarity of the microtubules. The plus-ends have a higher tendency to polymerize or depolymerize than the minus-ends (Radtke et al. 2006). Microtubules are found in cells in bundle like structures. They have an important role in moving organelles in the cytoplasm with the aid of motor proteins. The main microtubule associated motor proteins are dynein and kinesin. Within a cell, the minus ends of the microtubules are anchored to the microtubule-organizing center, the centrosome, whereas the plus ends are projected towards the cell surface. Microtubules form a mitotic spindle during cell mitosis as they separate and distribute the chromosomes to daughter cells (Cooper 2000). 2.3.3 Intermediate filaments Intermediate filaments (IF) comprise a big family of fibrous, non-polarized structurally and sequentially related proteins (Herrmann et al. 2009). They are coil-like polymers which mainly have a structural role within the cell. They participate in maintaining the cell shape and in anchoring cellular organelles by forming a network throughout the cell. The size of the IFs is 10 nanometers on average and each intermediate filament contains approximately eight protofilaments wound around each other to form a ropelike appearance. All IF proteins have a central α-helical rod domain. The expression and the types of IFs expressed are dependent on the cell function (Fuchs & Weber 1994). IFs are divided into six different groups (Table 3). Type I IFs are acidic keratins and type II basic or neutral keratins. Type III IFs include vimentin, desmin, glial fibrillary acidic protein and peripherin. Type IV IFs are neurofilament proteins and α-internexin. Type V nuclear lamins and type VI nestin (Doherty & McMahon 2008). IFs dynamics, organization and distribution are modified by phosphorylation (Omary et al. 2006). 14 Table 3. IF proteins and their distribution. IF protein Type Distribution Keratin I Epithelia Keratin (basic) II Epithelia Vimentin III Mesenchymal cell Glial Fibrillary Acidic Protein III Glial cells, astrocytes Peripherin III Peripheral neurons Neurofilament proteins IV Nervous system Lamins V Nuclear envelopes Nestin VI Embryonal cells Vimentin, highly conserved in all vertebrates, is the most widely expressed type III intermediate filament protein encountered in mesenchymal cells (Ivaska et al. 2007; Herrmann et al. 2009). Vimentin is usually found in fibroblasts, smooth muscle cells and white blood cells but is widely expressed in various other cell types as well (Katsumoto et al. 1990). The expression of vimentin varies during different developmental stages (Ivaska et al. 2007). Vimentin functions as an organizer in cell attachment, adhesion, migration and signaling and hence has a role in cell physiology, cellular interactions and organ homeostasis (Ivaska et al. 2007). Vimentin plays also a significant role in supporting and anchoring organelles in the cytosol since it is attached to the nucleus, endoplasmic reticulum and mitochondria (Katsumoto et al. 1990). Vimentin is regulated by phosphorylation and vimentin polymers are very dynamic in nature. The phosphorylation and dephosphorylation of vimentin leads to rapid exchange between assembled IF polymers and a small fraction of disassembled IF subunits (Eriksson et al. 2004). Rearrangement of vimentin in cells requires phosphorylation of the N-terminal domains by cellular kinases and leads to disassembly into tetrameric subunits (Eriksson et al. 2004). Phosphorylation is controlled by different protein kinase C (PKC) isoforms (Ivaska et al. 2007). Vimentin is phosphorylated at specific sites within the protein. Thr-457 and Ser-458 are targeted by mitotic p37 kinase (Chou et al. 1991) and Ser-55 by Cdc2 (Chou et al. 1990). Ser-38 and Ser-71 are targeted by protein kinase A (PKA) and Rho kinase, and Ser-72 by PKA. These sites have a big impact in maintaining and regulating vimentin structure (Eriksson et al. 2004; Goto et al. 1998). Major targets for PKC are Ser-4, 6, 7, 8 and 9 and Ser-33 and 50 (Eriksson et al. 2004; Ogawara et al. 1995) (Table 4). Table 4. Examples of vimentin phosphorylation sites. Protein kinase Ser phosphorylation site Reference Cdc2 55 (Chou et al. 1991; Tsujimura et al. 1994) PKC 33 (Ogawara et al. 1995; Takai et al. 1996) PKC 50 (Ogawara et al. 1995; Takai et al. 1996) Rho kinase, PKA 38 (Goto et al. 1998; Eriksson et al. 2004) Rho kinase, PKA 71 (Goto et al. 1998; Eriksson et al. 2004) PKA 72 (Eriksson et al. 2004) CaM kinase II 82 (Ogawara et al. 1995; Ando et al. 1991) 15 2.3.4 Viral delivery and the cytoskeleton The host cytoskeleton has an important role in the entry and replication of several different viruses (Smith & Enquist 2002). MFs and MTs have been proposed to participate in cooperation during endocytosis where MFs help in the uptake of ligands via endocytosis and MT are involved in the regulation of trafficking between peripheral early endosomes and juxtanuclear late endosomes (Durrbach et al. 1996). Many viruses take advantage of the cell cytoskeleton by either directly interacting with the proteins or re-arranging them (Radtke et al. 2006). Multiple viruses have been shown to utilize cellular actin and microtubules and their associated motor proteins for different steps during their life cycle (Mercer et al. 2010). Some viral infections such as that caused by the African swine fever virus has been shown to cause microtubule-mediated vimentin rearrangement (Stefanovic et al. 2005). Recently a link between parvovirus and the role of vimentin in the viral escape from endosomes and during endosomal trafficking was demonstrated (Fay & Panté 2013). Vimentin has been shown to have an important role in vesicular membrane traffic and it is also found important in endolysosomal vesicle transport and positioning (Styers et al. 2004). 2.4 HEPARAN SULFATE PROTEOGLYCANS Heparan sulfate proteoglycans (HSPGs) are a family of cell surface molecules which form the major components of the extracellular matrices (Kirkpatrick & Selleck 2007). HSPGs are also encountered in cell tissue compartments, intracellular granules and in the nucleus. HSPGs have a wide variety of functions and they take part in cell metabolism, transport, information transfer, support and regulation (Figure 6) (Sarrazin et al. 2011). HSPGs are composed of a core protein to which one or more long unbranched heparan sulfate (HS) glycosaminoglycan (GAG) disaccharide chains are covalently attached. The GAG chains are attached to the core protein onto specific serine residues through a tetrasaccharide linker. HS chains consist of approximately 40–300 sugar residues with a length of 20–150 nm (Sarrazin et al. 2011) and are composed of alternating N-acetylated or N-sulphated glucosamine units (N-acetylglucosamine [GlcNAc] or N-sulphoglucosamine [GlcNS]) and uronic acids (glucuronic acid [GlcA] or iduronic acid [IdoA]) (Bishop et al. 2007). Some HSPGs contain also chondroitin sulphate (CS)/dermatan sulphate that differs from HS in its sugars (N-acetylgalactosamine [GalNAc] and GlcA/IdoA), patterns of modification and types of glycans (N-linked and O-linked mucin-type chains) (Bishop et al. 2007). The number and sulfation state of the chains can vary according to the growth conditions, cell type and in response to growth factors (Sarrazin et al. 2011). The arrangement of negatively charged sulfate groups and the orientation of the carboxyl groups in the GAGs specify the location of ligand-binding sites (Sarrazin et al. 2011). HS is synthesized by glycosyltransferases of the exostosin family (Bishop et al. 2007) and the GAG chains are enzymatically polymerized in the Golgi and further modified by epimerization and sulfation. The processing of HS can also occur at the plasma-membrane with membrane bound endosulphatases (SULF1 and SULF2) which remove specific sulfate groups from the chains (Dhoot et al. 2001; Morimoto-Tomita et al. 2002). 16 Figure 6. Different functions of heparan sulfate proteoglycans in cells. HSPGs are involved in several different cellular functions such as: 1. Cell-cell cross talk, 2. Storage depot, 3. Barrier formation, 4. Basement membrane organization, 5. Cell adhesion and motility, 6. Cytoskeletal interactions, 7. Lysosomal degradation, 8. Endocytosis, 9. Secretory granules, 10. Transcellular transport, 11. Proteolytic shedding, 12. Heparanase cleavage, 13. Chemokine presentation, 14. Ligand-receptor clustering and signaling. Modified from (Sarrazin et al. 2011). HSPGs are divided in to three subfamilies: membrane-spanning proteoglycans (syndecans, betaglycan and CD44v3), glycophosphatidylinositol-linked proteoglycans (glypicans) and secreted extracellular matrix (ECM) proteoglycans (agrin, collagen XVIII and perlecan) (Bishop et al. 2007). Glypicans (GPCs) and syndecans (SDCs) are considered to form the two main families of HSPGs. Glypicans comprise of six members and carry only HS side chains. Glypicans are mostly found in epithelial cells and fibroblasts (Sarrazin et al. 2011). Whereas the GPC´s HS chains are attached to the core protein close to the plasma membrane, SDC´s HS chains are attached at more peripheral sites (Christianson & Belting 2013). Membrane HSPGs act as endocytic receptors for bound ligands and participate in endocytosis and vesicular trafficking by regulating the movement of molecules between intracellular and extracellular compartments (Kirkpatrick & Selleck 2007; Wittrup et al. 2009). They also act as coreceptors for various tyrosine kinase-type growth factor receptors and cooperate with integrins and other cell adhesion molecules to facilitate cell–cell interactions and cell motility. HSPGs can bind various cytokines, chemokines, growth factors and morphogens (Sarrazin et al. 2011) thus serving as receptors for multiple different ligands. These are e.g. the fibroblast growth factor family, the transforming growth factor beta family, bone morphogenetic proteins, Wnt proteins and interleukins, as well as enzymes and enzyme inhibitors, lipases, apolipoproteins, ECM and plasma proteins (Bishop et al. 2007). Although some ligands bind directly to the HSPG core proteins, the 17 vast majority of them interact with the sulfated domains of the HS chains (Sarrazin et al. 2011). 