Adenovirus Species B Interactions with CD46

Adenovirus Species B interactions with CD46

Dan Gustafsson

Institution of Clinical Microbiology, Department of Virology Umeå 2012

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-368-6 ISSN: 0346-6612 Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Print&Media Umeå, Sweden 2012

To my family!

Table of Contents

Table of contents ……………………………………………………...……… i Abstract ……………………………………………………………………...…. iii Abbreviations …………………………………………………………………. iv Summary in Swedish, Populärvetenskaplig sammanfattning på svenska ……………………………………………………………………… vi List of papers …………………………………………………………………………… 1 Aim of thesis ………………………………………...... 2 Introduction ……………………………………………………...... 3 History…………………………………………………………………………….. 3 Taxonomy…………………………………………………………………………. 3 Epidemiology and clinical features……………………………………………….. 5 Adenoviruses Structure ……………………………………………………. 6 General structure………………………………………………………………….. 6 The capsid………………………………………………………………………… 8 Major Proteins…………………………………………………………………….. 9 Hexon………………………………………………………………………………9 The Penton Base………………………………………………………………….. 10 The Fiber………………………………………………………………………….. 11 Minor Proteins…………………………………………………………………….. 14 Capsid proteins……………………………………………………………………. 14 Protein IIIa………………………………………………………………………… 14 Protein VI…………………………………………………………………………. 14 Protein VIII……………………………………………………………………….. 15 Protein IX…………………………………………………………………………. 15 Core proteins; pV, pVII, µ, pIVa2 and TP………………………………………... 16 Adenovirus Genome ………………………………………………………… 19 Early Genes……………………………………………………………………….. 20 E1A………………………………………………………………………………... 20 E1B………………………………………………………………………………... 20 E2…………………………………………………………………………………. 20 E3…………………………………………………………………………………. 21 E4…………………………………………………………………………………. 21 Delayed Early Genes……………………………………………………………… 22 Late Genes………………………………………………………………………… 22 Adenovirus lifecycle ………………………………………………………… 23 Receptors………………………………………………………………………..... 23 Coxsackie and Adenovirus Receptor (CAR)……………………………………... 23 Integrins…………………………………………………………………………... 24 CD46……………………………………………………………………………… 26 CD80/86…………………………………………………………………………... 31 Dipalmitoyl phosphatidylcholine (DPPC)………………………………………... 31 Coagulation Factors……………………………………………………………….. 31

i Heparan sulfate proteoglycans (HSPG)…………………………………………... 32 Major Histocompatibility Complex (MHC)-1……………………………………. 32 Vascular cell adhesion molecule 1 (VCAM-1)…………………………………… 32 Lactoferrin………………………………………………………………………… 33 Sialic Acid………………………………………………………………………… 33 Desmoglein 2 (DSG-2)…………..………………………………………………... 34 Internalization ………………………….…………………………………….. 34 Endocytosis of species B adenoviruses……………………….…………………... 35 Uncoating, endosomal release and intracellular trafficking………………………. 36 Transnuclear transport…………………………………………………………….. 37 Replication, assembly and release……………………………………………...…. 39 The Complement system …………………………………………...……… 40 Results …………………………………………………………………………... 44 Discussion ……………………………………………..……………………….. 51 Summary …………………………………………………………….…………. 55 Acknowledgments ……………………………………………………..…….. 56 References ……………………………………………………………...……… 59

ABSTRACT

Adenoviruses (Ad) are double-stranded (ds) DNA, non-enveloped viruses. There are seven species (A-G) of human Ads with 52 known serotypes to date. Human Ads cause a broad range of pathologies, ranging from upper respiratory tract infections to persistent urinary tract infections. The main determinant for Ads tropism in vitro is the protruding, antenna-like, fiber protein. The fiberknob is responsible for the main interaction with the attachment receptor of the host cell. Most Ad species use the coxsackie- adenovirus receptor (CAR) as their main attachment receptor. Most species B Ads, however use CD46. CD46 is a cell surface complement regulatory protein, which is expressed on all nucleated cells in humans. Species B Ads exhibit a low seroprevalenc in the human population, making these Ads promising vector candidates for gene therapy. We have studied human Ad species B members, serotypes 7 and 11 (Ad7 and Ad11), as well as their interaction with CD46. Our first experiments showed that all species B Ads use CD46 as their main attachment receptor, with the exception of Ad3 and Ad7. Second, we performed mutational studies of recombinant Ad11p fiberknobs. These studies showed that arginine 279 in the Ad 11 fiberknob is necessary for CD46 binding. Finally we studied the effect of Ad11 binding to CD46. The results indicate that CD46 is rapidly downregulated on the cell surface after Ad11 binding. These results may provide a further understanding of the basic biology and pathology of species B Ads and may also be useful in construction of gene therapy vectors based on species B Ads.

iii ABBREVIATIONS aa Amino acid Ad Adenovirus ADAM A disintegrin and metalloproteinase AIDS Acquired immune deficiency syndrome ATF1 Activating transcription factor-1 ATP Adenosine tri-phosphate CAR Coxsackie and Adenovirus Receptor CCP Complement control protein CK-2 Casein kinase-2 CHO Chinese Hamster Ovary CRM1 Chromosome region maintenance 1 CR-2 Complement receptor-2 CRP C-Reactive Protein DBP DNA-Binding Protein DNA Deoxyribonucleic Acid DPCC Dipalmitoyl phosphatidylcholine DSG Desmoglein ECM Extracellular Matrix EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growthfactor receptor GON Group of nine GPI Glycosylphosphatidylinisotol HSPG Heparan sulphate proteoglycan IC Intermediate chain ICTV International Committee on Taxonomy of Viruses IFN Interferon Ig Immunoglobulin IL-12 Interleukin-12 kDa Kilodalton LDV Leu-Asp-Val LIC Light intermediate chain MAC Membrane attack complex MAPK Mitogen activated protein kinase MASP Mannan-binding lectin-associated serine proteases MBL Mannose-binding lectin MHC Major histocompatibility complex

iv MMP Matrix metalloproteinases MTOC Microtubuli organizing center MV Measles virus NES Nuclear export signal NGFR Neural growthfactor receptor NLS Nuclear localization signal NPC Nuclear pore complex ORF Open reading frame PBMC Peripheral blood cells PML Promyelocytic leukemia protein PKA Protein kinase-A PKC Protein kinase-C PRM Pattern recognition molecule RBC Red blood cells RGD Arg-Gly-Asp RNA Ribonucleic acid SBAR Species B adenovirus receptor SB2AR Species B2 adenovirus receptor SCR Short consensus repeats STP Serine, threonine, proline-rich domain TP Terminal protein VCAM Vascular cell adhesion molecule

SUMMARY IN SWEDISH - POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA

Adenovirus (Ad) är icke höljeförsedda, dubbelsträngade DNA virus. Humana adenovirus delas upp i sju grupper (A-G) som innefattar i detta nu minst 52 kända serotyper. Humana adenovirus orsakar ett brett spann av olika sjukdomstillstånd, från övre luftvägsinfektioner till kroniska urinvägsinfektioner. Den största determinanten för adenovirus tropism (vilken cell viruset binder till) in vitro (i provrörsförhållanden) är fiberknoppen. Fibern är ett antenn likt protein som står ut från viruset. Dess yttersta del, fiberknoppen binder till receptorer på cellytan. De flesta adenovirus grupper använder sig av coxsackie-adenovirus receptorn (CAR) som sin primära bindningsreceptor. Grupp B adenovirus använder sig däremot av CD46. CD46 är ett protein på cellytan som uttrycks på alla humana celler med kärna och dess funktion är att reglera komplement systemet. Ganska få människor har antikroppar mot grupp B adenovirus, vilket gör dessa lämpade för genterapi. Vi har undersökt adenovirus grupp B interaktion med CD46. Vår första studie visar att alla grupp B adenovirus, förutom Ad3 och Ad7, använder CD46 som sin bindings receptor. I vår andra studie har vi utfört mutationsanalys av Ad 11 fiberknoppen för att bestämma vilken del av denna som är nödvändig för bindningen till CD46. Vi visar med våra experiment att en aminosyra, arginin vid position 279, avgör ifall Ad11 fiberkoppen binder CD46. Vi visar slutligen också att CD46 ned-regleras på cellytan som föjd av Ad11 bindning till CD46. Ovanstående resultat kan ge ytterligare insikt i den adenovirala grundbiologin. Dessa resultat kan även bidra med viktig kunskap vid konstruktionen av adenovirus vektorer för genterapi.

LIST OF PAPERS

I. CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7.

Marko Marttila, David Persson, Dan J Gustafsson, M Kathryn Liszewski , John P Atkinson, Göran Wadell, Niklas Arnberg.

Journal of Virology. 2005 Nov;79(22):14429-36

II. The Arg279Gln [corrected] substitution in the adenovirus type 11p (Ad11p) fiber knob abolishes EDTA-resistant binding to A549 and CHO-CD46 cells, converting the phenotype to that of Ad7p.

Dan J Gustafsson, Anna Segerman, Kristina Lindman, Ya-Fang Mei, Göran Wadell.

Journal of Virology . 2006 Feb;80(4):1897-905. Erratum in: Journal of Virology. 2006 May;80(10):5101.

III. Adenovirus 11p downregulates CD46 early in infection.

Dan J Gustafsson, Emma K Andersson, Yang-Ling Hu, Marko Marttila, Kristina Lindman, Mårten Strand, Li Wang, Ya-Fang Mei.

Virology . 2010 Sep 30;405(2):474-82. Epub 2010 Jul 16.

AIM OF THESIS

The aim of this thesis was to study the interactions of species B Ads with CD46. The goal was to determine which members of species B Ads use CD46 as a receptor, which part of the Ad11 fiberknob is responsible for binding and how this interaction affects CD46. The focus has been on the early events of infection.

INTRODUCTION History

Ads were isolated almost simultaneously by two separate researchers in 1953. Wallace Rowe noted that explants from surgically removed human adenoids underwent rounding and clustering, and suspected that this was caused by a microbe; the term “Adenoid-Degenerating” agent was thus coined (Rowe 1953). The same year Hilleman and Werner isolated a virus from tracheal cells of military recruits suffering from influenza-like symptoms (Hilleman and Werner 1954). The agents isolated by the separate investigators were related (Ginsberg, Gold et al. 1955). In 1962, Trentin et al. discovered the oncogenic potential of Ads in rodent cells, increasing interest in Ads (Trentin, Yabe et al. 1962). However, since then, Ads have been shown to not cause the transformation of human cells (Mende, Schneider et al. 2004). Ads have provided significant insight into the intracellular processes of the eukaryotic cell. Messenger RNA splicing was discovered using an Ad system in 1977 (Berget, Moore et al. 1977; Chow, Gelinas et al. 1977). In recent years, attention has been focused on Ad-based vector systems. Due to the tragic death of one individual during a phase I trial involving a Ad vector system, interest in the basic biology of the Ad lifecycle has increased (Raper 2005).

Taxonomy

Ads, belong to the family of Adenoviridae and are divided into four genera plus a fifth genus within the family according to the International Committee on Taxonomy of Viruses (ICTV) (Benkö 2000.) (Davison, Benko et al. 2003; King 2011). Mastadenoviruses and Aviadenoviruses originate from mammals and birds respectively (Davison, Benko et al. 2003). Sialoadenoviruses originate from birds and frogs while Atadenoviruses have been isolated from avian, reptilian, ruminant and marsupial species (Davison, Benko et al. 2003). The fifth genus has been isolated from fish (Davison, Benko et al. 2003). Human Ads, which belong to Mastadenoviruses , are the most studied within the family of Adenoviridae (Hall, Blair Zajdel et al. 2010). Within the genera of Mastadenoviruses there is a further subdivision of up to seven species (A-G) within each host species

(Davison, Benko et al. 2003; Hall, Blair Zajdel et al. 2010). The species classification is made according to the agglutination properties of red blood cells (RBCs), DNA homology, tissue tropism, pathology and oncogenic potential in rodents (Wadell, Hammarskjold et al. 1980). Currently the seven species encompass 52 different types of human Ads, which have been typed according to serum neutralization tests (Wadell 1980; Hall, Blair Zajdel et al. 2010). In addition adenovirus types 53, 54, 55 and 56 have been recognized (Walsh, Seto et al. 2010; Kaneko, Aoki et al. 2011; Kaneko, Suzutani et al. 2011; Robinson, Singh et al. 2011).

FIGURE 1. Summary of the phylogeny of Ad hexon genes. Genera are indicated by different colors, and serotypes belonging to the same species are grouped within the light blue ovals. Abbreviations (letters in italics): B, bovine;C, canine; D, duck; E, equine; F, fowl; Fr, frog; H, human; M, murine; O, ovine; P, porcine; Po, possum; Sn, snake; T, turkey;and TS, tree shrew. With permission from (Davison, Benko et al. 2003).