2.4.1 Syndecans Syndecans comprise a conserved family of heparan- and chondroitin-sulfate carrying type 1-transmembrane proteoglycans. Four syndecans are encountered in mammals; syndecans 1-4, whereas invertebrates have only one (Bernfield et al. 1999; Rapraeger 2001; Couchmann et al. 2001). Each syndecan is composed of a short cytoplasmic domain, a single-span transmembrane domain and an extracellular domain with attachment sites for three to five HS or CS chains (Sarrazin et al. 2011). SDC-2 and SDC-4 have 2–3 HS chains whereas SDC-1 and SDC-3 have 3–4 HS/1-2 CS chains (Sarrazin et al. 2011). HS and CS chains are on average 20 to 80 disaccharides long and the syndecan core protein is 20-45 kDa in size (Couchman 2003). HS binds several different types of ligands whereas the role of CS is less clear (Tkachenko et al. 2005). SDC-1 and -3 are considered to form a subfamily and SDC-2 and -4 another based on their protein sequence homology (Bernfield et al. 1999; Couchmann et al. 2001). The short cytoplasmic domain of syndecans is divided into three regions: conserved regions 1 and 2 (C1 and C2) and a variable (V) region. The C1 domain is nearly identical in all four mammalian syndecans (Figure 7). It is thought to be involved in syndecan dimerization and in binding of several intracellular proteins e.g. tubulin, ezrin, Src kinase and cortactin (Kinnunen et al. 1998; Granés et al. 2000). The conserved Cterminal C2 domain contains a postsynaptic density-95/disc large protein/zonula occludens-1 (PDZ)-binding site at the C-terminal end. Four syndecan-interacting PDZ domain–containing proteins are identified so far: syntenin, synectin, synbindin, and calcium/calmodulin-dependent serine protein kinase (Hsueh et al. 1998; Ethell et al. 2000; Shin et al. 2001; Gao et al. 2000; Grootjans et al. 1997; Cohen et al. 1998). The V domain is highly heterogeneous within the syndecan family and the syndecan-4 V-region includes e.g. a phosphatidylinositol-4,5-bisphosphate (PIP2) binding site that is involved in syndecan-4 dimerization (Shin et al. 2001). Figure 7. Different domains of syndecans. In addition to binding different ligands, syndecans can function also as coreceptors which attract and concentrate molecules and aid in the formation of ligand-receptor complexes either by a conformational change or by acting as a template which brings the ligand and its receptor together (Tkachenko et al. 2005). The syndecan transmembrane domains have a tendency to form dimers and/or oligomers. Although SDCs have a conserved transmembrane domain sequence, the homodimerization has been shown to be weak for SDC-1, strong for SDC-3 and SDC-4, and very strong for SDC-2 (Dews & Mackenzie 2007). Different levels of self-association suggest that dimerization of specific 18 SDC core proteins may play different roles in directing membrane clustering following ligand binding (Grembecka et al. 2006). The expression of syndecans varies depending on the cell type (Table 5) and the phase of development (Bernfield et al. 1992). SDC-1 is expressed early in development and is commonly found in epithelia and mesenchymal cells. It is also highly expressed in keratinocytes. SDC-2 is found on mesenchymal tissues, but also in liver and neuronal cells. SDC-3 is mainly expressed in neural cells but is also found in musculoskeletal tissues. SDC4 is highly expressed by epithelial and fibroblastic cells. It also represents the most widely expressed member being present in many different cell types (Kim et al. 1994). Table 5. Syndecans, the composition of their side chains and their distribution. Syndecan Side chains Distribution Syndecan-1 HS, CS Epithelial and mesenchymal cells Syndecan-2 HS Endothelial and neuronal cells, mesenchymal tissues Syndecan-3 HS, CS Neuronal and musculoskeletal tissue Syndecan-4 HS Ubiquitously expressed 2.4.2 Syndecan endocytosis and recycling Endocytosis can roughly be segregated into clathrin dependent and independent internalization mechanisms and the latter can be further divided into caveolin mediated endocytosis, macropinocytosis and caveolin-independent pathways involving lipid rafts (Howes et al. 2010; Mercer et al. 2010). Syndecans undergo constitutive as well as ligandinduced endocytosis (Williams & Fuki 1997; Belting 2003). The endocytosis appears to be clathrin, caveolin, and dynamin independent taking place in phospholipid- and cholesterolrich lipid-raft areas (Zimmermann et al. 2005; Wittrup et al. 2010; Fuki et al. 2000). Syndecan internalization is triggered by ligand or antibody clustering and it requires intact actin microfilaments as well as tyrosine kinases (Fuki et al. 1997). In the normal state, SDC is predominantly found in the nonraft compartments whereas clustering induces its movement to lipid rafts (Fuki et al. 1997; Tkachenko et al. 2004; Zimmermann et al. 2005; Tkachenko et al. 2006). The functions of the syndecans depend also highly on their cytoplasmic domains that generate connections with signaling and cytoskeletal molecules (Couchman, 2003) and with several PDZ proteins (Grootjans et al. 1997), kinases (Src and calcium/calmodulindependent serine protein kinase) (Chen & Williams 2013) and PIP2 (Alexopoulou et al. 2007). Often signaling occurs in collaboration with other cell-surface receptors (such as integrins) (Bishop et al. 2007). The raft-mediated endocytic pathway of SDC-1 is dependent on its MKKK motif located in the cytoplasmic domain. The MKKK motif also participates in ERK activation which further triggers the dissociation of SDC-1 from tubulin (Chen & Williams 2013). After the dissociation, Src kinases phosphorylate SDC-1 transmembrane tyrosyl residues and cytoplasmic regions. Tyrosine phosphorylation of SDC-1 triggers the recruitment of cortactin which further mediates actin-dependent endocytosis (Chen & Williams 2013). Although recycling of internalized syndecan has been observed, most syndecans end up in lysosomes where they undergo degradation by lysosomal proteases and exolytic glycosidases and sulfatases (Fransson et al. 1995; Zimmermann et al. 2005). Syndecan recycling is controlled by syntenin which interacts with the EFYA sequence in the C2 region of the syndecans (Grembecka et al. 2006). Syntenin PDZ domain interacts also with PIP2, which is involved in the regulation of the actin cytoskeleton and membrane trafficking. Syntenin PIP2 interaction controls Arf6-mediated syndecan recycling and is required for efficient exit of syndecan from Arf6 recycling endosomes. The interaction is also important 19 for the recycling of SDC/syntenin from the endosomal compartments to the cell surface (Zimmermann et al. 2005). 2.4.3 Viral interactions with syndecans Several different bacteria, parasites and viruses utilize heparan sulfate proteoglycans for their initial attachment to the cell membrane but also while entering the cells (Chen et al. 2008). The wide variety of HSPG binding viruses include herpes simplex virus (Shieh et al. 1992; WuDunn & Spear 1989), hepatitis E virus (HEV) (Kalia et al. 2009), vaccinia virus (Chung et al. 1998), noroviruses (Tamura et al. 2004), AAV (Summerford & Samulski 1998), hepatitis C virus (HCV) (Barth et al. 2003) and HIV-1 (Mondor et al. 1998). Although most of the viruses bind to the HS moieties, the core protein can also bind ligands (Kirkpatrick et al. 2006). Where many different bacteria have been shown to specifically utilize SDC-1 for their binding (Chen et al. 2008), only HCV (Shi et al. 2013), HIV-1 (Bobardt et al. 2003) and human papilloma virus (Krepstakies et al. 2012) have been so far shown to interact with SDC-1. The binding of viruses to HSPGs can be sulfation specific and occur to either N- or O-sulfated polysaccharides. Several viruses have been shown to specifically prefer a certain sulfation pattern. HCV utilizes N-sulfation (Barth et al. 2003), coxsackievirus N-and 6-Osulfation (Zautner et al. 2006) and HEV 6-O-sulfation (Kalia et al. 2009). 2.5 PROTEIN KINASE C The protein kinase C family consists of serine/threonine kinase enzymes that control the function of other proteins by phosphorylating the hydroxyl groups of specific amino acid residues. PKCs have an important role in many signaling cascades and they are generally activated by diacylglycerol (DAG) or calcium ions (Ca 2+). There are over ten known isozymes of PKCs in humans (Mellor & Parker 1998; Ohno & Nishizuka 2002) which are divided into 3 different subfamilies based on their structural and biological properties (Table 6). These are the classical or conventional PKC isotypes (cPKC), the novel isotypes (nPKC) and the atypical isotypes (aPKC). All PKCs have highly conserved catalytic domains but they differ in their regulatory domains (Mellor & Parker 1998). The classical isotypes of PKCs are PKCα, βI, βII and γ. They are activated by phospholipids such as phosphatidylserine (PS) in a Ca2+-dependent manner and they also bind DAG (Takai et al. 1979). Classical PKCs can also be activated with phorbol esters, such as phorbol 12myristate 13-acetate (PMA) (Castagna et al. 1982). The use of phorbol esters results in a prolonged activation of PKC (Newton 1995). Novel PKCs include PKCδ, ε, η and θ. They are not sensitive to Ca2+ but are activated by DAG or phorbol esters in the presence of PS (Ono et al. 1988). The last group of PKCs are the atypical kinases which include PKCs ζ and ι. These PKCs do not require Ca2+ and they do not respond to PMA or DAG but are activated by PS (Ono et al. 1989). Table 6. Protein kinase C isotypes and their activation. Isotype PKCs Activation Classical α, βI, βII, γ PS, Ca2+, DAG Novel δ, ε, η, θ PS, DAG Atypical ζ, ι PS Protein kinase C members are formed from a single polypeptide which consists of a Nterminal regulatory region (approx. 20–40 kDa) and a C-terminal catalytic region (approx. 45 kDa) (Newton 1995) (Figure 8). The C1 domain of the classical and novel PKCs forms the DAG/phorbol ester binding site whereas the C2 domain contains the recognition site for PS 20 and in some PKCs, the Ca2+ -binding site. All PKCs also have a pseudosubstrate region in the regulatory domain (Steinberg 2008). PKC resides in an inactive conformation due to interaction of the regulatory domain with the catalytic domain through pseudosubstrate sequences. The binding of cofactors, such as Ca2+ and DAG/phorbol esters to the regulatory domain induces a conformational change in the enzyme exposing the substrate-binding site and the catalysis then takes place. Activation of PKC is usually associated with membrane translocation and prolonged cellular exposure to PKC activators can cause its degradation or downregulation. Members of the PKC superfamily play key roles in several cellular processes. They are involved in receptor desensitization, modulating membrane structural events, regulating transcription, mediating immune responses and in regulating cell growth among multiple other functions (Newton 1995). Figure 8. Different domains of PKCs. Gray represents the pseudosubstrate domain. 2.5.1 PKCα Along with other PKCs, PKCα is also a lipid sensitive enzyme that is activated by growth factor receptors that stimulate phospholipase C (PLC). PLC hydrolyzes PIP2 generating DAG, which activates PKCand inositol trisphosphate, which further causes mobilization of intracellular calcium (Steinberg 2008). PKCα is ubiquitously expressed in all tissues. It is involved in multiple cellular functions such as proliferation, apoptosis, differentiation, motility and inflammation. When PKCα is activated, it is translocated from the cytosol to e.g. the nucleus, focal adhesions and caveolae. The activation of PKCα occurs as a result of multiple different kinds of stimuli ranging from receptor activation and cell contact to physical stress. The kinase activity of PKCα is regulated by phosphorylation of three conserved residues located in its kinase domain: the activation-loop site Thr-497, the autophosphorylation site Thr-638 and the C-terminal site Ser-657 (Parker & Bornancin 1997; Hansra et al. 1999). The integrins are a family of heterodimer forming transmembrane glycoproteins. 18 αand 8 β-integrin genes encode polypeptides that form altogether 24 αβ receptors encountered in mammals (Hynes 2002). The extracellular domains of integrins interact with multiple ligands such as ECM glycoproteins and cell surface proteins (Humphries et al. 2006). The cytoplasmic domains of integrins interact with components of the actin cytoskeleton. Integrins play a role in tissue structure and cell migration (Bökel & Brown 2002) and participate in cell adhesion, haemostasis, thrombosis, inflammation, wound healing and neoplasia. PKCα has been shown co-localize with β1-integrin and regulate its trafficking. The expression of PKCα leads to upregulation of cell surface β1-integrin, whereas the stimulation of PKCα leads to β1-integrin internalization (Ng et al. 1999). Integrins and syndecans act in co-operation to regulate cytoskeletal dynamics. Thus, PKCα has also been linked to interact with SDC-4 and PIP2 binding to the V region of SDC-4 has been shown to have a crucial role in the binding and activation of PKC(Oh et al. 1997). 21 Also, the activity of SDC-4 depends on multimerization, which takes place via recruitment of PIP2, activation of PKCα, and downstream signaling through the RhoA pathway (Sarrazin et al. 2011; Oh 1998; Oh et al. 1997). 2.5.2 PKCɛ PKCε belongs to the novel class of PKCs and is, like the other members of the subfamily, sensitive to DAG/phorbol esters and PS. PKCε participates in numerous signaling systems and regulates various physiological functions including the activation of nervous, endocrine, exocrine, inflammatory and immune systems (Akita 2002). Among all the PKCs, PKCε is the only isozyme that has been considered to be oncogenic by contributing to malignancy either by enhancing cell proliferation or by inhibiting cell death. PKCε is expressed in several tissues and cells, but widely in neuronal, hormonal and immune cells (Ono et al. 1988). PKCε requires phosphorylation at three conserved sites to become responsive to second messengers: Thr-566 in the activation loop, Ser-729 in C-terminal hydrophobic site and Thr-710 at an autophosphorylation site (Takahashi et al. 2000). Phosphorylated PKCε is activated by several different second messengers: DAG, phosphatidylinositol 3,4,5-triphosphate and fatty acids. The second messenger bound to the Cl domain determines the subcellular localization of the kinase. PKCε translocates to the plasma membrane and/or cytoskeleton in response to DAG and tridecanoic acids, whereas it translocates to Golgi in response to arachidonic and linoleic acids (Shirai et al. 1998). PKCɛ has been shown to control the transport of endocytosed β1-integrins to the plasma membrane. If the kinase function of the PKCɛ is inhibited, β1-integrin and PKCε become reversibly trapped in a tetraspanin (CD81)-positive intracellular compartment (Ivaska et al. 2002). β1-integrin recycling is controlled by phosphorylation of vimentin by PKCɛ in integrin containing intracellular vesicles and the PKCɛ-mediated phosphorylation of vimentin regulates the exit from the vesicles to the plasma membrane (Ivaska et al. 2005). 22 23 3 Aims of the Study Insect pathogenic baculoviruses are widely encountered in nature. They have been used in recent decades in the production of recombinant proteins but also as gene delivery vehicles in mammalian cells. Although some of the events taking place in non-host mammalian cells during baculovirus transduction are understood, the exact receptor to which the virus binds to and the cellular factors influencing virus transduction are mainly unknown. Gaining knowledge of virus transduction starting from the viral interaction with the surface of the cells and intracellular events enabling efficient transduction can aid in the development of improved viral vectors and in selecting the most suitable target cells for baculovirus-mediated gene delivery. The aim of this study was to investigate: I) The effect of different cell culture media on baculovirus-mediated transgene delivery and the mechanism behind the phenomenon in association with the cell cytoskeletal network. II) The cellular factors leading to efficient baculovirus transduction and the factors contributing to the low transduction efficiency in poorly permissive cells. III) The interaction of baculovirus with the cell surface HSPGs and the identification of the member(s) participating in the binding and entry of the virus into mammalian cells. 24 25 4 Materials and Methods The materials and methods used in the articles I‐III are summarized in the tables shown below (Tables 7-12) and are described in more detail in the original publications. 4.1 METHODS Table 7. Methods used in studies I-III. Methods Description Used in Production of viruses Concentration and production of baculoviruses I-III Analysis of viruses Titering and immunoblotting I-III In vitro experiments Cell culture I-III BV transduction I-III BV binding and internalization assays II, III Drug treatments II, III DNA transfection II, III SiRNA transfection II, III Receptor clustering II, III Sulfation studies III Flow cytometry analysis I-III Immunoblotting I, III Cytotoxicity assay (MTT) II, III Immunofluoresence labeling I-III Quantification of microscopic data II, III Proteome Profiler Human Phospho-Kinase Array Unpublished data BCA Protein assay kit Unpublished data Light and fluorescent microscopy I-III Confocal microscopy I-III Mean±SEM, unpaired t-test I-III Analytical experiments Microscopy Statistical methods 26 4.2 MATERIALS 4.2.1 Cell lines Table 8. Cell lines used in studies I-III. Cell line Source Descriptions Used in 293(T) ATCC: CRL-11268 Human embryonic kidney I, III HepG2 ATCC: HB-8065 Human hepatocarcinoma cells I-III MG-63 ATCC: CRL-1427 Human osteosarcoma cells I, II Ea.hy926 University of North Carolina, Hybridoma of human lung epithelium I-III Department of Pathology, NC, USA and human umbilican vein endothelial cells RaaSMC (Ylä-Herttuala et al. 1995) Rabbit aortic smooth muscle cells I HeLa ATCC: CCL-2 Human cervical cancer cells I Sf-9 Invitrogen Spodoptera frugiperda IPLB-Sf-21-AE I-III cells 4.2.2 Viral vectors Table 9. Viral vectors used in studies I-III. Vector name Description Used in Ba-CAG-EGFP/WPRE (Mähönen et al. 2007) I-III Vp39EGFP (Kukkonen et al. 2003) I p24Cherry (Laakkonen et al. 2009) II, III LV-GFP VSVG-pseudotyped lentivirus I LV-Gp64 Gp64-pseudotyped lentivirus I EV1 (Marjomäki et al. 2002) II 27 4.2.3 Antibodies Table 10. Antibodies used in studies I-III. Antibodies Source Used in Anti-vimentin Novocastra I Anti-α-tubulin Abcam I Anti-nuclear lamins A/C Novocastra I Anti-vimentin S72 Abcam I Anti-vimentin S38 Abcam I Anti-vimentin S82 Abcam I Anti-beta actin Cell Signalling I Anti-A211E10 (α2-integrin) From F. Berditchevski II Anti-AcMNPV From L. Volkman II, III Anti-Arf6 Thermo Fisher Scientific III Anti-Cd59 SantaCruz Biotechnology II, III Anti-Cd81 SantaCruz Biotechnology II Anti-EEA-1 Transduction Laboratories II Anti-EV1 (Marjomäki et al. 2002) II Anti-gp64 From L. Volkman II, III Anti-PIP2 Molecular Probes II Anti-PKCα Transduction Laboratories II Anti-PKCε SantaCruz Biotechnology II Anti-pPKCα Upstate Biotechnology II Anti-pPKCε Upstate Biotechnology II Anti-syndecan-1 SantaCruz Biotechnology II, III Anti-syndecan-2 SantaCruz Biotechnology II, III Anti-syndecan-3 SantaCruz Biotechnology II, III Anti-syndecan-4 SantaCruz Biotechnology II, III Anti-vimentin Leica Microsystems II Anti-vp39 From L. Volkman II, III 4.2.4 Constructs and siRNAs Table 11. Constructs and SiRNAs used in studies I-III. Construct Description Used in Arf6 WT Wild type II PH-GFP PIP2 PH-domain binding II PKCα DN Dominant negative II PKCα WT Wild type II PKCε k/m Kinase dead II PKCε WT Wild type II Sdc-1 WT Wild type I-III Syntenin siRNA II Vimentin siRNA II 28 4.2.5 Reagents and drugs Table 12. Reagents and drugs used in studies I-III. Construct Description Source Used in Desulfated heparins 2-O, 6-O and N-desulfated heparins Iduron III Phorbol 12-myristate 13-acetate Activator of PKCs Sigma-Aldrich II PI-PLC Removal of GPI-anchored proteins Sigma-Aldrich II Recombinant SDC-1 Competition assay Abcam III Sodium chlorate Prevention of CAG sulfation Sigma-Aldrich III TRITC-phalloidin Filamentous actin probe Sigma-Aldrich II 4.2.6 Statistical analysis An unpaired t‐test, performed with PrismTM from GraphPad Software, Inc., was used to determine the statistical significance of the data (in Article I, II and III). 29 5 Results and Discussion The following chapter presents the main results of studies I–III (publications identified by a Roman numeral). Unpublished data is also shown. 5.1 BACULOVIRAL INTERACTION WITH HSPGS (II, III) Baculoviruses have been considered to have a wide tropism in mammalian cells in vitro and in vivo. This is thought to be explained by the fact that the virus binds to a general cell surface molecule, such as a heparan sulfate proteoglycan or a phospholipid (Duisit et al. 1999; Tani et al. 2001). However, though some preliminary data exists to support the role of HSPGs, no identification of a specific HSPG family member has been previously reported. The purpose of the study was to investigate in detail the role of HSPGs in the binding and entry of baculoviruses into mammalian cells. Different types of cells were selected, permissive 293T and HepG2 cells and poorly permissive MG-63 and Ea.hy926 cells in which the intracellular virus trafficking has previously shown to be non-functional (Kukkonen et al. 2003). 5.1.1 Baculovirus interacts and colocalizes with SDC-1 HSPGs have been suggested to serve as attachment factors for baculoviruses in mammalian cells. However, whether any particular HSPGs can serve as a receptor is unknown. So, we first studied the two main families, syndecans and glypicans. In order to define if the glypican family was involved in the binding, we performed an enzymatic PI-PLC treatment for HepG2 and Ea.hy926 cells. The treatment results in the loss of cell surface glypicans. The loss was verified by staining the cells with CD59-antibody, which recognizes a member of the GPI-anchored protein family. The treatment did not however result in a statistically significant decrease in baculovirus binding to the surface of the studied cells (III, Fig. 3) nor in the baculovirus transduction (data not shown) thus indicating that the glypican family is not involved in the interaction of the baculovirus virion and the cell surface. The results are in line and support the previously published data (Laakkonen et al. 2009) and also the fact that most of the interactions of pathogens with HSPGs are shown to take place with the syndecan family (Chen et al. 2008). Since glypicans did not show a role in the interaction, we next assessed the expression of syndecans at the surface of HepG2, Ea.hy926, MG-63 and 293T cells. The study was done by immunofluorescence staining the surface of the cells with antibodies against the different members of the syndecan family. The results were analyzed by confocal microscopy. The levels of syndecans-2, -3 and -4 seemed to be more uniform in all cells whereas there was more variation detected with syndecan-1 expression (III, Fig. 4). The amount of bound baculovirus on the surface of the cells was determined and the binding showed a trend that followed SDC-1 expression. This suggested that SDC-1 could be involved in the binding of baculovirus onto the cells. Interestingly, baculovirus is known to transduce well cells and tissues which have high expression of SDC-1 (Airenne et al. 2013; Su et al. 2004, www.proteinatlas.org). The involvement of syndecans in the virus-cell interaction was further studied with colocalization studies in HepG2 and Ea.hy926 cells. In practice, baculoviral virions were 30 allowed to bind and enter the cells, and baculoviruses and syndecans were then immunostained and imaged by confocal microscopy. As a result, no or minimal colocalization of baculovirus was detected with syndecan family members 2, 3 and 4. However, a clear colocalization was seen with the virus and SDC-1 at several time points (III, Fig. 5). This suggests that baculovirus does not only bind to SDC-1 at the cell surface but also interacts with it during the entry process. Though the cells examined were of a very different phenotype, the common factor was their SDC-1-mediated interaction with the baculovirus. 5.1.2 SDC-1 expression regulates baculovirus transduction efficiency In order to further study the role of SDC-1 in baculovirus transduction, we transfected HepG2 cells with a SDC-1 encoding plasmid. The transfection resulted in upregulation of SDC-1 at the surface of the cells (data not shown). Transduction after transfection led to a significantly improved transduction efficiency in flow cytometer analysis (III, Fig. 6D). The results thus suggest, that when the expression of cell surface SDC-1 was elevated, the viral binding and entry improved which further led to enhanced marker gene expression. However, the level of SDC-1 expression is not always directly proportional to the level of viral transduction since in some cells expressing SDC-1, e.g. Ea.hy926, viral trafficking can be halted (Kukkonen et al. 2003). An alternate approach was also used where HepG2 cells were pre-treated with SDC-1 antibody before baculovirus transduction. The transgene expression was then analyzed 48 h later with a flow cytometer. The antibody decreased baculovirus transduction efficiency compared to the control cells (III, Fig. 6A). Also, a negative effect of the antibody on virus entry was observed with confocal microscopy (III, Fig. 6B). This suggests that the receptor masking caused by the antibody is able to prevent virus binding which is seen as decreased transduction efficiency. A similar type of antibody inhibition approach led to the same outcome with HSV-1 in HeLA cells (Bacsa et al. 2010). A competition assay with recombinant SDC-1 protein was also carried out. The recombinant protein was added in varying concentrations to HepG2 cells before the addition of the virus and marker gene expression was analyzed with flow cytometer 48 h later. As a result, a dose-dependent inhibition of virus-mediated transgene expression was seen (III, Fig. 6C). Although the recombinant SDC-1 protein was not glycosylated it could still interfere with transduction. This could be taking place either by SDC-1 directly binding to the baculovirus virions and thus competing with the cell surface SDC-1, or by its interference with essential intra- and intermolecular syndecan interactions. The latter is supported by the fact that for example SDC-1-mediated regulation of αvβ5-integrin activity in cell attachment and spreading does not require the HS-GAG chains of SDC-1 (McQuade et al. 2006). However, the exact mechanism, and demonstration of direct binding between non-glycosylated SDC-1 and baculovirus remains to be verified. 