Epidemiology and clinical features

Infections caused by Ads in humans are very common in children. Approximately 5-15% of upper respiratory and 5% of lower respiratory tract infections are caused by Ads during childhood (Gardner 1968; Edwards, Thompson et al. 1985; Kim, Lee et al. 2000; Hong, Lee et al. 2001). Ad infections in humans occur most frequently during the first 5 years of life (Pacini, Collier et al. 1987; Hong, Lee et al. 2001). Ads are transmitted among humans through respiratory droplets the fecal-oral and conjunctival routes. The incubation period generally ranges from 2-14 days (Pacini, Collier et al. 1987; Hong, Lee et al. 2001). Ads cause a broad range of disease; respiratory tract disease is caused by species B1, C and E (Wadell 2000 ). Gastrointestinal infections are caused by species A and F (Allard, Albinsson et al. 1992; Wadell 2000 ) (Uhnoo, Wadell et al. 1984). Keratoconjunctivitis is caused by species D types 8, 19a, 37, 53 and 54 and conjunctivitis by species B1, D and E (Wadell 2000 ). Urinary tract infections are caused by species B2 (Wadell 2000 ). Ad infections are usually not fatal; however, fatal outcomes have been reported among immunocompromised patients, such as AIDS and transplant recipients (Hierholzer 1992) (Kojaoghlanian, Flomenberg et al. 2003).

Species B1 (Ad3, 7, 16, 21, 50) cause primarily respiratory disease in children (Wadell 2000 ). Interestingly Ad3, 7, 14 and 21 are associated with severe respiratory disease and sometimes fatal outcomes in healthy children (Kajon, Mistchenko et al. 1996) (Kajon and Suarez 1990). Species B2 (11, 34, 35) cause urinary tract infections, associated with persistency and prolonged shedding in urine (Wadell 2000 ). Ad11 can also cause hemorrhagic cystitis in young, immunocompetent, pre-pubertal males (Numazaki, Shigeta et al. 1968; Wadell 2000 ). In immunocompromised patients, such as transplant patients and AIDS patients, Ad11, Ad34 and Ad35 are frequently isolated (Hierholzer 1992).

FIGURE 2. Table modified from (Hall, Blair Zajdel et al. 2010) depicting commonly associated diseases and attachment molecules for different Ad species.

ADENOVIRUSES, STRUCTURE General Structure

Human Ads have a naked icosahedral protein capsid, which is composed of three major proteins: the penton base, the hexon and the fiberknob (Ginsberg, Pereira et al. 1966) (Brenner and Horne 1959; Valentine and Pereira 1965). Furthermore, a number of minor proteins are associated with the capsid: IIIa, VI, VIII and IX (Vellinga, Van der Heijdt et al. 2005). The division into major and minor proteins was based on their decresing apperent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of virions and soluble antigen characteristics of Ad infections (Ginsberg, Pereira et al. 1966). The double-stranded (ds), linear-DNA genome resides inside the core of the capsid and is covalently linked via its 5` termini to the terminal protein (TP) (Rekosh, Russell et al.

1977). The size of the particle ranges from 0.065 to 0.080 µm in diameter, contains more than 2700 polypeptides and has an approximate molecular weight of 150 x 10 6 kDa (Bondoc 1998; Vellinga, Van der Heijdt et al. 2005).

FIGURE 3. General structure of the Ad virion. Reproduced with permission from (Russell 2009).

The Capsid

The capsid is composed of 20 triangular facets that are each formed by 12 hexon trimers (Stewart, Burnett et al. 1991). The penton base (5 subunits) and the trimeric fiber (3 monomers) form complexes termed pentons at each of the angles of the triangular facets (van Oostrum and Burnett 1985; Stewart, Burnett et al. 1991). This gives rise to a icosahedral symmetry with protrusion of the antenna-like fiber protein at each of the twelve vertices of the capsid (Horne 1959) (Rux and Burnett 2004). The major protein components, of the particle, according to size, are the hexon, penton base and fiber (Ginsberg, Pereira et al. 1966). The four minor proteins (IIIa, VI, VIII and IX) are located within the triangular facet, and interact with the major proteins (Stewart and Burnett 1995; Rux and Burnett 2004). The Group of Nine (GON) is located at the center of the triangular facet (Furcinitti, van Oostrum et al. 1989; Stewart, Burnett et al. 1991). The GON is made up of nine hexon trimers connected by four protein IX trimers (Furcinitti, van Oostrum et al. 1989; Stewart, Burnett et al. 1991). When schematically viewed from the top, the GON forms a hexagon, by associating further with three additional hexon trimers and three protein IIIa monomers (Furcinitti, van Oostrum et al. 1989; Stewart, Burnett et al. 1991; Stewart, Fuller et al. 1993). The hexagon has three long edges (composed of three hexon trimers) and three short edges (composed of two hexon trimers). With each of the long edges of the hexagon, two hexon trimers, one penton (three penton monomers and one fiber trimer) and one protein VI monomer are associated in a triangular form, thus completing the arrangement of the triangular facet (Rux and Burnett 2004; Russell 2009).

Figure 4. Depiction of the Ad facets. External view (a), internal view (b) and intenal view of the apex (c). Reproduced with permission from (Russell 2009).

MAJOR PROTEINS The Hexon

The 109-kDa hexon was the first animal virus protein to be crystallized (Pereira, Valentine et al. 1968). The size of the hexon molecule varies with different serotypes (Russell 2009). The viral particle contains 240 hexon trimers comprising 63% of the total protein mass (van Oostrum and Burnett 1985). The hexon monomers form extensive intermolecular contacts at the top and base of the pseudohexagonal trimeric structure (Rux and Burnett 2004), which leads to an exceptionally stable trimer (Shortridge and Biddle 1970). The loop and two eight-stranded jellyrolls at the base of each hexon facilitate interaction with neighboring capsomers, likely through charged residues in the interacting loops (Russell 2009). Extended loop structures are formed between the D-E and F-G strands of the β- barrel in each of the hexon subunits (Rux and Burnett 2004). One loop (DE1, FG1 or FG2) from each subunit (three in total) wrap around each other to form a “three tower” structure at the top of the hexon

(Crawford-Miksza and Schnurr 1996; Saban, Silvestry et al. 2006; Pichla-Gollon, Drinker et al. 2007). These hypervariable loops at the top of the molecule form the type-specific antigenic determinants (Crawford-Miksza and Schnurr 1996) (Rux and Burnett 2004). The pseudo-hexagonal shape of the molecule is formed by two similar β- barrel (V1 and V2) domains arranged in a 3-fold repetition at the base of each subunit (Rux, Kuser et al. 2003). This (pseudo-hexagonal) shape occurs in a number of viruses infecting all forms of life (Eukarya, Bacteria and Archea) (Benson, Bamford et al. 2004). There are four groups of hexons: H1, H2, H3 and H4 (Burnett 1985). H1 hexons associate with the pentons at the apices of the capsid (peripentonal hexons), whereas the remaining hexons (H2, H3 and H4) form the GON structure (Russell 2009).

The Penton Base

The Ad2 penton base is a 571-aa, 63296-Da pentameric molecule situated at the twelve vertices of the viral capsid (Rux and Burnett 2004). Together, the penton base and the fiber form the penton complex (Rux and Burnett 2004). The penton base is highly conserved; the difference in sequence is expressed in the hyper variable region (Zubieta, Schoehn et al. 2005). Crystallization studies of the Ad2 penton have shed light on the detailed structure of the penton (Zubieta, Schoehn et al. 2005). The topology of the monomeric penton resembles that of an inverted hexon (Zubieta, Schoehn et al. 2005). At the base of the molecule is a jelly-roll β-barrel domain formed by 2 four-stranded, antiparalell β-sheets (Zubieta, Schoehn et al. 2005). The second distal domain of the penton contains irregular folds formed by two insertions from the lower jellyroll strands, which culminate on the top in the hyper variable RGD loop (Russell 2009). Combined the five monomers form a bucket-shaped molecule, with five RGD loops on top of the bucket. In Ad2 the pentamer forms an intimate shape that buries approximately 26% of the surface area of each monomer (Russell 2009). Stability is provided by burying the hydrophobic surfaces (Zubieta, Schoehn et al. 2005; Russell 2009). The central cavity of the pentamer was initially thought to accommodate the penton-binding motif (FNPVYPY) of the fiber shaft (Rux and Burnett 2004; Zubieta, Schoehn et al. 2005). However, crystallization studies have shown that the diameter of the cavity at the base is 15-20Å and tapers to 5-10 Å at the top (Zubieta, Schoehn

et al. 2005). The diameter thus prevents insertion of the fiber shaft into the central cavity of the penton. The N-terminal tail portion of the fiber, however, appears to bind to a hydrophobic groove between two monomers. The interaction seems to be mediated by a number of hydrogen bonds and salt bridges (Zubieta, Schoehn et al. 2005). Three fiber tail peptides would thus interact with the grooves on the top of the penton, spreading horizontally and vertically and forming the base of the protruding antenna-like fiber protein (Zubieta, Schoehn et al. 2005; Russell 2009).

The Fiber

The fiber trimer can be divided into three domains; the tail, shaft and knob domain (Chroboczek, Ruigrok et al. 1995). Crystal structures for the fibers of Ad2, Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21 and Ad35 have been determined (Xia 1995; van Raaij 1999.; Durmort 2001; Persson BD 2007 ; Pache, Venkataraman et al. 2008; Pache, Venkataraman et al. 2008; Cupelli, Muller et al. 2010). The C- terminal, globular, knob domain has a propeller-like symmetry when viewed from the top (Durmort 2001). The three monomers composing the knob form an eight-stranded antiparalell beta-sandwich structure (Durmort 2001). The knob domain is the main contact domain in the fiber-receptor interaction. Most trimeric proteins are composed of α- helical coiled-coils, in which three amphipathic α-helixes are wrapped around one another forming a supercoil structure (Stehle 2003.). The Ad fiber shaft, however, is composed of repetitive triple β-spiral folds; the only protein known to share this property is the Reo virus attachment protein σ1 (Stehle 2003.). The motif can be described as a spiral staircase in which each step is composed of a β-repeat. The number of repetitive triple β-spiral shaft motifs ranges from 3 to 23 (Xia 1995; Durmort 2001) . The variation in number of motifs creates a difference in shaft length between different Ad serotypes (Wu, Pache et al. 2003). Insertions of two to four residues in the β-turn of the repetitive triple β-spiral folds motifs create a “hinge” function (Lecollinet, Gavard et al. 2006) (Nicklin, Wu et al. 2005). This structure probably provides more flexibility to the shaft, creating additional binding opportunities for the knob and the RGD motifs of the penton (Wu, Pache et al. 2003; Nicklin, Wu et al. 2005). The flexible region is located in the third β-repeat from the penton (Wu, Pache et al. 2003). In addition there is a conserved KLGXGLXFND/N

sequence motif proximal to the knob domain that is also thought to also increase flexibility (Wu, Pache et al. 2003). Only species D fibers lack both the flexible region and the KLGXGLXFND/N sequence motif (Wu, Pache et al. 2003). The N´terminal tail domain contains a conserved sequence (FNPVYPY) responsible for binding to the penton base (Zubieta, Schoehn et al. 2005).

Hexon Penton

Fiber knob viewed

from the top

Fiber

FIGURE 5.Space-filling models of the Ad5 hexon, penton and Ad2 fiber. A ribbon model of the Ad35 fiberknob is depicted. The RGD site, the binding sites for receptors and the nomenclature of the fiberknob loops are indicated. Adapted with permission from (Russell 2009).

MINOR PROTEINS

Capsid proteins

Protein IIIa

Protein IIIa is a 63.5-kDa protein, which is cleaved from the N´terminus of its 67-kDa precursor by the viral protease (Chroboczek, Viard et al. 1986). IIIa is conserved in all Ads studied (San Martin, Glasgow et al. 2008). The monomer of protein IIIa stretches from the outside of the capsid to the inner space (Vellinga, Van der Heijdt et al. 2005). Protein IIIa is localized to the facets of the capsid, where it probably has a stabilizing function (Rux and Burnett 2004). Coimmunoprecipitation experiments have shown that protein IIIa interacts with protein VII (Boudin, D'Halluin et al. 1980; Lemay, Boudin et al. 1980; Stewart, Fuller et al. 1993). Protein IIIa is required for the assembly of virions. Heat-sensitive mutations affecting splicing of protein IIIa generate empty virus particles at the non- permissive temperature (Boudin, D'Halluin et al. 1980; Chroboczek, Viard et al. 1986). Overexpression of IIIa has an inhibitory effect on L1 mRNA and an upregulating effect on IIIa mRNA (Molin, Bouakaz et al. 2002).