5.1.3 Baculovirus utilizes specific HSPG sulfation HSPGs are highly sulfated molecules and most of their interaction with other molecules takes place with the aid of sulfated residues. Our purpose was to study if HSPG sulfation is also important in baculovirus binding and transduction. In practice, HepG2 and Ea.hy926 cells were treated with different concentrations (0-75 mM) of sodium chlorate which leads to undersulfated GAGs at the cell surface. As an outcome of the treatment, both binding and transduction of baculovirus clearly decreased, consistent with sulfation being important in the baculoviral cell interaction (III, Fig. 2A, B). The same treatment has also been shown to decrease AAV-2 transduction (Qiu et al. 2000). Since some viruses have been shown to bind specifically to sulfated residues, we also wanted to assess if the same applied to baculoviruses. For that purpose, we used differentially desulfated heparins; basic 31 heparin, N-desulfated, 2-O-desulfated and 6-O-desulfated heparins, which were incubated with viruses before the viruses were added and allowed to transduce 293T and HepG2 cells. The transduction rate was analyzed 48 h later with a flow cytometer. Pretreatment of baculoviruses with both heparin and 2-O-desulfated heparin had an inhibitory effect on the transduction rate in both cell lines. However, 6-O-desulfated and N-desulfated heparins were not able to influence the baculovirus transduction rate indicating that these sulfations are important in the viral interaction with HSPGs (III, Fig. 2C). It can thus be concluded that the interaction between baculovirus particles and the cell surface is not solely based on random electrostatic interactions, but is indeed specific and requires a specific sulfation pattern. N- and 6-O-sulfation of HSPGs have also been seen to be important to coxsackie B virus and HIV-1 (Zautner et al. 2006; de Parseval et al. 2005). 5.1.4 SDC-1 and baculovirus virions form aggregates in poorly permissive cells Since it was evident that SDC-1 had a role in baculovirus binding and transduction, we further investigated the role of SDC-1 in the poorly permissive context of EA.hy926 and MG-63 cells and assessed at which stage the virus was blocked along its internalization pathway. For that reason, internalization and differential labeling assays were performed and the results were visualized by confocal microscopy. According to the results, the majority of the viruses remained on the Ea.hy926 and MG-63 cell surface in large clusters even 5 h after virus addition (II, Fig. 2A). The virus was found to be associated with the cell surface SDC-1 and not with intracellular SDC-1 (II, Fig. 2B) indicating that the complex had not entered the cells. Further studies done in Ea.hy926 cells showed that the colocalization of baculovirus particles with SDC-1 stayed constant, whereas colocalization of SDC-1 with the baculovirus particles grew over time (II, Fig. 2C), suggesting that baculovirus clusters gathered more SDC-1 over time. In permissive HepG2 cells, no large extracellular virus– SDC-1 clusters were observed (II, Fig. 2B). The results thus indicate that baculovirus binding to SDC-1 on poorly permissive cells leads to its aggregation at the cell surface and inability to enter the cells. This also explains the poor transduction efficiency. The exact reason behind the aggregation remains to be determined. However, it can be due to several factors, e.g. the lack or non-functionality of additional molecule(s) involved in efficient entry or defective activation of signaling route which prevents the virus/SDC-1 complex entering the cells. 32 5.2 CELLULAR FACTORS AFFECTING EFFICIENT BACULOVIRUS TRANSDUCTION AND ECHOVIRUS-1 INFECTION (II) In order to characterize and discriminate cell specific features which affect efficient virus transduction, we investigated several cell lines that are known to be permissive and poorly permissive to baculoviruses. The human pathogen echovirus-1 (EV-1) was also used in these studies. Factors known to affect baculovirus, EV-1 and their receptors were selected for investigation. Flow cytometry and confocal microscopy was used to analyze the results. 5.2.1 The expression levels of viral receptor does not explain the inefficient transduction/infection To assess if there was a correlation with the expression level of the baculovirus receptor, SDC-1, and the EV-1 receptor, α2β1-integrin, for successful viral entry, we immunostained the receptors in question in EA.hy926, HepG2 and MG-63 cells and visualized the results with the aid of confocal microscopy. All cell lines showed high expression levels of SDC-1 (II, Fig. 1C) which correlated well with baculovirus binding (II, Fig. 1D). Similar results were gained with EV-1 and its receptor α2β1-integrin (II, Fig. 1E, F). Transduction/infection assays performed in the same cell lines showed, however, that the level of transduction/infection was low in the poorly permissive cells (II, Fig. 1A, B). This demonstrates that these cells are deficient for baculovirus transduction and EV-1 infection, although they express large amounts of viral receptors at their surfaces and the viruses bind efficiently to the cells. In permissive HepG2 cells, both viruses showed high transduction and infection levels despite having similar expression levels of the viral receptors compared to poorly permissive cells. The data suggests that expression of the cell surface receptor does not directly correlate with the efficiency of transduction/infection implying that other factors are responsible for the outcome. The reason for the inefficient infection of EV-1 in poorly permissive cells was further studied by investigating the entry of antibody-clustered α2β1-integrin. It has been previously shown that antibody-clustered α2β1-integrin follows the same path as EV1clustered α2β1-integrin (Upla et al. 2004; Karjalainen et al. 2008; Karjalainen et al. 2011). No block in integrin internalization was detected and clear intracellular clusters were observed in all studied cell lines (II, Fig. 2E). Also, no difference was observed between the levels of internalized and cell surface integrin in the cells (II, Fig. 2F), leading to the conclusion that internalization of α2β1-integrin was not affected in poorly permissive Ea.hy926 and MG-63 cells. Interestingly, this further suggested that the block in these cells is after the internalization step of the EV-1 infection pathway. This implies that there are multiple different stages in the cells where the viral trafficking can be halted. 5.2.2 Cellular differences in poorly permissive and permissive cells To investigate further the possible factors affecting baculovirus and EV-1 trafficking, we studied features that differ between permissive and poorly permissive cell lines by confocal microscopy. Proteins were selected under investigation by their association to baculovirus, EV-1, SDC-1 or α2β1-integrin trafficking. We first studied the involvement of filamentous actin (F-actin) in poorly permissive MG-63 and EA.hy926 cells. According to the results, the levels of F-actin in HepG2 cells were high, whereas in poorly permissive cells, the levels were clearly lower (II, Fig. 3A). Due to the fact that RhoA has been shown to regulate 33 baculovirus uptake (Laakkonen et al. 2009) and actin dynamics, we also investigated the involvement of RhoA in poorly permissive cells. Studies performed with RhoA wt, dominant negative and constitutively active (CA) constructs showed that RhoA had no apparent effect on baculovirus transduction in Ea.hy926 cells (data not shown), suggesting that though F-actin levels were low, changing the actin dynamics with RhoA was not the determining factor. However, also other factors could influence actin organization and the expression of G-actin can differ between cell lines. To further investigate the role of cell cytoskeleton in baculovirus transduction, we studied vimentin distribution in HepG2, EA.hy926 and MG-63 cells. According to the results, there were considerable differences between these cell lines. The overall levels of vimentin were higher in poorly permissive cells compared to the permissive cells. A difference was also seen in the form of vimentin within the cells. The poorly permissive cells showed a complex vimentin network whereas the permissive cells showed no clear network and within those cells, vimentin was seen in vesicle-like structures. This suggested that in HepG2 cells vimentin is possibly more in monomeric rather than in the filamentous form (II, Fig. 3A). This could thus possibly be one of the crucial factors affecting efficient baculovirus transduction and EV-1 infection since infection of some viruses, e.g. African swine fever virus (Stefanovic et al. 2005), have been shown to be associated with vimentin rearrangement. Due to the importance of syntenin in syndecan recycling (Zimmermann et al. 2005) we also studied the role of syntenin in more detail in HepG2, EA.hy926 and MG-63 cells. According to our data, syntenin expression was high in Ea.hy926 cells whereas in MG-63 cells, the expression of syntenin was lower but still higher than in HepG2 cells (II, Fig. 3A). It could thus be concluded that high syntenin expression levels possibly correlate negatively with efficient baculovirus transduction and EV-1 infection. Although EV-1 does not require SDC-1 for its infection, syntenin is a multifunctional protein that interacts with various other transmembrane and cytoplasmic proteins (Sarkar et al. 2004). Both Arf6 and PIP2 are known to regulate syndecan and/or integrin recycling as well as actin organization (Macia et al. 2008; Powelka et al. 2004; Zimmermann et al. 2005). Our previously shown involvement of Arf6 in the regulation of baculovirus uptake in permissive cells (Laakkonen et al. 2009) led us to further investigate the role of Arf6 in poorly permissive Ea.hy926 cells. According to the results, the overexpression of Arf6 wild type or a CA mutant could not induce baculovirus transduction. No changes were seen in PIP2 distribution or dynamics as monitored by a PH-GFP-expressing construct in either of the poorly permissive or permissive cells. PH-GFP is a fusion protein which consists of GFP and the pleckstrin homology (PH) domain of PLCδ1. The domain is known to bind PIP2 with high affinity and specificity (Stauffer et al. 1998). Also, no colocalization was observed with baculovirus or EV-1 and Arf6 or PIP2 (data not shown). These results thus indicate that though Arf6 and possibly PIP2 have a role in baculovirus entry, neither Arf6 nor PIP2 is associated with the inefficient viral internalization seen in Ea.hy926 cells. PKCα has been shown to be associated with β1-integrin trafficking (Ng et al. 1999), EV-1 infection (Upla et al. 2004) and SDC-4 interaction (Keum et al. 2004). Hence, both the phosphorylated form and the total pool of PKCα were studied in the cells. No significant differences were observed between the cell lines in the total pool of PKCα (data not shown), whereas the phosphorylated form showed distinctive distribution patterns in the poorly permissive cells. In those cells PKCα was mostly seen as cytoplasmic and aggregated in the perinuclear compartment. In the permissive HepG2 cells, pPKCα was evenly distributed throughout the cell (II, Fig. 3B). As the EV-1 internalization assay was performed, it led to a reorganization of the pPKCα pool in HepG2 cells. In Ea.hy926 cells, there was no reorganization seen thus implying that though EV-1 is able to internalize, the internalization does not lead to efficient infection due to the different pPKCα function/response in the cells. Since the activation of PKCα is linked to SDC-4 (Keum et al. 34 2004), our data suggests that SDC-1 might also be involved. In conclusion, we observed differences in F-actin, syntenin, vimentin and pPKCα. These changes might further explain the difference in baculovirus transduction and EV-1 infection in permissive and poorly permissive cell lines. 5.3 CELL CULTURE MEDIUM HAS AN EFFECT ON VIRUS TRANSDUCTION/INFECTION (I, II) It is known that baculovirus transduction is not only dependent on the cell or tissue type, but also on the conditions of the transduction. The purpose of our study was to determine if different cell culture media had an impact on baculovirus-mediated transgene expression. In addition, we wanted to further investigate what could explain the favorable effect that was observed with RPMI 1640 medium. 5.3.1 Baculovirus transduction efficiency is culture medium dependent In order to investigate the effect of different cell culture media on baculovirus transduction, permissive as well as poorly permissive cells were chosen for the study (HepG2, 293T, HeLa, MG-63, RaaSMC and Ea.hy926). Baculoviral particles were used to transduce the cells in different media and the data was analyzed with fluorescence microscopy and flow cytometry. According to the results, the effect of the cell culture medium was clear in all cell lines tested. The amount of EGFP expressing cells was clearly lower in DMEM compared to the results gained from RPMI 1640 cultured cells. Even the low gene expression normally achieved in DMEM cultured MG-63 and Ea.hy926 cells could be improved when the cells were cultured in RPMI 1640 (I, Fig. 1A). A further comparison done between several other culture media (F-12, DMEM, MEM, Optimem, RPMI 1640 and McCoys 5A) revealed that DMEM was the poorest and RPMI 1640 the best medium in respect to baculovirus transduction efficiency (data not shown). The effect of the increased expression was also evident with all MOIs used and the increase in the expression from DMEM to RPMI 1640 was 2.5-fold in HepG2 cells, 9-fold in EA.hy926 cells and 11-fold in MG-63 cells (I, Fig. 1B). The results thus suggest that culturing poorly permissive cells in RPMI 1640 leads to cellular changes that enable improved baculovirus transduction and enhanced transgene expression. The effect of different cell culture media on baculovirus transduction was also studied in 293T suspension cells. The results supported the result that RPMI 1640 is one of the most optimal media for baculovirus transduction (Lesch et al. 2011). What the exact components and mechanisms are behind the phenomenon, needs to be further determined. 5.3.2 Cell culture medium also affects transduction/infection of other viruses To study does the medium affect also transduction efficiency of other viruses, gp64- and VSVG-pseudotyped lentiviruses were used to transduce DMEM and RPMI 1640 cultured cells. The data was in line with the baculoviral results and both pseudotyped viruses showed higher transduction efficiency in RPMI 1640 medium than in DMEM (I, Fig. 2). The increase in the expression was 2-fold for gp64-pseudotyped lentiviruses in 293T cells and 5.8-fold in HeLa cells. VSVG-pseudotyped lentiviruses showed a 1.6-fold increase in the expression in 293T cells and 1.8-fold in HeLa cells. The same effect was studied with EV-1. RPMI 1640 and DMEM grown Ea.hy926, MG-63 and HepG2 cells were infected with the virus (II, Fig. 4A). The infection efficiency doubled in RPMI 1640 medium. This indicates 35 that the medium-effect is not virus specific but rather related to cellular responses to the extracellular milieu. 5.3.3 RPMI 1640 leads to improved nuclear entry of baculoviruses Since the effect of RPMI 1640 on baculoviral transduction was clear, the next step was to analyze baculoviral nuclear entry and intracellular localization in the transduced Ea.hy926 and HepG2 cells with the aid of confocal microscopy. Nuclear lamins and the nucleus were stained to distinguish the cytoplasm from the nucleus. Differences in the amount of EGFPtagged baculoviral nucleocapsids (Kukkonen et al. 2003) located in the nucleus between RPMI 1640 and DMEM cultured cells was observed (I, Fig. 4). In the RPMI-cultured cells, the nucleocapsids were detected all around the cytoplasm and in the nucleus, whereas in DMEM, the nucleocapsids were located mainly outside the nucleus (I, Fig. 4A) suggesting that RPMI 1640 medium leads to enhanced nuclear entry of baculoviral nucleocapsids. The increased amount of nucleocapsids was also observed in MEM compared to DMEMcultured cells (I, Fig. 4B). This suggests that DMEM somehow inhibits virus entry to the nucleus and thus causes most of the viruses to remain in the cytosol. 5.3.4 RPMI 1640 leads to changes in vimentin network Cell culture medium has been previously shown to have an effect on the IF network of cells (Giese & Traub 1988). For that reason we also studied the effects of RPMI 1640 on the cell cytoskeletal network. The experiments were performed by immunostaining IFs (vimentin), microtubules and actin filaments of MG-63 and Ea.hy926 cells cultured in DMEM or RPMI 1640 followed by an analysis with confocal microscopy (I, Fig. 5A–C). Reorganization of the vimentin network was observed in RPMI 1640 cultured cells (I, Fig. 5A). The cells showed a loose, faint and reorganized vimentin network whereas in DMEM-cultured cells, the network was denser. No clear rearrangement of MTs (I, Fig. 5B) or MFs (I, Fig. 5C) was detected in any of the cell lines indicating that the main effect of RPMI 1640 on the cell cytoskeletal network was through vimentin. Vimentin is regulated by phosphorylation and dephosphorylation which leads to an exchange between assembled IF polymers and disassembled IF subunits (Eriksson et al. 2004). We thus studied the presence or absence of phosphate groups on three individual serine phosphorylation sites of vimentin (S38, S72 and S82) in MG-63 and Ea.hy926 cells by Western blotting (I, Fig. 6). Since phosphorylation of Ser-38 and Ser-72 by cAMP-dependent kinase (PKA) has been shown to lead to decelerated assembly kinetics of vimentin subunits in vivo, these sites were selected (Eriksson et al. 2004). Ser-38 and Ser-72 have not only been shown to be important in determining the assembly state but also in regulating the structure of vimentin (Eriksson et al. 2004). The total level of vimentin was high and remained fairly constant in both media (I, Fig. 6A). When comparing DMEM vs. RPMI 1640 cultured cells, there were changes in the phosphorylation patterns of the individual serine sites. The phosphorylation of S38 and S72 was changed in RPMI 1640 cultured Ea.hy926 cells (I, Fig. 6B), whereas in RPMI 1640 cultured MG-63 cells, S82 was altered (I, Fig. 6C). This suggests that RPMI 1640 leads to changes in vimentin phosphorylation and thus affects its assembly kinetics and structure regulation. The results also suggest, that a more phosphorylated and looser vimentin network leads to less of a mechanical barrier for viruses and thus enables efficient intracellular viral trafficking. What are the exact events and cascades leading to increased vimentin phosphorylation, needs to be further determined. 36 5.3.5 RPMI 1640 does not change SDC-1 expression or enhance EV-1 binding To determine if the medium change from DMEM to RPMI 1640 affects EV-1 internalization or boost the binding of the virus on cells, we measured the binding of EV-1 onto the cells grown in either media. There was no difference seen in virus binding (data not shown), suggesting that RPMI 1640 medium helps virus trafficking rather than binding to the cells. We also wanted to see if the amount of SDC-1 was affected when the cells were grown in the different media. RPMI 1640 and DMEM cultured cells were stained with SDC-1 antibody and analyzed with flow cytometry. There was no significant difference in the amount of cell surface SDC-1 (Figure 9) (unpublished data). Altogether this data implies that the enhanced transduction/infection of the viruses in RPMI 1640 is not dependent on the amount of viral receptors expressed at the cell surface nor the improved virus binding, but improved intracellular trafficking. As seen in Ea.hy926 cells, the aggregation of SDC-1 and baculovirus at the cell surface in DMEM is bypassed when RPMI 1640 is used. The factors behind this need to be further determined. Figure 9. The effect of cell culture medium on SDC-1 expression. PE stands for the percentage of phycoerythrin expressing cells. 