Protein VI

Protein VI is a 22-kDa monomeric protein (Everitt and Philipson 1974). Protein VI resides inside the capsid, probably close to the hexons (Everitt, Lutter et al. 1975; Stewart, Burnett et al. 1991). The basic region of protein VI may interact with viral DNA (Stewart, Fuller et al. 1993). Endosome disruption is pH-dependent and induced by protein VI, thus aiding viral escape (Wiethoff, Wodrich et al. 2005). An amphipathic α-helix in the N-terminus of protein VI is critical for membrane disruption (Maier, Galan et al. 2010). The precursor of protein VI has two NLS and two NES sequences (Vellinga, Van der Heijdt et al. 2005). NLS-2 and NES-2 are cleaved off by proteolysis (Vellinga, Van der Heijdt et al. 2005). Protein VI facilitates nuclear import of the hexon (Wodrich, Guan et al. 2003). It has been suggested that the hexon sterically masks the remaining NES sequence on the mature form of protein VI during interaction (Wodrich, Guan et al. 2003).

Protein VIII

The 15.3-kDa protein VIII is the least-studied of the Ad minor proteins. It is located in dimerized form on the inner surface of the capsid facets and interacts with the hexons (Stewart, Fuller et al. 1993). Protein VIII has a role in virion stability (Liu, Babiss et al. 1985). The precursor of Protein VIII is only present in empty virus particles, making it an assay marker for empty virions in vector preparations (Vellekamp, Porter et al. 2001).

Protein IX

Protein IX is unique to the mastadeovirus genera and is not found in other Ads (Vellinga, Van der Heijdt et al. 2005). Together, the 14.3- kDa protein IX and the hexons form the GONs (Everitt and Philipson 1974). Protein IX likely forms trimers in the cavities between hexon tops (Furcinitti, van Oostrum et al. 1989). Deletion studies have shown that protein IX is dispensable in virion formation (Boulanger, Lemay et al. 1979; Vellinga, van den Wollenberg et al. 2005). However, virions lacking protein IX do not form GONS and are more heat-sensitive than wild-type (wt) virions (Boulanger, Lemay et al. 1979). Protein IX has a conserved leucine-zipper domain in the C- terminus that permits trimerization (Vellinga, van den Wollenberg et al. 2005). The packing capacity of viral DNA into the capsid is more efficient when protein IX is present; deletion of protein IX reduces the DNA packing capacity by approximately 2 kilo-bases (kB) (Ghosh- Choudhury, Haj-Ahmad et al. 1987). During infection, protein IX forms nuclear inclusion bodies containing promyelocytic leukemia protein (PML) (Rosa-Calatrava, Grave et al. 2001), which is interesting because PML is involved in regulating the cellular antiviral response (Rosa-Calatrava, Grave et al. 2001; Rosa-Calatrava, Puvion- Dutilleul et al. 2003). The conserved leucine-zipper domain in the C- terminus of protein IX is involved in the formation of inclusion bodies (Rosa-Calatrava, Grave et al. 2001).

In some studies, protein IX has been shown to stimulate the transcription of E1A and E4 and major late promoter activity (Lutz, Rosa-Calatrava et al. 1997; Rosa-Calatrava, Grave et al. 2001). Other studies have shown only a modest effect of protein IX on E1A and E4 promoter activity (Sargent, Ng et al. 2004).

Core proteins: pV, pVII, µ, pIVa2, protease and terminal protein (TP)

Protein V (pV) is unique to the mastadenoviruses (Chatterjee, Vayda et al. 1985) and plays a role in the viral nucleocapsid structure by making contacts between pVI and pIII (Chatterjee, Vayda et al. 1985). Each virus particle contains 157 copies of pV (van Oostrum and Burnett 1985). pV is associated non-specifically with the viral dsDNA (Chatterjee, Vayda et al. 1986). During viral infection, pV localizes to the nucleus and nucleolus, resulting in nucleolin and nucleophosmin redistribution to the cytoplasm (Matthews 2001). The deletion of pV results in reduced infectivity and thermo-stability, although viral replication remains (Ugai, Borovjagin et al. 2007). pVII is the major core protein; 800 copies are found in each viral core (Stewart and Burnett 1995; Rux and Burnett 2004). pVII is a basic alanine/arginine-rich protein around which the viral DNA is wrapped into adenosomes (Chatterjee, Vayda et al. 1986; Spector 2007). It has been postulated that pVII is a template for the initial stages of transcription in vivo (Chatterjee, Vayda et al. 1986) (Haruki, Okuwaki et al. 2006). After endocytosis, pVII directs the viral core to the host cell nucleus (Wodrich, Cassany et al. 2006). During virus assembly pVII forms a complex with pIVa2 and L2 52/55K (Zhang and Arcos 2005). pIVa2 is only present in a few copies and is attached to the viral DNA (Russell 2009). pIVa2 plays a major role in viral encapsidation and regulation of late transcription. Packing of viral DNA involves pIVa2 in complex with L1-52/55K and L4-22K and pVII (Russell 2009). Furthermore, regulation of the major late promoter (MLP) is thought to be mediated through dimerization of pIVa2 and the non-structural protein L4-33K (Ali, LeRoy et al. 2007).

Mu ( µ) is a 36-aa basic protein. Mu (also called pX) is initially formed from a 79-aa precursor that is cleaved both N- and C-terminally (Anderson, Young et al. 1989). Both Mu and its precursor, contain an NLS (Lee, Lawrence et al. 2004). There are approximately 100 copies of µ per virus particle. Mu is a protamine-like protein and there is no knowledge about its distribution around the viral DNA (Russell 2009). However, Mu has been shown to precipitate with DNA in vitro,

leading to speculation that Mu may help condense viral DNA (Anderson, Young et al. 1989).

The last structural component of the viral core is the terminal protein, which is covalently attached to the viral DNA (Rekosh, Russell et al. 1977). The terminal protein is cleaved by viral protease from its precursor (pTP), a step vital for viral replication. pTP, along with viral polymerase and NF-1 forms the viral transcription complex (Webster, Leith et al. 1997) (Liu, Naismith et al. 2003). TP is approximately 55- kDa in size and consists of approximately 500-aa (Rux and Burnett 2004). Circularization of the viral genome may also be facilitated by TP (Ruben, Bacchetti et al. 1983).

FIGURE 6. Table showing the different components of the Ad virion, their molecular masses, location and general function. Modified with permission from (Rux and Burnett 2004).

ADENOVIRUS GENOME

The Ad genome consists of linear double-stranded DNA covalently connected at its 5´ end to TP (Wadell 2000 ; Rux and Burnett 2004). Ads are classified into the family of small DNA viruses and the genome varies between 34-36 kB in size depending on serotype (Sambrook 1980; Wadell 2000 ) (Chroboczek, Bieber et al. 1992). The genome encodes approximately 40 different proteins (Wadell 2000 ). Transcription occurs from both stands and both directions during replication (Wadell 2000 ). The genome is divided into early, delayed early and major late genes (Sambrook 1980; Wadell 2000 ). The early genes consist of E1A, E1B, E2, E3 and E4. (Chroboczek, Bieber et al. 1992; Kidd, Garwicz et al. 1995). The functions of the early gene products are to induce viral transcription, prime the host cell for S-phase, inhibit apoptosis, enhance viral DNA replication, modulate host cell immune response, regulate viral transcription/translation and facilitate viral mRNA export from the nucleus (Sambrook 1980) (Wadell 2000 ; Hall, Blair Zajdel et al. 2010). The delayed early genes mediate packing of viral DNA and are required for assembly of the virion (Sambrook 1980) (Wadell 2000 ; Hall, Blair Zajdel et al. 2010). The delayed early genes are IX, IVa2 and E2 late (Wadell 2000 ; Hall, Blair Zajdel et al. 2010). The late genes are L1, L2, L3, L4 and L5. The late genes encode the structural proteins that comprise the viral capsid (Wadell 2000 ; Hall, Blair Zajdel et al. 2010).

FIGURE 7. Adenovirus genome organization. Black indicates early genes, blue indicates delayed early genes and green indicates major late genes. Red indicates VA-RNA. With permission from (Hall, Blair Zajdel et al. 2010).

EARLY GENES E1A

The E1A region lies at the 5´ terminus of the viral genome close to the ITR (Mei, Skog et al. 2003). E1A encodes two major proteins, 243R and 289R (Wadell 2000 ; Mei, Skog et al. 2003). E1A transcription is induced by host cell transcription factors as well as auto regulatory positive feedback loops (Kovesdi, Reichel et al. 1987; Kirch, Putzer et al. 1993). E1A-encoded proteins are non-specific transcriptional modulators that impact host cell transcription by interacting with sequence specific host cell transcriptional regulators, such as ATF1, CREB and c-Jun (Hiebert, Blake et al. 1991; Hall, Blair Zajdel et al. 2010).

E1B

The E1B region is located downstream of E1A in the same transcriptional direction. The region encodes two proteins, 19K and 55K. The 19K protein is a functional analogue of Bcl-2 and thus inhibits host cell apoptosis (Chiou, Tseng et al. 1994; Zhao and Liao 2003). 19K binds and inhibits mediator proteins downstream of p53 (Zhao and Liao 2003). These include Nbk/Bik, Bax and Bak (Sundararajan, Cuconati et al. 2001). The 55K protein binds and sequesters p53 in the cytoplasm, promoting p53 degradation in the proteasome, in complex with E4ORF6 (Punga and Akusjarvi 2003). Both E1B gene products are required to counteract host cell apoptosis (Sundararajan, Cuconati et al. 2001; Harada, Shevchenko et al. 2002).

The E2 region lies between the E1B and E2B regions and is transcribed in a “leftward direction” (Mei, Skog et al. 2003). It encodes 3 proteins: adenovirus DNA polymerase (Adpol), preterminal protein (pTP) and DNA-binding protein (DBP) (Liu, Naismith et al. 2003). Adpol is a highly conserved protein belonging to the Pol-α family of protein-primed DNA polymerases (Enomoto, Lichy et al. 1981; Ikeda, Enomoto et al. 1981; Hall, Blair Zajdel et al. 2010). Adpol has intrinsic 3´-5´proofreading exonuclease activity (Field, Gronostajski et al. 1984).

pTP binds viral DNA in a sequence-specific manner while forming a heterodimer with Adpol to initiate viral DNA replication (Webster, Leith et al. 1994). pTP is required for viral DNA replication (Webster, Leith et al. 1994). DBP enables viral DNA replication and elongation. DBP has an ATP- independent helix-destabilizing function and a high affinity for ssDNA (Evans and Hearing 2003).

The E3 region encodes 5-9 different polypeptides depending on the Ad species (Lichtenstein, Toth et al. 2004; Windheim, Hilgendorf et al. 2004). The proteins encoded by the E3 region seem to function in enabling the virus to avoid the host cell immune system (Lichtenstein, Toth et al. 2004; Windheim, Hilgendorf et al. 2004). However, the region is not essential for virus replication in vitro (Lichtenstein, Toth et al. 2004). The proteins of the E3 region are predicted to be transmembrane proteins (with the exception of 12,5K, 14.7K and Ad4-6.3K (Windheim, Hilgendorf et al. 2004). The majority also contain possible transport motifs, and it has been speculated that they may interfere with the host cell protein sorting machinery (Windheim, Hilgendorf et al. 2004). E3-6.7K, -10.4K, -14.4K and -14.7K each inhibit host cell apoptosis (Benedict, Norris et al. 2001). The E3 region also encodes the Ad death protein (ADP), or E3-11.6K (Tollefson, Scaria et al. 1996). E3-11.6K has been proposed to interact with MAD2B and induce cell lysis (Ying and Wold 2003; Windheim, Hilgendorf et al. 2004). Alternative mechanisms to E3-11.6K- mediated cell lysis must exist because species B Ads do not encode E3-11.6K.

The E4 region encodes six known protein products (Leppard 1997). The major function of these is modulation of DNA replication, apoptosis, mRNA nuclear export and transcriptional/translational regulation (Leppard 1997; Branton and Roopchand 2001). The protein products are named after the open reading frames (ORFs): ORF1, ORF2, OF3, ORF4, ORF6 and ORF6/7 (Leppard 1997). ORF3/ORF6 viral double null mutants are severely defective in cell culture growth (Bridge and Ketner 1989). ORF3 augments viral DNA replication

whereas ORF6 forms a complex with E1B55K, enhancing viral gene expression while inhibiting cellular mRNA export (Leppard and Everett 1999; Branton and Roopchand 2001).

DELAYED EARLY GENES

The delayed early genes encode two protein products; pIVa2 and pX. These are discussed under the heading Minor Proteins.

LATE GENES

The late genes include six ORFs (L1-L5 (L6)) (Mei, Skog et al. 2003). The protein products encoded by the late genes compromise the bulk of the viral structural proteins and proteins needed for encapsidation, post-translational modification, scaffolding and transcriptional activation of major late transcription unit (MLTU), excluding pIX and pIVa2 (Greber 1998; Balakirev, Jaquinod et al. 2002; Farley, Brown et al. 2004).

The structural proteins encoded by the late genes are the penton base, precursor pVII, precursor pX (all within the L2 ORF), hexon, precursor pVI (L3 ORF), precursor pVIII (L4 ORF) and the fiber protein (L5 ORF). (Wadell 2000 ; Balakirev, Jaquinod et al. 2002; Mei, Skog et al. 2003). The non structural proteins that are needed for assembly are 52/55K, precursor pIII a (L1 ORF), 23K cysteine protease (L3 ORF), 100K and 33K (L4 ORF) (Oosterom-Dragon and Ginsberg 1981; Gustin, Lutz et al. 1996; Greber 1998; Wadell 2000 ). In species B Ads (Ad7, Ad11 and Ad35) there is a putative ORF located downstream of L5 (Mei, Skog et al. 2003). The L6 ORF may encode a gene product designated agnoprotein, but its function or structure is unknown (Mei, Skog et al. 2003).

ADENOVIRUS LIFE CYCLE

RECEPTORS

Human Ads are transmitted through droplets, the fecal-oral route, latent viruses originating from lymphocytes in the transplant setting and through iatrogenic spread (infected instruments) (Foy, Cooney et al. 1968; Fox, Brandt et al. 1969; Jernigan, Lowry et al. 1993; Lichtenstein and Wold 2004). Human Ads seem to utilize a number of different cell surface receptors, the best-known of which are discussed below.

Coxsackie and Adenovirus Receptor (CAR)

In 1976, Lonberg-Holm discovered that some coxsackie B viruses and group C Ads competed for the same receptor site (Lonberg-Holm, Crowell et al. 1976). In 1997 and 1998 two separate groups isolated the common receptor and named it the coxsackie adenovirus receptor (CAR) (Bergelson, Cunningham et al. 1997; Roelvink, Lizonova et al. 1998). CAR is a type I transmembrane protein that belongs to the immunoglobulin super family (Wang and Bergelson 1999). The size of CAR varies between 40-46 kDa, probably due to different glycosylation patterns (Tomko, Xu et al. 1997). CAR consists of two extra cellular N-terminal immunoglobulin (Ig) domains (D1 and D2), a transmembrane helix domain and a C-terminal intracellular domain (Freimuth, Springer et al. 1999). The physiological function of CAR seems to be inter cellular adhesion between epithelial cells through formation of CAR D1 homodimers (Cohen, Gaetz et al. 2001; Cohen, Shieh et al. 2001; Philipson and Pettersson 2004). CAR is expressed in the epithelial linings of the human brain, lung, heart, liver, pancreas, colon, small intestine, prostate and testis (Philipson and Pettersson 2004). CAR is not expressed in the spleen, placenta, thymus, ovary and skeletal muscle and is expressed to a limited extent on hematopoietic cells (Philipson and Pettersson 2004). Ad serotypes belonging to species A, C, D, E and F have been shown to bind CAR in vitro ; however, species B Ads do not exhibit this binding (Roelvink, Lizonova et al. 1998).

The binding of Ad12 to CAR has been resolved by crystallization studies that demonstrate the interaction of the CAR D1 domain with lateral loops AB, CD, and DG of one fiberknob monomer and the FG loop of another adjacent fiberknob monomer (Bewley, Springer et al. 1999). The cleft formed between the AB and DE loops of the fiberknob is responsible for the majority of the interaction, which is probably mediated by water molecules in the cleft (Bewley, Springer et al. 1999). In the context of the binding of the entire adenovirus virion to CAR, the interaction between CAR and the lateral portion of the fiberknob poses some steric difficulty for the interaction of the RGD domain of the penton base with the integrins on the cell surface (van Raaij 1999.; Wu, Pache et al. 2003). Therefore, fiber shaft length and flexibility affect the ability to bind CAR (Wu, Pache et al. 2003; Zubieta, Schoehn et al. 2005). C-terminus-truncated or GPI-anchored CAR mediates Ad uptake as efficiently as wt CAR, suggesting that CAR is only an attachment receptor (Bergelson 1999; Wang and Bergelson 1999). Interestingly CAR is expressed laterally and basolaterally in polarized epithelial cells (Walters, Grunst et al. 1999). CAR mRNA expression poorly matches in vivo tropism and removal of Ad CAR binding capacity does not change the distribution of the vector in vivo (Tomko, Xu et al. 1997; Alemany, Gomez-Manzano et al. 2001). This has led to questioning of the role of CAR as the primary attachment receptor in vivo and to the suggestion that CAR instead facilitates viral escape from the infected tissue (Walters, Freimuth et al. 2002). Viral production of excess fibers that are secreted into the tight junctions could dissociate intercellular CAR homodimers, thus opening the tight junctions and facilitating viral escape (Walters, Freimuth et al. 2002). Counterarguments to this hypothesis have also been raised; in the case of airway epithelia, it has been suggested that Ads could infect specialized non-polarized cells that express CAR on the luminal membrane instead of relying on CAR in the tight junction as an attachment receptor (Arnberg 2009). Furthermore, lesions (e.g. Influenza A virus infection) of the airway epithelia could expose CAR at the basolateral membranes (Meier and Greber 2004).

Integrins

Integrins are members of a family of transmembrane intercellular and matrix adhesion molecules that are composed of two subunits, α and β (Hynes 2002). Eighteen α subunits and eight β subunits have been identified in mammals (Hynes 2002). The subunits can heterodimerize to form at least 24 different heterodimers (Luo, Carman et al. 2007). Integrins mediate binding to extra cellular matrix (ECM) molecules, such as vitronectin, fibronectin, collagens, laminins and proteoglycans and serve as receptors for a variety of pathogens (Ruoslahti and Pierschbacher 1987; Arnaout 2002; Arnaout, Goodman et al. 2002; Luo, Carman et al. 2007). In addition, integrins play a critical role in regulating cellular adhesion, migration and proliferation (Gonzalez, Devoto et al. 2001; Gonzalez, Gonzales et al. 2002). Ligand binding to integrins is dependent on divalent cations (Ruoslahti and Pierschbacher 1987). Integrin binding motifs are the RGD and Leu- Asp-Val (LDV) motifs (Ruoslahti and Pierschbacher 1987). The Ad penton base contains the exposed RGD motif, which is conserved in all human Ads except Ad40 and 41, which belong to species F (Albinsson and Kidd 1999; Zubieta, Schoehn et al. 2005). Integrins are required for the internalization of Ads (Wickham, Mathias et al. 1993). Ad 40 and 41, which lack the RGD motif, exhibit delayed uptake in epithelial cells (Albinsson and Kidd 1999; Zubieta, Schoehn et al. 2005). The function of the interaction of integrins with Ads seems to be that of a co-receptor mediating uptake (Wickham, Mathias et al. 1993). αvβ1, αvβ3, αvβ5, αMβ2 and α3β1 integrins have all been shown to bind the Ad penton RGD motif (Mathias, Wickham et al. 1994; Huang, Kamata et al. 1996; Salone, Martina et al. 2003). Ligand binding to integrins causes receptor clustering on the cell surface and a change in the conformation of the integrin from an unfolded to a folded state (Arnaout 2002; Arnaout, Goodman et al. 2002; Hynes 2002). In turn, this activation causes a conformational change in the cytoplasmic tail, inducing interactions with downstream signaling molecules (Arnaout 2002; Arnaout, Goodman et al. 2002; Takagi, Suzuma et al. 2002). The Ad-integrin interaction triggers the mitogen- activated protein kinase (MAPK) and phosphoinoside-3-kinase (PI3K) pathway, thus affecting the actin cytoskeleton through Rho, Rac and Cdc42 (Li, Stupack et al. 1998; Greber 2002).

CD46

CD46 is a complement regulatory protein that is expressed on all nucleated cells (Liszewski, Post et al. 1991; Liszewski, Kemper et al. 2005). CD46 is also a transmembrane type I glyco protein (Barilla- LaBarca, Liszewski et al. 2002). C3b and C4b are natural ligands of CD46 (Cattaneo 2004). CD46 mediates their breakdown and protects host cells from homologous complement attack by acting as a cofactor for serine protease factor I, which mediates the cleavage of C3b and C4b (Cattaneo 2004). CD46 was identified as a C3b- and C4b-binding protein in co- precipitation studies of extracts from human peripheral blood cells (PBMC) (Liszewski and Atkinson 1996; Riley-Vargas 2004). Human CD46 is alternatively spliced, thus creating a distinctive 51-58-kDa and 59-68-kDa “double band” on western blots (Liszewski, Kemper et al. 2005). The gene encoding CD46 lies at 1q3.2 within the regulators for complement (RCA) cluster (Liszewski and Atkinson 1996). CD46 is composed of an extra cellular domain, a transmembrane domain and an intracellular domain (Riley-Vargas 2004). The extra cellular domain consists of 4 apical repetitive sequences termed short consensus repeats (SCR) or complement control protein (CCP) and a basal serine, threonine, proline-rich domain (STP) that is heavily O- glycosylated (Seya, Taniguchi et al. 1999). The 3 exons encoding the STP domain give rise to the A, B and C isoforms (Liszewski, Kemper et al. 2005). N-glycosylation motifs (Asn-X-[Thr/Ser]) are located in all 4 SCR domains (Maisner, Alvarez et al. 1996). There are two intracellular domains due to alternative splicing termed CYT1 (C1) and CYT2 (C2), with distinctive properties and variation in amino acid length (Liszewski, Kemper et al. 2005). The C1 domain consists of 16 amino acids, which have a putative tyrosine phosphorylation motif for protein kinase C (PKC) and casein kinase 2 (CK-2) (Wang, Liszewski et al. 2000). The C2 domain consists of 23 amino acids and is tyrosine phosphorylated by Src family kinases in T-cells (Wang, Liszewski et al. 2000). CD46 is highly expressed on the epithelial linings of exocrine glands and ducts, as well as- on kidney tubules and glomerular epithelium (Johnstone, Loveland et al. 1993). Alternative splicing of CD46 gives rise to a total of 6 isoforms: ABC1, ABC2, BC1, BC2, C1 and C2 (Seya, Taniguchi et al. 1999; (Liszewski, Kemper et al. 2005). Of these the most abundantly expressed are BC1, BC2, C1 and C2 (Johnstone, Loveland et al. 1993) (Russell, Johnstone

et al. 1992; Russell, Loveland et al. 1992; Russell, Sparrow et al. 1992). Most cells express all four isoforms; however, some tissue discrepancies have been noted in kidney and fetal heart (predominance of BC), brain (predominance of C2) and spermatozoa (predominance of hypoglycosylated C2) (Johnstone, Loveland et al. 1993) (Russell, Johnstone et al. 1992; Russell, Loveland et al. 1992; Russell, Sparrow et al. 1992; Seya, Hara et al. 1993) (Riley, Tannenbaum et al. 2002).

SCR

STP

CYT

FIGURE 8. Schematic structure of CD46; Short consensus repeats (SCR), serine-threonine-proline rich domain (STP), transmembrane (TM) and cytoplasmatic domain (CYT) are indicated.

CD46 is constitutively recycled from the cell surface via clathrin coated pits and transported to perinuclear multivesicular bodies (Crimeen-Irwin, Ellis et al. 2003). The half-life of CD46 is 8-12 h in human cells (Crimeen-Irwin, Ellis et al. 2003). Antibody cross-linking of CD46 induces macropinocytosis followed by rapid intracellular degradation (Crimeen-Irwin, Ellis et al. 2003).

CD46 has also been shown to be released on vesicles from tumour cells, but also in a soluble form (probably processed by ADAM family MMPs) (Hakulinen, Junnikkala et al. 2004). CD46 has been termed a pathogens magnet; HHV-6, the Edmonston strain of the measles virus, BVDV, N. gonorrhoeae , N. meningitidis , S. pyogenes and group B adenoviruses use CD46 as a receptor (Segerman 2003; Cattaneo 2004.; Marttila, Persson et al. 2005). Measles virus (MV), Ad11 and Ad35 binding to CD46 leads to downregulation of cell surface CD46 (Galbraith, Tiwari et al. 1998; Sakurai F 2007; Gustafsson et al. 2010). MV binding to CD46 on epithelial cells leads to rapid internalization (Galbraith, Tiwari et al. 1998). MV-mediated CD46 downregulation alters MHC class I or class II presentation of antigens (Gerlier, Trescol-Biemont et al. 1994; Gerlier, Trescol-Biemont et al. 1994). Piliated Neisseria gonorrhoeae induces shedding and surface redistribution of CD46 (Gill, Koomey et al. 2003; Gill DB 2005). Ad35 binding to CD46 has been shown to mediate the reduction of IL-12 levels in IFN- or lipopolysaccaride- stimulated human peripheral blood mononuclear cells (Iacobelli- Martinez, Nepomuceno et al. 2005). The biological implications of CD46 modulation are not fully understood (Liszewski, Kemper et al. 2005). Studies in transgenic mice have shown that human CD46 CYT1 inhibits, and CD46 CYT2 increases the contact hypersensitivity reaction in mouse T-cells (Marie, Astier et al. 2002). In addition CD46 internalization renders cells more susceptible to complement-mediated lysis (Schnorr, Dunster et al. 1995; Schneider-Schaulies, Schnorr et al. 1996). Ad species B binds CD46 at the SCR1 and SCR2 N-terminal repeats (Fleischli C 2007 ). Ad11 can bind three CD46 molecules per fiberknob (Persson, Reiter et al. 2007). The contact region of Ad11 and CD46 can be divided into three parts (Persson, Reiter et al. 2007). In the first part the HI loop of Ad11 contacts side residues of SCR1. A large van der Waals force is created by Tyr36 which increases shape complemetarity between the fiberknob and CD46 (Persson, Reiter et al. 2007). Hydrogen bonds and the polar interaction between (Ad11) Asp284 and (CD46) His34 also contribute to interactions in this region (Persson, Reiter et al. 2007). The second area of interactions is formed by the interaction of the HI and DG loops with SCR1 and SCR2 (Persson, Reiter et al. 2007). A salt bridge between (Ad11) Arg280 and (CD46) Glu63 is formed, and the hydrophobic portion of (Ad11) Arg280 docks against the (CD46) Phe35 side chain in the

SCR1 domain (Persson, Reiter et al. 2007). The region of contact is formed by the IJ loop and the SCR2 domain creating two hydrogen bonds (Persson, Reiter et al. 2007). The Ad11-CD46 interaction creates an alteration in the angle between the SCR1 and SCR2 domains, reducing the bend from approximately 60º to 0º. This is the first interaction between virus and receptor known to result in a conformational change of the receptor (Persson, Reiter et al. 2007).