5.3.6 NaHCO3 does not explain the RPMI 1640 effect Since it was previously suggested that the amount of NaHCO 3 in DMEM can inhibit baculovirus transduction (Shen et al. 2007), we further studied the role of NaHCO3 by increasing the amount of NaHCO3 in RPMI 1640 to the level found in DMEM, and tested the effect on the level of transduction in HepG2, EA.hy926 and MG-63 cells. The addition of NaHCO3 into RPMI 1640 resulted in a 2.3-fold decrease in the marker gene expression only in Ea.hy926 cells (I, Fig. 3). The effect was mild and statistically insignificant in HepG2 and MG-63 cells. The data thus suggests that the decrease in the transduction efficiency seen in DMEM is not solely explained by the HCO3− ion concentration or that the RPMI 1640 media is able to overcome the negative effect associated with the HCO3− ion. 5.3.7. RPMI 1640 changes the properties of the cells To study the possible factors affecting baculovirus and EV-1 trafficking in different media, we studied again the proteins that have previously been associated with baculovirus, EV-1, SDC-1 or α2β1-integrin trafficking in context of the medium change to RPMI 1640. Since the trafficking of baculovirus (Ohkawa et al. 2010) as well as syndecan (Tkachenko et al. 2004; 37 Fuki et al. 1997) has been shown to be actin dependent, we studied the difference of F-actin in poorly permissive MG-63 and EA.hy926 cells and permissive HepG2 cells in the two different media. No difference in F-actin was observed (II, Fig. 4B), whereas the expression levels of syntenin, a syndecan binding protein important in syndecan recycling (Zimmermann et al. 2005), changed in response to the culture medium. In HepG2 cells the effect was less clear, whereas in poorly permissive cell lines (II, Fig. 4B), an apparent decrease in the syntenin level was detected in RPMI 1640 medium. Further assessment of the role of syntenin was performed with a knockdown experiment using a syntenintargeted smart pool siRNA. Though the syntenin level was efficiently downregulated (II, Fig. 4C) the siRNA treatment had no effect on baculovirus transduction or EV-1 infection, implying that syntenin is not a crucial factor in the cell susceptibility to these processes (II, Fig. 4D). The results also suggest that since the expression of syntenin itself is not solely important, the factors behind the change might be. Because PKCα is shown to be important in β1-integrin trafficking (Ng et al. 1999) as well as in EV-1 internalization (Upla et al. 2004), the role of pPKCα in the medium phenomenon was determined by confocal microscopy. The change of culture medium affected both pPKCα distribution and its phosphorylation status within the cells. In RPMI 1640 medium cultured MG-63 and EA.hy926 cells, pPKCα was translocated from the cytosol to plasma membrane. Also the overall pPKCα expression levels were higher (II, Fig. 4B). The induction seen in the phosphorylation levels was also verified with Western blotting (II, Fig. 4E). This data together indicates that the enhanced activation of PKCα in RPMI 1640 could at least partly explain the improved EV-1 infection. Previously, it was shown that the use of a phorbol ester led to a similar type of translocation of PKCα from the nucleus to the plasma membrane (Li et al. 2002). Since PKCα is activated by Ca2+, DAG and PS, changes in the amount of these activators in the cells in response to RPMI 1640 could account for the results. What the exact mechanism is, needs to be further determined. Further investigation of the role of the vimentin network was also carried out. According to our observations, the vimentin network changed in response to the culture medium in poorly permissive cell lines. The network was tight and compact in DMEM-cultured cells whereas in RPMI-cultured cells, the network was looser. We also observed more filamentous vimentin in the cells cultured in DMEM than in RPMI 1640. No changes were detected in HepG2 cells (II, Fig. 4B). Since PKCε affects both vimentin dynamics and β1integrin trafficking (Ivaska et al. 2005) we wanted to study its possible involvement. No changes in the level of total PKCε between the media was detected whereas a clear reduction in the phosphorylated form of PKCε in cells cultured in RPMI 1640 was seen (II, Fig. 5A). When the pPKCε distribution was monitored after baculovirus internalization, an association of pPKCε with baculovirus-syndecan-1 aggregate on the cell surface was detected (II, Fig. 5B). However, no association between EV-1 and pPKCε was seen (data not shown) suggesting that EV-1 infection does not share the same mechanism as baculovirus transduction. A clear downregulation of pPKCε was, however, seen with Western blotting in EV-1-treated Ea.hy926 cells in response to EV-1 internalization (II, Fig. 5C) implying that changes in the PKCε phosphorylation could regulate EV-1 internalization. When the same effect was studied with baculoviruses, no difference in the amount of pPKCε was seen (II, Fig. 5C) suggesting that lack of pPKCε downregulation might possibly lead to deficient baculovirus internalization. It has been previously shown, that inhibition of PKCε can lead to a blockage in α2β1integrin recycling and further to its entrapment in CD81-positive vesicles (Ivaska et al. 2002). Thus the association of CD81 with the baculovirus aggregates was studied. As a result, no CD81 was found in the aggregates (data not shown), implying that the CD81 positive vesicle structures previously reported are not the same seen with baculovirus. 38 5.3.8 PKC activation with PMA leads to different changes in permissive and poorly permissive cells Both novel and classical PKCs can be activated with phorbol esters, such as PMA. Due to the fact that PKCα and PKCε were shown to play a role in the culture medium phenomenon, we investigated the effects of PMA treatment on both baculoviral transduction as well as EV-1 infection in MG-63, EA.hy926 and HepG2 cells cultured in RPMI 1640 and DMEM. The effect of PMA on baculovirus transduction was first monitored with the aid of flow cytometer. PMA was able to increase the transduction efficiency in HepG2 cells cultured in DMEM, whereas in MG-63 and EA.hy926 cells, the transduction efficiency was not significantly changed (II, Fig. 6A). In RPMI 1640 however, the transduction efficiency decreased in MG-63 and EA.hy926 cells whereas in HepG2 cells there was no significant change. EV-1 infection levels determined by confocal microscopy during PMA treatment revealed a similar kind of pattern. In permissive cells, PMA increased the infection rate in both media, and in poorly permissive cells, PMA reduced it (II, Fig. 6B). The effect of PMA treatment on PKC activation and syntenin expression as well as the effects of syntenin knockdown on PKCα activation were also studied with confocal microscopy. According to the results, no interdependent regulation was observed (II, Fig. 6C). The effect of PMA and culture media on the vimentin network were also studied since PKCε is activated by PMA. In addition, the medium change to RPMI as well as EV-1 internalization lead to vimentin downregulation. According to the results, the vimentin network was more loosely packed in RPMI-cultured Ea.hy926 cells (II, Fig. 7A). In both media, PMA treatment resulted in a more compact vimentin network. However, in HepG2 cells, neither culture media nor the PMA treatment had any effects on vimentin (II, Fig. 7B), suggesting that the changes in vimentin dynamics were possibly responsible for the decreased transduction and infection levels seen after PMA treatment in poorly permissive cells. As RPMI medium was shown to downregulate pPKCε in the poorly permissive cells, these results also imply that PKCε activation by PMA treatment leads to changes in vimentin organization in poorly permissive cells. These changes are then able to overcome the positive effect of PKCα activation on virus internalization, resulting thus in reduction in baculovirus transduction and EV-1 infection. In addition to filamentous versus monomeric forms of vimentin observed in the poorly permissive and permissive cells, poorly permissive cells also expressed higher levels of vimentin than the permissive cells. In order to see if a reduction of vimentin levels had any effect on baculovirus transduction and EV-1 infection, we used a vimentin targeted smart pool siRNA approach and monitored the transduction and infection levels with confocal microscopy. Vimentin knockdown did not have an obvious effect on EV-1 infection or baculovirus transduction in poorly permissive cells (II, Fig. 7C). This suggests that instead of the overall vimentin level, the organization of vimentin is more crucial in the virus transduction and infection. To confirm that vimentin overexpression was not the restricting factor in PMA-treated cells, vimentin siRNA treatment was performed along with PMA treatment (II, Fig. 7B). The success of vimentin knockdown with the siRNA treatment was verified by confocal microscopy. Although the treatment did not reduce vimentin expression to zero (77% knockdown), it was able to significantly diminish vimentin expression (II, Fig. 7D). However, vimentin overexpression was not the restricting factor in PMA-treated cells. The results thus suggest that the unfavorable effects of the vimentin network on cellular entry and intracellular trafficking of viruses is not due to the vimentin levels, but rather changes in the PKCε regulated vimentin organization. What factors are leading to the changes in the PKCε regulation needs to be further studied. In conclusion, the key factors affecting baculovirus transduction and EV-1 infection in different cell culture media are PKC subtypes α and ε together with vimentin reorganization. 