FIGURE 9. Ribbon drawing of the Ad11 fiberknob in complex with CD46 SCR1-2. The subunits of the Ad11 fiberknob are in green, grey and blue. CD46 SCR1-2 is depicted in red. With permission from (Persson, Reiter et al. 2007).

a b c

FIGURE 10.The three major contact regions between the Ad11 fiberknob and CD46 (a-c) are depicted.Structural changes of CD46 upon binding the Ad11 knob (d): the unbound state is indicated in blue, the bound state in red.The arrow shows the rearrangement of SCR1 upon Ad11 knob binding. With permission from (Persson, Reiter et al. 2007).

CD80/86

CD80 and CD86 are both co-stimulatory ligands expressed on antigen-presenting cells (APC) that augment the T-cell response through interaction with CD28 (Linsley, Brady et al. 1991) or inhibit T-cell responses by interacting with CTLA-4 (Walunas, Bakker et al. 1996). Both are type 1 transmembrane proteins and have been implicated in signalling to T-cells (Doty and Clark 1996; Orabona, Grohmann et al. 2004). Species B1 and B2 Ads (Ad 3, 7, 11 and 35) have been shown to infect CD80/86- expressing CHO cellines (Short, Vasu et al. 2006). However another study reported very low infection of Ad3 in CD80/86-expressing CHO cell lines (Hall, Blair Zajdel et al. 2009). Thus, the importance of CD80/86 as species B Ad receptors remains unclear.

Dipalmitoyl phosphatidylcholine (DPPC)

DPCC is a component of lung surfactant, corresponding to approximately 50% of the total phospholipd content (Veldhuizen, Batenburg et al. 2000). DPCC covers the inner surface of the lung alveoli and is secreted by alveolar type II epithelial cells (Veldhuizen, Batenburg et al. 2000). DPCC has been shown to bind the Ad2 hexon protein (Balakireva, Schoehn et al. 2003). Surfactant has been shown to promote Ad5 vector transfection of A549 cells and both rat and rabbit lung tissue (Jobe, Ueda et al. 1996; Katkin, Husser et al. 1997; Balakireva, Schoehn et al. 2003). This is interesting in the setting of Ad-induced pneumonia, because DPCC is re-circulated, and returns to the alveolar epithelium during surfactant turnover (Balakireva, Schoehn et al. 2003), which provides a possible viral entry mechanism.

Coagulation Factors

Factors IX, VII and X and protein C have been shown to increase Ad5 hepatocyte transduction (Shayakhmetov, Gaggar et al. 2005; Parker, Waddington et al. 2006). Ad5 was shown to bind heparin sulfate proteoglycans (HSPG) using FIX and C4BP as a “molecular bridge” (Shayakhmetov, Gaggar et al. 2005). Furthermore the depletion of

vitamin K-dependent zymogens by warfarin reduced Ad5 transduction of hepatocytes in vivo (Parker, Waddington et al. 2006). FX was shown to bind the central depression of the Ad5 hexon (Waddington, McVey et al. 2008). The hexon-FX binding appears to be calcium- dependent. The efficacy of transduction mediated by FX was superior to that of other coagulation factors (Waddington, McVey et al. 2008). Extrahepatic tropism does not seem to be determined by the hexon-FX interaction in the case of intramuscular injections, but FX has been shown to increase the transduction of respiratory tumours transplanted in nude mice (Gimenez-Alejandre, Cascallo et al. 2008). Whether the increased transduction of the implanted tumors is a reflection of the in vivo situation or simply a consequence of the model system used, remains to be determined.

Heparan sulfate proteoglycans (HSPG)

HSPG is a glycosaminoglycan abundantly present on a number of human cell types (Raman, Venkataraman et al. 2006; Sasisekharan, Raman et al. 2006). The KKTK motif in the shaft of the Ad fiber is a theoretical HSPG-binding motif (Smith, Idamakanti et al. 2003). Direct binding between the KKTK motif and HSPG has not been demonstrated. Fiberknobs Ad35 and Ad3 also interact to some extent with HSPG (Tuve, Wang et al. 2008).

Major Histocompatibility Complex (MHC)-1

Ad5 has been shown to bind the α2 domain of the MHC-1 heavy chain (Hong, Karayan et al. 1997). Transfection of Daudi cells with the MHC-1 α2 domain significantly increased Ad5 attachment (Hong, Karayan et al. 1997). CHO-MHC-1α2 cells were transduced more efficiently by Ads (McDonald, Stockwin et al. 1999). However the role of MHC-1 as a receptor for adenoviruses is controversial (Davison, Kirby et al. 1999).

Vascular cell adhesion molecule 1 (VCAM-1)

VCAM-1 belongs to the same immunoglobulin superfamily as CAR; its main function is as a receptor for leukocytes carrying the α4β1 integrin (Elices, Osborn et al. 1990; Hemler, Elices et al. 1990).

Expression of VCAM-1 on NIH-3T3 cells increases Ad5 transduction, possibly because of the homology between CAR D1 and the VCAM-1 D7 domain (Chu, Heistad et al. 2001). The role of VCAM-1 expressed apically on endothelial cells as a receptor for Ads in vivo is unclear (Arnberg 2009).

Lactoferrin

Lactoferrin is an iron-binding, 80-kDa, single polypeptide chain glycoprotein, found in mucosal secretions but also present in neutrophilic granules (Baggiolini, De Duve et al. 1970; Baker and Baker 2005; Legrand, Pierce et al. 2008) (Arnberg 2009). Lactoferrin has direct antimicrobial activity against Gram-negative bacteria, partly through its iron and non iron-dependent bacteriostatic/bactericidal properties (Tomita, Bellamy et al. 1991; Bellamy, Takase et al. 1992; Bellamy, Takase et al. 1992). Lactoferrin is abundant in respiratory mucosa and tear fluid but is only found in concentrations of 0.2-0.6 µg/ml in blood (Erga, Peen et al. 2000). Lactoferrin promotes the infection of respiratory mucosa and conjunctiva (even CAR of negative cells) by species C Ads (Garnett, Erdman et al. 2002). Lactoferrin appears to be a non CAR-dependent bridging molecule between Ads and the host cell (Johansson, Nilsson et al. 2007).

Sialic Acid

Sialic acid is the unifying name of a large group of saccharides with a neuraminic acid backbone (Angata and Varki 2002). Because sialic acid is located on the terminal ends of glycan chains, the sialic acid moieties are readily available on the cell surface (Angata and Varki 2002). Species D Ads, specifically Ad8, Ad19 and Ad37, use sialic acid as a receptor (Arnberg, Mei et al. 1997; Arnberg, Edlund et al. 2000; Arnberg, Kidd et al. 2000). All three Ads have positively charged fiberknobs (Arnberg, Mei et al. 1997; Arnberg, Kidd et al. 2000). The fiberknob of Ad37 has been co-crystallized with sialyl lactose. The results suggest that Lys245 in the fibeknob interacts via a salt bridge with the carboxy acid group of sialic acid (Burmeister, Guilligay et al. 2004).

Desmoglein 2 (DSG-2)

The most recently identified Ad receptor is desmoglein 2 (DSG-2) (Wang, Li et al. 2011). DSG-2 is a part of the desmosome (Delva, Tucker et al. 2009). The desmosome is an adhesive intercellular junction, found in tissues subjected to mechanical stress, such as skin, bladder, gastrointestinal mucosa and myocardium (Delva, Tucker et al. 2009). DSG-2 functions as an intercellular adhesion molecule (Chitaev and Troyanovsky 1997). DSG-2 belongs to the cadherin protein family and is a calcium-binding transmembrane glycoprotein (Delva, Tucker et al. 2009). There are four known human desmoglein isoforms (DSG1-4) (Delva, Tucker et al. 2009). DSG-2 is expressed in all desmosome-containing tissues (Delva, Tucker et al. 2009). DSG-2 is also over-expressed in a number of epithelial malignancies (Wang, Li et al. 2011). Wang et al . identified DSG-2 as an attachment receptor for Ad3, Ad7, Ad11 and Ad14 (Wang, Li et al. 2011). Interestingly binding of Ad3 to DSG-2 seems to result in a transient opening of intercellular junctions (Wang, Li et al. 2011). Because there are indications that CD46 may be trapped in intercellular junctions, at least in polarized epithelial cancer cell cultures, DSG-2 binding may expose of CD46 to species B Ads (Wang, Li et al. 2011).

INTERNALIZATION

Viruses have been shown to utilize a number of naturally occurring endocytic pathways to gain access to the host cell after attachment to their receptor/receptors. Ads have been shown to use two different internalization processes thus far: clathrin-mediated endocytosis (Ad2, Ad5) and macropinocytosis (Ad3, Ad35) (Greber, Willetts et al. 1993; Amstutz, Gastaldelli et al. 2008; Kalin, Amstutz et al. 2010). Ad2/5 binding to the cell surface is mediated by the fiber-CAR interaction, followed by the association of integrins with the pentonbase RGD motif (Li, Stupack et al. 1998). Binding of the penton base RGD motif to the αvβ integrins leads to a conformational change in the integrins, which in turn leads to the activation of downstream signalling molecules such as PI3K (Li, Stupack et al. 1998). This signaling cascade probably leads to the local recruitment of clathrin by Hip1/Hip1R, followed by the actin-dependent formation of flattened coated pit zones at the plasma membrane (McPherson 2002). Ad5 internalization activates PI3K which in turn leads to

activation of Rab5, Rac1, Cdc42 and PKC (Zerial and McBride 2001). The downstream signaling events induce polarization of the actin filaments, a process vital for clathrin-mediated endocytosis (Wang, Huang et al. 1998). The clathrin-coated vesicles fuse into endosomes and the virions are released into the cytosol approximately 15min after binding to the cell surface receptors (Greber and Kasamatsu 1996; Greber, Webster et al. 1996).

Endocytosis of species B adenoviruses

In the case of species B Ads (Ad3, Ad35), uptake is mediated through macropinocytosis in epithelial and hematopoietic cells (Greber, Willetts et al. 1993; Amstutz, Gastaldelli et al. 2008; Kalin, Amstutz et al. 2010). Macropinocytosis leads to the formation of large vacuoles at the cellular periphery, sometimes preceded by membrane ruffling (Doherty and McMahon 2009). Macropinocytosis can be divided into a recycling and processive form (Meier and Greber 2004). Processive macropinosomes shrink and mix with late endosomes and lysosomes (Racoosin and Swanson 1993). Recycling macropinosomes are formed in response to ligand binding to receptors, such as EGFR or NGFR, and can regulate surface receptor concentrations (Hewlett, Prescott et al. 1994). Both forms share several common features, such as F-actin surface ruffling, PKC dependence and the formation of large vesicles: however, progressive macropinocytosis can be initiated by ανβ3/β5 and α5β1 signaling and is not dynamin-dependent (Boleti, Benmerah et al. 1999; May 2001; May and Machesky 2001; May and Machesky 2001). Macropinocytosis induction by Ad3 has been shown to require both CD46 and αν integrins (Amstutz, Gastaldelli et al. 2008). Integrin signaling has been implicated in macropinocytosis, and antibody cross-linking of CD46 induces the same endocytic pathway (Crimeen- Irwin B 2003 ; Wu, Booth et al. 2006). Ad3 appears to use macropinocytosis as a major endocytic pathway, while also utilizing a clathrin and dynamin-dependent pathway in some cell types (Amstutz, Gastaldelli et al. 2008). Endocytic uptake of Ad3 is dependent on F- actin, Rac1, PAK1, CtBp1, PKC and the sodium-proton exchanger 1 (Amstutz, Gastaldelli et al. 2008). Ad35 also induces macropinocytotic uptake upon binding CD46 and integrin co-receptors (Kalin, Amstutz et al. 2010). The use of both macropinocytosis and clathrin-mediated endocytosis by species B Ads

to gain entry to the host cell appears to follow the constitutive recycling of CD46 and macropinocytosis triggered by ligand cross- linking of CD46 (Crimeen-Irwin, Ellis et al. 2003; Amstutz, Gastaldelli et al. 2008).