39 5.4 CELL SIGNALLING DURING BACULOVIRUS TRANSDUCTION (unpublished data) The aim of this study was to investigate the intracellular differences in kinase phosphorylation between poorly permissive (EA.hy926; DMEM and RPMI 1640) and permissive (HepG2; MEM) cells cultured in different media after baculovirus transduction. No previous data of protein kinase activities in mammalian cells during baculovirus transduction exists. The commercially available antibody based Proteome Profiler Human Phospho-Kinase Array (R&D Systems) was used to measure phosphorylation differences in protein content normalized samples. A multiplicity of infection of 1000 and time points of 0.5 h and 4 h post transduction (pt) were selected since the majority of the virus should be at those time points in the entry pathway, early endosomes or in the nucleus (Salminen et al. 2005; Matilainen et al. 2005). The signal from the phosphorylated kinases was developed by Super signal West Dura Extended Substrate and the intensities of the detected signals were measured by Nonlinear Dynamics Totallab TL120 software. The presented results are replicates of two independent experiments. 5.4.1 Different kinases and pathways are active during baculovirus transduction in different media Phosphokinase activation was studied in MEM cultured HepG2 cells and DMEM/RPMI 1640 cultured EA.hy926 cells. In EA.hy926 cells cultured in DMEM, no significant increase or decrease of phosphorylation was observed in most of the kinases within the two different time points when compared to the untransduced cells (Figure 10). Only two kinases, paxillin and FAK were detected to be significantly upregulated at 4 h pt compared to 0.5 h pt (Table 13, *p<0.05). The results thus indicate, that as previously seen in DMEMcultured EA.hy926 cells, the viruses remain mainly at the cell surface (II) or in the cytosol (I). The lack of a kinase response also possibly explains the halted virus trafficking seen in EA.hy926 cells. In HepG2 cells, several kinases showed increased phosphorylation states during baculovirus transduction compared to mock treated cells (Figure 11). It was also observed that as the transduction proceeded, activation of kinases increased. In HepG2 cells, which are one of the most permissive cells for baculovirus, the virus transduction thus clearly leads to intracellular changes through the activation of kinases which probably accounts for the efficient virus delivery to the nucleus. P3 ER 8 a K JN 1/2 K G pa SK n p5 -3a 3( /b S3 92 ) TO C R R E H B S p7 P A M 27 0S P 6 b K -c Ka2 in a as te e nin (T p5 38 3 9) p2 (S1 7( 5) T1 Pa 9 8 ) xi lli n Fy n Ye s Fg p7 ST r 0S A 6 K ST T 3 in A as T e( 5b T2 R 29) SK 1/ c- 2 Ju n Py k2 Pixel Density P3 ER 8 a K JN 1/2 G Kp a SK n p5 -3a 3( /b S3 92 ) TO R C R E H B S p7 P A M 27 0S P 6 b K -c Ka2 in a as te e nin (T p5 38 3 9) p2 (S1 7( 5) T1 Pa 9 8 ) xi lli n Fy n Ye s Fg p7 ST r 0S A 6 K ST T 3 in A as T e( 5b T2 R 29) SK 1/ c- 2 Ju n Py k2 FA K Pixel Density 40 50000 EA.hy926 DMEM 40000 Control 0.5 h 4h 30000 20000 10000 0 Kinases Figure 10. Phosphorylation state of different kinases in DMEM cultured and baculovirus transduced EA.hy926 cells. HepG2 MEM 150000 Control 0.5h 4h 100000 50000 0 Kinases Figure 11. Phosphorylation state of different kinases in MEM cultured and baculovirus transduced HepG2 cells. 41 Since the use of RPMI 1640 medium during baculoviral transduction clearly leads to enhanced nuclear transport of viral nucleocapsids in poorly permissive cells (I), it was reasonable to expect some changes also in kinase activity. When we compared the different time points after baculovirus transduction (0.5 h and 4 h) in RPMI 1640 grown EA.hy926 cells (Figure 12), we saw an increase in the phosphorylation of 27 kinases (Table 13) compared to non-treated cells. Among these kinases, increased phosphorylation of 21 kinases was statistically significant indicating that RPMI 1640 medium affects the activation of kinases. EA.hy926 RPMI 30000 Pixel Density Control 0.5h 4h 20000 10000 L K ST ck in A as e ( ST T 2 T4 A 21 T5 /S a R 424 SK ) p2 1/2 PL 7 /3 C (T1 ga 5 m 7) m a1 H c C k hk -2 FA S K ST TAT A 6 T5 a ST /b A T ST 1 A T eN 4 O S P7 0S 6 M EK M 1/2 SK 1 A /2 M A kt Pa1 (S A 47 kt 3 (T ) p5 305 3( ) S4 6) Sr c Ly n 0 Kinases Figure 12. Phosphorylation state of different kinases in RPMI 1640 cultured and baculovirus transduced EA.hy926 cells. The kinase phosphorylation pattern seen in RPMI 1640 cultured EA.hy926 after baculovirus transduction resembled the one in MEM-cultured highly permissive HepG2 cells (Table 13) indicating that the cell response to RPMI 1640 culture medium was favorable for baculovirus entry. 42 Table 13. Comparison of kinase responses between control and baculovirus transduced cells. Yes indicates upregulated phosphorylation compared to control cells. Ns indicates statistical non-significance and * means statistical significance (at least *p<0.05). HepG2 EA.hy926 EA.hy 926 Kinase Phosphorylation site MEM RPMI DMEM P38a T180/Y182 Yes - Yes (ns) ERK1/2 T202/Y204, T185/Y187 Yes - - JNKpan T183/Y185, T221/Y223 Yes Yes (ns) Yes GSK-3a/b S21/S9 Yes - - MEK1/2 S218/S222, S222/S226 Yes* Yes* - MSK1/2 S376/S360 Yes* Yes* - Akt S473 Yes - - Akt(T305) T308 Yes Yes* Yes p53(S46) S46 - - - TOR S2448 Yes* Yes* - CREB S133 Yes Yes* - HSP27 S78/S82 Yes* Yes* - AMPKa2 T172 Yes Yes (ns) - Yes Yes (ns) - b-catenin p53 S15 - Yes* - p27 T198 Yes Yes* - Paxillin Y118 Yes* Yes (ns) Yes* Src Y419 Yes* Yes* - Lyn Y397 Yes(ns) Yes* - Lck Y394 Yes(ns) Yes* Yes P70S6 Kinase T421/S424 Yes Yes* - RSK1/2/3 S380/S386/S377 Yes Yes* - p27 T157 Yes Yes* - PLC gamma-1 Y783 Yes* Yes* Yes Fyn Y420 Yes* Yes* - Yes Y426 Yes* Yes* - p70S6 Kinase T229 Yes Yes (ns) - RSK1/2 S221/S227 Yes Yes* - c-Jun S63 Yes - - Pyk2 Y402 Yes Yes* Yes Hck Y411 Yes Yes (ns) - Chk-2 T68 Yes* Yes* Yes FAK Y397 Yes* Yes* Yes* eNOS S1177 Yes - Yes In order to find out the kinase signaling cascades involved in baculovirus transduction, we fitted the different activated kinases into two available signaling pathway databases (http://www.genome.jp and http://www.cellsignal.com). Four signaling pathways; the MAPK signaling pathway, the mTOR signaling pathway, the integrin signaling pathway and the PI3K signaling pathway (Figure 13) were activated during baculovirus transduction. We then further investigated pathways in which the key kinases were shared by both cell types. Accordingly, the MAPK signaling pathway was ruled out since the cells did not share some of the key kinases involved in the cascade (Figure 13), e.g. there was a significant increase in the phosphorylation of MEK1/2 in Ea.hy926 cells, whereas in ERK1/2 there was not, although it is a key downstream effector of MEK1/2 . As a result, it could be 43 concluded that the integrin (Fyn, Akt, FAK and Src), PI3K (Akt, Fyn, PLC) and mTOR (mTOR, RSK1/2, p70S6K) pathways might have a role in baculovirus entry into mammalian cells. Figure 13. Involvement of different possible signaling pathways in baculovirus transduction. Black circles represent HepG2 cells and blue RPMI-1640 cultured EA.hy926 cells. Classification of kinases is based on the KEGG pathway database (http://www.genome.jp) and (Pelkmans et al. 2005). However, the presented preliminary results are not conclusive since many of the kinases have differential roles in several pathways. The study covers thus far 34 different kinases and gives a partial clue about the possible pathways involved. Altogether, the data presented here demonstrates that baculoviruses utilize 6-O and N-sulfated syndecan-1 for its binding and entry. The use of optimized cell culture medium leads to improved transduction efficiency and intracellular changes in the cells. These changes include the activation of PKCα, deactivation of PKCε and a looser vimentin network (Figure 14). In conclusion, the data presented in this study can give important insights about the factors affecting baculovirus transduction and may be used in the future to develop improved viral vectors for gene transfer purposes. 44 Figure 14. Factors affecting efficient baculovirus transduction. Baculoviral particles bind and enter into cells with N- and 6-O-sulfated syndecan-1. Also additional receptors might be involved. Factors that affect efficient baculovirus transduction within the cells include the activation of PKCα, deactivation of PKCε and a looser vimentin network. Signaling cascades that seem to be involved in the transduction process include the integrin, PI3K and mTor cascades. 45 6 Summary and Conclusion The main conclusions of the thesis are: I Cell culture medium has an effect on baculovirus‐mediated gene delivery. The use of an optimized medium leads to enhanced nuclear entry of baculoviruses and efficient transgene expression. Changes associated with the culture medium are seen in the vimentin network. II Virally poorly permissive cells show differential expression of F-actin, vimentin, PKCα and PKCε compared to permissive cells. 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