UNCOATING, ENDOSOMAL RELEASE AND INTRACELLULAR TRANSPORT

The escape of Ads from clathrin-coated pits or macropinosomes is poorly understood (Meier and Greber 2004). The most-studied Ads are members of species C. However, the escape of Ads (species A, C, D, E and F) from the endosome occurs rapidly after attachment. After 15 minutes, fiberless virions can be detected in the cytosol; however species B Ads can remain in the endosomal pathway for up to 8 hours (Greber and Kasamatsu 1996; Miyazawa, Leopold et al. 1999; Miyazawa, Crystal et al. 2001). After endocytosis the goal of infection is to deliver the viral genome to the nucleus of the host cell so that viral replication can occur. There are three events that must precede viral replication: viral uncoating, endosomal escape and transport to and through the nuclear membrane. Viral uncoating occurs in steps, the first of which is the dissociation of the fiber or the entire penton from the capsid (Prage, Pettersson et al. 1968; Greber, Willetts et al. 1993; Nakano, Boucke et al. 2000). After penton dissociation the peripentonal hexons , IIIa, VIII, IX and protein VI are released from the capsid (Greber, Willetts et al. 1993). Uncoating appears to be linked to endosomal penetration (Greber, Willetts et al. 1993). It has been shown that acidic pH in conjunction with penton binding to ανβ5 integrins permeabilizes endocytic vesicles (Wickham, Filardo et al. 1994; Wang, Guan et al. 2000). In addition, immunodepletion of protein VI eliminates nearly all Ad membrane penetration capability (Wiethoff, Wodrich et al. 2005). Protein VI contains a N-terminal 80-aa amphipathic helical structure that is highly conserved among Ads (Wiethoff, Wodrich et al. 2005). The observed pH dependency during endosomal escape may be related to uncoating, while membrane penetration is likely to be facilitated to 95% by protein VI (Smith, Wiethoff et al. 2010). One theory is that integrin binding exposes a cleavage site on protein VI. Inside the reducing environment of the endosome the Ad 23K cysteine protease becomes activated and cleaves protein VI, thus activating its membrane lytic properties. Protein VI degradation would then enable

the escaped remnant of the Ad capsid to dissociate from the viral DNA during docking with the nuclear pore complex (Greber and Kasamatsu 1996; Greber 1998; Smith, Wiethoff et al. 2010).

TRANSNUCLEAR TRANSPORT

After endosomal escape, transport of the post-endosomal capsid is bidirectional along the microtubules (Suomalainen, Nakano et al. 1999; Leopold, Kreitzer et al. 2000). Accumulation of post-endosomal capsids can be observed at the nuclear pore complexes (NPC) 1 hour p.i (Suomalainen, Nakano et al. 1999; Leopold, Kreitzer et al. 2000). Transport along the microtubule is mediated by cytoplasmic dynein and probably kinesin (Leopold, Kreitzer et al. 2000; Scherer and Vallee 2011). The hexon mediates binding to the intermediate chain (IC) and light intermediate chains (LIC) of dynein after exposure to pH 4.5-5.4 (Scherer and Vallee 2011)(Sherer 2011). Protein VI also exposes a PPXY motif during capsid disassembly, which recruits Nedd4 E3 ubiquitin ligases (Wodrich, Henaff et al. 2010). This results in ubiquitylation of protein VI and rapid microtubule-dependent trafficking towards the nucleus (Wodrich, Henaff et al. 2010). Activation of the PKA and p38/MAPK cascade promote the MT- dependent dynein/casein transport towards the nucleus (Meier and Greber 2004). The speed of movement (approximately 1 µm/min) depends on the integrin-p38/MAPK pathway (Suomalainen, Nakano et al. 1999; Suomalainen, Nakano et al. 2001). When the capsid remnants arrive at the microtubule-organizing center (MTOC) detachment from MT is likely mediated by chromosome region maintenance 1 (CRM1) a nuclear export factor or a nuclear factor exported by CRM1 (Strunze, Trotman et al. 2005). The NPC serves as a docking site for the capsid remnants, harbours capsid dissociation activity and imports viral DNA and associated protein VII to the host cell nucleus (Greber, Suomalainen et al. 1997). Docking occurs between the hexon and the NPC receptor CAN/Nup214 (Trotman, Mosberger et al. 2001). Further disassembly/import occurs at the NPC (Greber, Suomalainen et al. 1997). Soluble histone H1 then binds to a loop of the adenovirus hexon, thereby enabling the histone H1 import factor Imp7/Imp β to bind histone H1 already bound to the hexon (Suomalainen, Nakano et al. 1999; Saphire, Guan et al. 2000; Suomalainen, Nakano et al. 2001) (Leopold, Kreitzer et al. 2000; Trotman, Mosberger et al. 2001; Mabit,

Nakano et al. 2002). Hsc 70 has also been shown to interact with the capsid during NPC docking(Saphire, Guan et al. 2000). The interactions at the NPC lead to a conformational change of the capsid remnants, enabling protein VII to interact with transportin and leading to the import of the histone H1-AdDNA-proteinVII complex into the nucleus (Trotman, Mosberger et al. 2001; Hindley, Lawrence et al. 2007).

Attachment Endocytosis

Uncoating Nuclear import

Endosomal escape

Microtubuli transport

FIGURE 11. General cell entry pathway for adenoviruses. Adapted with permission from (Nemerow, Pache et al. 2009).

REPLICATION, EXPRESSION, ASSEMBLY AND RELEASE

Upon Ad DNA entry, nuclear transcription begins at the periphery of the genome, followed by the center of the DNA structure (Lechner and Kelly 1977). E1, E3 and E4 are transcribed first, which activates transcription, induces S-phase and mediates mRNA nuclear export and transcriptional and translational regulation (Binger and Flint 1984). This enables transcriptional activation of the E2 early promoter, leading to transcription of the E2 gene products Adpol, TP and DBP (Bagchi, Raychaudhuri et al. 1990; Raychaudhuri, Bagchi et al. 1990). Adpol, using TP and cytosine monophosphate as primers, initiates viral DNA replication approximately 5-8 hours post infection (Lechner and Kelly 1977). Viral DNA replication is a two-step process, Type I and Type II (Lechner and Kelly 1977). Type I replication utilizes only one strand of the DNA duplex as a template, resulting in a replication product consisting of a duplex and a single strand of DNA (Lechner and Kelly 1977). Type II replication is the second stage of DNA replication in which the single strand of DNA is circularized by annealing to its own self- complementary termini, forming a circular panhandle structure (Lechner and Kelly 1977). The duplex structure at the panhandle is recognized by the virus replication machinery and replication is initiated. Transcription of late genes begins after DNA replication. Hexon monomers assemble into trimers with the aid of the L4-100K scaffold protein (Hong, Szolajska et al. 2005; Franqueville, Henning et al. 2008). The hexon trimers must associate with protein VI to gain access to the nucleus after translation and trimerization (Kauffman and Ginsberg 1976). In distinction to the hexon, both the fiber and penton contain a NLS signal and are likely imported independently into the nucleus (Horwitz, Scharff et al. 1969; Fender, Ruigrok et al. 1997). Trimerization of the fiber and pentamerization of the pentonbase occurs outside of the nucleus, while pentons probably form inside the nucleus (Fender, Boussaid et al. 2005). Inside the nucleus, L4-33K acts as a scaffolding protein during the formation of the procapsid (Fessler and Young 1999). After capsid formation viral DNA is directed into the capsid, which is probably mediated by protein IVa2 (Ostapchuk and Hearing 2005; Ostapchuk, Yang et al. 2005). L1-

52/55K and L4-22K promote encapsidation and bind to the packaging sequence of the DNA (Perez-Romero, Tyler et al. 2005; Ewing, Byrd et al. 2007). After DNA has been incorporated into the capsid, it is sealed, and the final maturation is achieved trough the activity of the L2-23K cysteine protease (Mangel, McGrath et al. 1993; Gupta, Mangel et al. 2004). L2-23K cleaves the precursor for protein Mu, TP, VI, VII and VIII (Zhang and Imperiale 2003). During the late stages of species C Ad infection, E3-11.6K accumulates and localizes to the nuclear membrane, the Golgi apparatus and the endoplasmatic reticulum (Tollefson, Ryerse et al. 1996; Tollefson, Scaria et al. 1996). E3-11.6K then comes in contact with MAD2B (Ying and Wold 2003). The exact mechanism of the E3-11.6K-MAD2B interaction is poorly understood; however, the L3 protease cleaves cytokeratin, priming the cell for lysis (Chen, Ornelles et al. 1993). Species B Ads do not encode E3-11.6K; therefore, their mechanism of cellular lysis has not been determined (Hall, Blair Zajdel et al. 2010).

THE COMPLEMENT SYSTEM

The complement system was discovered over 100 years ago by Ehrlich, Bordet and Nuttall, who initially observed the heat-sensitive bactericidal effect of blood on Anthrax bacteria (Ehrnthaller, Ignatius et al. 2011). In 1899, heat-labile alexin was renamed “complement” (Ehrnthaller, Ignatius et al. 2011). Complement is a part of the innate immune system and consists of 30 soluble and membrane-bound proteins (Kim and Song 2006). The complement cascade protects the host from infections by lysing target cells, facilitating opsonization leading to phagocytosis of pathogens and clearance of antibody-antigen complexes. This provides a bridge between the innate and adaptive immune systems by binding opsonized antigens via C3d to the complement receptor 2 (CR2) on B-cells, thus facilitating antibody production and differentiation of memory B-cells (Ehrnthaller, Ignatius et al. 2011). Circulating complement proteins are activated by proteolytic cleavage and the resulting products in turn activate further downstream complement proteins (Oksjoki, Kovanen et al. 2007). The sequential activation leads to an expanding cascade in which a few activated proteins subsequently generate thousands of active downstream mediators (Oksjoki, Kovanen et al. 2007; Ricklin, Hajishengallis et al.

2010). Three different pathways of complement activation have been described: the classical pathway, the lectin-activated pathway and the alternative pathway (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). The classical pathway is activated when the pattern recognition molecule (PRM) C1q recognizes the Fc region of antigen-bound IgM or IgG antibodies (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). In addition C-reactive protein (CRP) binding to microbes activates C1q-mediated cleavage of C4 and C2 to form the C3 convertase complex (C4b2b) (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). Cleavage of C4 into C4a and C4b exposes a hidden thiolester group which leads to the covalent deposition of C4b on surfaces close to the cleavage site. C4b deposition leads to opsonization (Oksjoki, Kovanen et al. 2007; Ricklin, Hajishengallis et al. 2010; Ehrnthaller, Ignatius et al. 2011). The lectin pathway activates the complement cascade after plasma mannan-binding lectins (MBL), acting as PRMs, bind carbohydrate groups on bacteria or micro organisms (Oksjoki, Kovanen et al. 2007; Ricklin, Hajishengallis et al. 2010; Ehrnthaller, Ignatius et al. 2011). Mannan-binding, lectin-associated serine proteases (MASP) cleaves C4 and C2 generating C4b2b (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). The activation of complement- mediated lysis via the alternative pathway is somewhat different than the classical and lectin pathways. In the alternative pathway there is a constant, low-grade “tick-over” activation of C3 (Ricklin, Hajishengallis et al. 2010). A fraction of C3 is hydrolyzed, bound by factor B, and then cleaved by factor D. This generates a solvent-based C3 convertase that generates C3a and C3b. C3b is quickly regulated on human cells, but leads to complement attack on foreign cells (Ricklin, Hajishengallis et al. 2010). If C3b forms a complex with C3 convertase, C4b2b3b and C3bC3bBb (C5 convertases) are formed (Oksjoki, Kovanen et al. 2007; Ricklin, Hajishengallis et al. 2010; Ehrnthaller, Ignatius et al. 2011). These two C5 convertases cleave C5 generating C5a and C5b. C5b in conjunction with C6, C7, C8 and C9 form the (C5b-9) membrane attack complex (MAC) (Oksjoki, Kovanen et al. 2007; Ricklin, Hajishengallis et al. 2010; Ehrnthaller, Ignatius et al. 2011). Because the activation of complement is an expanding cascade and “tick-over” activation is constantly occurring, tight regulation of the complement cascade is needed to avoid attacking on healthy host cells

(Ricklin, Hajishengallis et al. 2010). Complement regulating proteins are found in the fluid phase and on cell membranes (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). These proteins exert such an efficient regulation of the complement cascade that 30- 100 molecules of C3 must be cleaved in order to cleave C5 and 10 C5 molecules must be cleaved to generate one C5b-9 (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). The membrane-bound proteins responsible for regulating complement function are CD46, CD55, CD59, complement receptor 1 and CRIg (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). In the fluid phase, the complement-regulating proteins are C1 inhibitor, C4-binding protein, Factor H, Factor I, S protein, and clusterin (Oksjoki, Kovanen et al. 2007; Ehrnthaller, Ignatius et al. 2011). Complement function not only has a major impact on infections in the host but has also been shown to have a role in a variety of conditions ranging from paroxysmal nocturnal hemoglobinuria to traumatic brain injury and stroke (Ehrnthaller, Ignatius et al. 2011).

FIGURE 12. Overview of the complement system, activation, effectors and regulators . With permission from (Oksjoki, Kovanen et al. 2007).

RESULTS

Paper I CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7.

Marko Marttila, David Persson, Dan J Gustafsson, M Kathryn Liszewski , John P Atkinson, Göran Wadell, Niklas Arnberg.

Journal of Virology. 2005 Nov;79(22):14429-36

In this study, species B Ads that could use CD46 as a functional receptor were identified. Segerman et al. had previously shown that Ad11p binds CD46 (Segerman 2003). Interestingly Ad7 binds CD46- transfected CHO cells, but does not infect these cells to the same extent as Ad11p (Segerman 2003).

The initial experiments showed that all species B serotypes, except Ad3 and Ad7, infected CD46 (BC1 isoform) -expressing CHO cells. Ad50 and Ad11 were most efficient in infecting CD46-expressing CHO cells. To further validate the findings A549 cells were infected with species B serotypes in the presence or absence of rabbit serum against CD46. The antiserum completely inhibited A549 infection with serotypes Ad16, Ad21, Ad50 Ad 34 and Ad35 (species B1). The Ad11 and Ad14 (species B2) infectivity of A549 cells was reduced but not completely inhibited. Control serotypes from species C, E and F were not significantly affected by the CD46 antiserum, while, surprisingly Ad31 (species A) infected A549 cells more efficient in the presence of the anti-CD46 serum. Because the fiber is the major determinant of Ad biding to the cellular receptor, we wanted to compare the sequences of the species B serotype fiberknobs. The Ad50 (species B1) fiber was the only fiber not sequenced previously. Aligning the knob sequences from species B Ads and comparing the sequences revealed a common feature among species B Ads compared with other Ad species. The AB loop contained a common 3 amino- acid insert in all species B serotypes, that was not found in Ad knobs of other serotypes. Furthermore, two amino acid (leucine and valine at positions 240 and 296, respectively) residues differentiate the knob sequences of Ad7 and Ad3 from the rest of the species B serotypes. Other species B Ads contain charged or hydrophilic amino acids at

these positions. In Ad12, position 240 corresponds to the CAR- binding site, whereas position 296 in Ad37 corresponds to the sialic acid- binding site.

Paper II The Arg279Gln [corrected] substitution in the adenovirus type 11p (Ad11p) fiber knob abolishes EDTA-resistant binding to A549 and CHO-CD46 cells, converting the phenotype to that of Ad7p.

Dan J Gustafsson, Anna Segerman, Kristina Lindman, Ya-Fang Mei, Göran Wadell.

Journal of Virology . 2006 Feb;80(4):1897-905. Erratum in: Journal of Virology. 2006 May;80(10):5101.

In the second paper, the region of the Ad11p fiberknob responsible for binding to CD46 was determined. Previously Segerman et al. (Segerman, 2003) had shown that divalent cations affected Ad7p but not Ad11p binding to A549 cells. At the time of the study it was hypothesized that there were two species B receptors, sBAR and sB2AR (Segerman, 2003). sB2AR is used by Ad7p and Ad11p and the interaction was shown to be independent of divalent cations (Segerman, 2003). Previously, an interaction between Ad11p and CD46 had also been demonstrated (Segerman 2003). Sequence alignment of the Ad11 and Ad7 knob domain revealed a high homology, in which only 13 amino acids differed at the same positions. Given that Ad11p but not Ad7p had been shown to bind CD46, a number of chimerical recombinant fiberknobs were constructed by inserting Ad7p sequences into an Ad11p backbone. To confirm that the wt fiberknobs displayed the same binding phenotype previously shown for virions, an in vitro system was established that consisted of A549, CHO wt and CD46-transfected CHO cells. The binding of wt Ad11p, Ad7p, Ad37 and Ad5 virions to the different cell types was assessed by flow cytometry. The cells were pre-treated with EDTA only (to chelate divalent cations) or recovered with cell media (to restore cations). The fiberknobs of Ad7p and Ad11p exhibited the same binding phenotype with the cell lines as the whole virions. Interestingly, the tentative negative control, the Ad37 fiberknob, did not exhibit any binding to CHO-CD46 cells. To determine if CD46 corresponded to sB2AR A549, CHO wt and CD46-transfected CHO cells were preincubated with EDTA and then further incubated with polyclonal rabbit anti-CD46 serum. The antiserum and EDTA treatment reduced Ad11p fiberknob binding by

95%, while Ad37 fiberknob binding remained unaffected. Having established that the model system with recombinant fiberknobs corresponded to binding studies with whole virions, the chimeric fiberknob constructs were evaluated. Binding experiments with chimeric Ad11/7p indicated that the HI and I domains (positions 274- 294) in the C-terminus of the Ad11p fiberknob were responsible for the CD46 interaction. Having narrowed down the binding region, a number of point mutations were constructed in an Ad11p fiberknob backbone. Ad7p amino acids that differed from the corresponding sequence of the Ad11p fiberknob were inserted between positions 274 and 294. One point mutation in the Ad11p fiberknob backbone stood out, namely Arg279Gln residing in the exposed HI loop. This single mutation gave the Ad11p fiberknob backbone essentially the same CD46 binding phenotype as the wt Ad7p fiberknob, which indicated that Arg279 was crucial for CD46 binding. To determine if Arg279, alone, was also sufficient for CD46 binding, an Ad7p fiberknob with the reverse mutation (Gln279Arg) and an Ad7p fiberknob in which the entire HI loop was swapped for that of the wt Ad11p fiberknob sequence (Gln279Arg, Leu282Iso, Asn284Asp) were constructed. The single substitution (Gln279Arg) exhibited significantly improved binding to CD46 compared with the wt Ad7p fiberknobs. Substituting the entire HI loop with that of the Ad11p sequence in the Ad7p fiberknob backbone further increased CD46 binding.

Paper III Adenovirus 11p downregulates CD46 early in infection.

Dan J Gustafsson, Emma K Andersson, Yang-Ling Hu, Marko Marttila, Kristina Lindman, Mårten Strand, Li Wang, Ya-Fang Mei.

Virology. 2010 Sep 30;405(2):474-82. Epub 2010 Jul 16.

In the third paper the effect of Ad11 binding to CD46 was investigated. It has been shown that CD46 binding by Ad35 or measles virus (MV) leads to the downregulation of CD46 (Lecouturier, Rizzitelli et al. 1999; Sakurai, Akitomo et al. 2007). CD46 is also constitutively recycled via clathrin-coated pits but cross- linking by antibodies induces macropinocytosis and rapid CD46 degradation (Crimeen-Irwin, Ellis et al. 2003). The biological effects of CD46 downregulation are, however, poorly understood. In 2007 Fleischii et al . published findings of a low-affinity binding site for Ad7 and Ad3 on CD46, which was shared by other CD46- binding adenoviruses (Fleischli C 2007 ). This finding led to an investigation of whether CD46 was downregulated by Ad11 binding and if a possible low-affinity binding of Ad7 to CD46 would have the same consequence. CD46 is one of a number of cell surface regulators of complement activity. A review of the literature showed that downregulation of CD46 in lung cancer cell lines did not expose cells to autologous complement lysis to the same extent as inactivation of CD59 or combined CD55 and CD59 inactivation (Ajona D 2007 ). The effect of Ad11 on the cell surface complement regulators CD55 and CD59 was evaluated to provide a more balanced picture of the complement regulatory potential on the target cells. The effects of Ad11 and Ad7 binding to CD46 were investigated in two cell lines, K562 and A549. The primary focus was whether anti- CD46 serum would downregulate CD46 on K562 cells to establish that the same mechanism of CD46 downregulation that was noted by Crimeen-Irwing et al . (Crimeen-Irwin, Ellis et al. 2003) was present in the K562 clone used in this study. K562 cells were exposed to anti- CD46 rabbit serum for 4 h at 37ºC or on ice. Cell surface CD46 levels were analyzed by flow cytometry. The results showed a clear

downregulation of CD46 on the K562 cell surface after 4 h at 37ºC. The ice and unimmunized serum control did not affect CD46 cell surface levels. The ability of Ad11 virions to downregulate CD46 on K562 cells was next investigated. Downregulation of CD46 was measured by flow cytometry 30 min and 4 h after incubation of Ad11 with K562 cells. CD46 was downregulated in a dose-dependent fashion. The maximum downregulation at both time points was observed at a concentration of 8x10 4 virions / cell. Flow cytometry showed that Ad11 caused an initial rapid downregulation, within 5 min, of CD46 on K562 cells. The MEM-258 antibody used to detect CD46 interacts with the SCR1 region of CD46. To ensure that the observed downregulation of CD46 was not simply a consequence of Ad11/MEM-258 competition for the same binding site, a control experiment was performed in which polyclonal serum (H-294) or monoclonal antibodies (E4.3) was used to detect CD46 levels. CD46 downregulation on K562 cells after a 30 min Ad11 incubation was also observed in this experiment. The post-incubation time point at which CD46 was downregulated was then determined. K562 cells were incubated with Ad11 virions for 5 min, 30 min and 4 h. CD46 cell surface levels were then analyzed by flow cytometry and epifluorescence microscopy. In both assays, CD46 was already significantly downregulated already after 5 min at 37ºC and continued to be downregulated in a time-dependent manner. Previous publications have shown that CD46, CD55 and CD59 downregulation sensitizes cells to complement lysis (Krantic S 1995 ; Ajona D 2007 ). Fleischli et al . have published data suggesting a low- affinity binding of Ad3 and Ad7 to CD46. It has also previously been shown that Ad7 fiberknobs do not bind CD46 in the same fashion as Ad11 fiberknobs (Gustafsson, Segerman et al. 2006). It was therefore of interest to investigate the biological consequence, in the context of CD46 downregulation, of a possible Ad7-CD46 low- affinity binding. To address this question, two cell lines were studied, A549 (non-small cell lung cancer cell line) and K562 (established from chronic myelogenous leukemia in terminal blast crisis). First, the cell surface expression of CD46, CD55 and CD59 was analyzed in both cell lines by flow cytometry. All three complement regulatory molecules were expressed on both cell lines. Ad7 or Ad11 virions were then incubated with K562 or A549 cells at 37ºC for 30 min, 4 h or 8 h. Incubation of K562 cells with Ad11p virions under the above

conditions caused a significant downregulation of CD46. Ad7 virions did not downregulate CD46. Ad11p also caused downregulation of CD55 and CD59 at 4 h and 8 h. However the downregulation of CD55 and CD59 was much less than that of CD46. Ad7 did not downregulate CD46 or CD55 to a significant extent; however, some downregulation of CD55 was noted at 4 h and 8 h. In A549 cells, the same phenomenon of CD46 downregulation was observed; however, a general downregulation of CD55 and CD59 was observed at 8 h. This downregulation was mediated by both Ad7 and Ad11.

The main determinant for Ad binding to its receptor is the fiberknob protein. It was hypothesized that Ad recombinant fiberknobs could be sufficient to downregulate CD46. wt and recombinant Ad7 and Ad11 fiberknobs (Ad7pwt-rFK and Ad11pwt-rFK) were tested. A previously constructed Ad11 fiberknob mutant (Ad11p-R279Q-rFK) that did not bind CD46 or bound CD46 with the same low affinity as wt Ad7 fiberknobs was included.

Fiberknob constructs were incubated with K562 cells for 30 min or 8 h at 37ºC. CD46 cell surface levels were analyzed with flow cytometry, as previously described. wt Ad11p fiberknobs (Ad11pwt- rFK) but not Ad7p fiberknobs (Ad7pwt-rFK) caused a significant downregulation of CD46 levels already at 30 min and 8 h. Incubation of Ad11p-R279Q-rFK did not cause any significant reduction of CD46 levels at 30 min; however, at 8 h, a small reduction was observed. This fiding indicates that the Ad11p fiber alone is sufficient to cause the downregulation of CD46.

DISCUSSION

The results in the first study (Marttila, Persson et al. 2005) suggest that CD46 is the major receptor for all species B serotypes except Ad3 and Ad7. We also noted that Ad37 that had been reported to bind CD46 did not infect CHO-CD46 cells to any greater extent that non- CD46-expressing CHO cells. Sequence alignment showed that species B serotypes have a 3-aa insert in the AB loop of the fiberknob domain. Leucine 240 and Valine 296, distinguish Ad3 and Ad7 from the other species B serotypes. However, the sequence difference does not alter the theoretically calculated isoelectric points for the knobs of these two serotypes compared with the other species B knobs. We concluded that none of the analyzed species C, D, E or F serotypes infected target cells using CD46 as the receptor.

In the second study, (Gustafsson, Segerman et al. 2006), it was concluded that sB2AR and CD46 represent the same entity. The results also implicate Arg279 as the critical determinant, in the context of the HI loop, for Ad11p fiberknob binding to CD46. Interestingly one previous study showed that the Arg279Gln mutation abolished the ability of the Ad11p fiberknob to hemagglutinate monkey erythrocytes (Mei 1993 ). In contrast to human erythrocytes, CD46 is expressed on rhesus monkey erythrocytes. The data of Mei et al. (Mei 1993) seems to further corroborate our finding that Arg279 (in the context of the HI loop) is important for Ad11-CD46 binding.

In the final study, (Gustafsson, Andersson et al. 2010) it was shown that Ad11 virions and fiberknobs downregulate CD46 early in infection. The downregulation occurs on both A549 and K562 cells. Ad7 virions seem to contribute to a small downregulation of CD46 after 8 h, which was most evident on A549 cells. Ad11 also appears to downregulate cell surface levels of CD55 and CD59 after 8 h.

In 2007, Fleischli et al. showed that there is a low-affinity binding of Ad3 and Ad7 to CD46 (Fleischli C 2007 ). Persson et al . have also shown that there is a low-affinity binding of Ad7 to CD46 (Persson, Muller et al. 2009). We initially interpreted our results in a way that led us to believe that Ad7 does not bind CD46 at all. The studies by Fleischli et al. and Persson et al . demonstrated that this conclusion may not be accurate (Fleischli C 2007 ; Persson, Muller et al. 2009).

The findings of Persson et al . corroborate the importance of Arg279 in the Ad11 fiberknob HI loop (Gustafsson, Segerman et al. 2006; Persson, Muller et al. 2009). However, unlike our first hypothesis, this study suggests that Arg279 is not a contact residue. Instead, Arg279 appears to correctly position Arg280 to establish binding between Ad11 and CD46 (Persson, Muller et al. 2009). Persson et al . also showed that the Arg279Gln mutant decreases the interaction between Ad11 and CD46 by approximately 60% (Persson, Muller et al. 2009). Conversely the reverse mutation in Ad7 (Gln279Arg) increased binding by 60% (Persson, Muller et al. 2009). This result is in line with our findings (Gustafsson, Segerman et al. 2006), particularly the improved CD46 binding of the mutant in which the entire HI loop in the Ad7 fiberknobs was replaced with the Ad11 sequence relative to Gln279Arg. A recent publication fills another piece of the Ad-CD46 puzzle. Wang et al . have identified DSG-2 as a receptor for Ad3, Ad7 Ad11 and Ad14 (Wang, Li et al. 2011). Wang et al . also proposed a division of Ad species B into 3 groups: Group 1 (Ad 16, 21, 35, 50), which use only CD46; group 2 (Ad3, 7, 14), which use DSG-2 as a receptor; and group 3 (Ad11), which use CD46 preferentially but can also use DSG-2 (Wang, Li et al. 2011). The use of DSG-2 as a receptor for species B adenoviruses fits our previous observations, especially because DSG-2 is a calcium-binding transmembrane glycoprotein (Wang, Li et al. 2011). At least in epithelial ovarian cancer cells CD46 is trapped inside tight junctions that are inaccessible to CD46-binding Ads (Strauss, Sova et al. 2009). DSG-2 is found in cell membranes of normal epithelial foreskin and colon tissues (Wang, Li et al. 2011). However, in polarized colon cancer (T82 and CaCo-2) cells, DSG-2 appears to be expressed on the distal end of the intercellular junction (Wang, Li et al. 2011), which has prompted the speculation that binding to DSG-2 may open intercellular junctions and make CD46 more accessible to Ad virions (Wang, Li et al. 2011). This could also contribute to lateral viral spread and viremia. Interestingly the authors show that the subviral dodecahedral particles formed during replication by Ad3, Ad7, Ad11 and Ad14 trigger opening of intercellular junctions (Wang, Li et al. 2011). A self-dimerizing protein containing the Ad3 fiberknob triggers the intermittent opening of intercellular junctions and increases access to receptors within the tight junctions (Wang, Li et al. 2011), which can be used as an adjuvant to increase the efficacy of trastuzumab on Her2/neu-positive breastcancer cells (Wang, Li et al.

2011). Because DSG-2 has been shown to be a receptor for Ad7, the role of CD46 as a receptor for Ad7 appears somewhat unclear. The dissociation constant (K d) for Ad11 fiber binding to CD46 is 11-13 nM, and the corresponding K d for Ad7 fiber binding to CD46 is 35 µM (Persson, Muller et al. 2009). This raises the question of whether Ad7-CD46 binding is biologically significant? One recent publication brings further clarity to this discussion: Trinh et al. have shown that DSG-2 is the major attachment receptor for Ad3, corresponding to approximately 90% of the binding to the cell surface. The remaining 10% of bound Ad3 particles would use CD46 as their attachment molecule (Trinh, Lesage et al. 2012). It is also shown that Ad7 binding to CD46 is dependent of the amount of CD46 cell surface expression. Biacore experiments in this study show that binding of Ad3 or 7 fiberknobs to immobilized CD46 is dependent on CD46 density (Trinh, Lesage et al. 2012). This data indicates that there is an avidity mechanism leading to interaction between Ad3/Ad7 and CD46 (Trinh, Lesage et al. 2012). In the final study of this thesis (Gustafsson, Andersson et al. 2010), we show that CD46 is downregulated on both A549 and K562 cells by Ad11 virions and fiberknobs as early as 5min after incubation. Contrary to Ad11, Ad7 virions and fiberknobs do not affect CD46 levels in the same manner. A small downregulation of CD46 is observed after 8 h on both A549 and K562 cells after incubation with Ad7 virions. The differences between Ad11 and Ad7 in the context of CD46 downregulation could be explained by the different dissociation constants. Could Ad7 use both DSG-2 and CD46 as receptors? The difference between Ad11 and Ad7 attachment appears to be the downregulation of CD46. Whether this leads to a difference in intracelular signalling remains to be seen. CD46 has been shown to affect T-cell polarity, and Ad35 can suppress dendritic cell (DC) induced activation of naïve CD4 + T-cells (Hawkins and Oliaro 2010; Adams, Gujer et al. 2011). The CYT-1 and CYT-2 domains of CD46 give rise to different responses, increasing the complexity of CD46 signaling (Marie, Astier et al. 2002). CD46 CYT-1 ligation inhibits the T-cell-mediated inflammatory response, while CD46 CYT-2 enhances it (Marie, Astier et al. 2002).

Differences between Ad7 and Ad11 pathology could perhaps partly be explained by different immunomodulatory stimuli; however, attachment represents only one part of the complex mechanism of

infection in vivo . In the case of Ad7 DSG-2 could serve as the main attachment receptor. Alternatively Ad7 and Ad3 would bind and infect host cells that express a high density of CD46 on their membranes. Because CD46 would not be downregulated and may not be as efficiently stimulated as with Ad11, complement clearance of the infected cells would not be as effective. Lateral virus spread through opening of intercellular junctions via DSG-2 could perhaps partly explain the aggressive respiratory pathology of Ad7. In the case of Ad11, the initial infection could utilize both DSG-2 and CD46. DSG- 2 would permit access to CD46 within the intercellular junctions. In the initial infection the majority of infected cells may be cleared through complement-mediated lysis. Alternatively these cells could initially be rescued by CD55 and CD59 cell surface levels. Perhaps the Ad11 stimulation of CD46 leads to an impaired T-cell response, which may result in the observed persistent infections of the urinary tract caused by Ad11.

Data from other publications point indirectly to a biological significance of Ad3 and Ad7 low-affinity binding to CD46. The data in this thesis shows that CD46 is a major receptor for species B Ads, that Arg279 in the Ad11 fiberknob is important for CD46 binding and that CD46 is downregulated by Ad11. In conclusion, the results in this thesis, in conjunction with recent publications, shed light on the receptor interactions of species B Ads.

In summary the results of this thesis are as follows :

-Species B Ads, except Ad3 and Ad7, use CD46 as one of their high affinity receptors.

-Mutation of Arg279 in the Ad11p fiberknob significantly reduces binding to CD46.

-CD46 is rapidly downregulated by Ad11 virions and fiberknobs.

ACKNOWLEDGEMENTS

Till att börja med skulle jag vilja tacka mina handledare; Göran och Ya-Fang! Ni har båda två varit en oerhörd källa till inspiration och kunskap!

Göran: din kunskap inom fältet och din förmåga att citera referenser samt din allmänbildning inom molekylärbiologin är lika sporrande och inspirerande som den är skrämmande! ☺ Tack för alla råd, synpunkter och framför allt friheten till eget ansvar!

Ya-Fang: Ditt idoga hårda arbete och din noggrannhet är en inspiration för oss alla som jobbat för dig! Tack för all hjälp, alla råd och all klokhet!

Utan er två skulle den här boken aldrig blivit färdig! Tusen tack!!!

Prof. Elgh; tack för stödet under färdigställandet av denna avhandling!

Till Katrin; Tack, tack och åter tack!!! Alla småsaker som blir helt plötsligt så krångliga löser du galant! Tack för glädjen, för att du håller reda på allt och att du håller ordning på oss!!

Kickan och Karin; Tack för allt ni gjort och all ni gör! Ni två är helt enkelt helt underbara! Från att lyssna på vardagsbekymmer till att fixa en buffert, hålla celler gående eller upprepa FACS experiment… det verkar som om ni två kan fixa allt! Tusen och åter tusen tack för allt!!!!

To Bengt and Ruth: Thanx for introducing me into the world of molecular biology trough your groups! I will always remember getting caught with my finger stuck in the glass tube precipitating DNA for the first time! Positive and negative controls –will NOT forget! Thanx!!

Till alla i labbet: nuvarande och före detta:

Johan: Din ”drive” är lika inspirerande som dina kunskaper och din positiva attityd! Tack för alla diskussoner, fester och luncher!!

Nitte: Dude-sällan har vårtor rockat lika fett! Riktigt roligt att du är tillbaka! Jag tänkte mången gång på dig då jag svettades med denna kappa! Tack för allt!

Marko: Min fåordiga, hårdhudade landsman med Nature pek i bältet.. Du är den enda jag hittills sett som fått subkonjuktivala blödningar efter dina pass i gymet.. Respekt!

David: Tack för att du kristalliserade fiberknoppen!! ☺

Tjej gänget: Cissi, Emma N och Emma A; Sällan skådad ordning och reda i ert labb, givetvis väldigt frestande att gå och ”låna” pipetter därifrån.. Men allvarligt tack för det goda sällskapet och den inspiration och goda stämning ni sår omkring er!

Jim, Micke, Calle, Iréne, Mari och Mårten; Tack för all hjälp och allt trevligt sällskap!

To Emma, Mårten, Yang-Ling Hu and Li Wang: thank you all for the work done on the last paper!

Niklas, Magnus och Annika: Tack för all kunskap, inspiration, klokhet och visdom ni ”äldre” ger oss ”ungdomar”! ☺

Tack till alla på den tidigare ”landstingsdelen” för alla fikastunder och intressanta diskussioner!

Urban och Pär: Tack för den delade erfarenheten, förmedlad via de intressantaste fika diskussioner jag deltagit i hittills!

To Dr Bowald: Thank you for the inspirational guidance in the science and craft of surgery!

Anders ”the rocking GP” L. och Pawel ”the spine” G.

Kära vänner, jag kan inte föreställa mig hur det skulle varit att plugga medicin utan ert sällskap!! Jag lyfter på hatten för er avspända attityd men ändå brillianta intellekt! Fast ni luktar fortfarande illa.. Skämt åsido.. Ett stort tack till er båda för allt!!

To my comrades during the SKJA rotation. My respect and appreciation for you is far greater than words can or are allowed to speak!

To “the team”: It has been an honour to serve with you!

Mats och Anu med familj, Jesse och Tiina, Kalle och Mika; Tack för vänskapen och alla galna och härliga stunder under tiden i Umeå!

Pär och Anna, Jun och Karolina med familjer och Magnus och Isabell; Tack för alla trevliga stunder, på jobbet, på fritiden, i köket, i gymet, på backen och på sjön! Ni är fantastiska! Lycka till med allt som ni någonsin åtar er!

Till min Mor och Far: Ni är de bästa föräldrar som finns! Utan er skulle detta aldrig blivit verklighet. Tack för allt stöd, all uppmuntran och all kärlek!

Till Ida: Tack för att du och Julia kommit in i mitt liv! Du är min andra halva; Tillsammans rullar vi genom livet med kärlek och glädje! Jag beundrar dig, du är fantastisk! Jag älskar dig!

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