Cellular Receptors for Viruses with Ocular Tropism

Home , Ichtadenovirus, Picornavirus, Siadenovirus

Cellular receptors for viruses with ocular tropism

Emma Nilsson

Department of Clinical Microbiology Umeå University 2011

Copyright © Emma Nilsson ISBN: 978-91-7459-182-8 ISSN: 0346-6612-1414 Front cover: Sonja Nordström, Print & Media. Picture of Ad37 fk in complex with GD1a, kindely provided by Johannes Bauer, University of Tübingen, Germany. Printed by: Print & Media, Umeå University Umeå, Sweden 2011

To my loved ones

Table of Contents

Abstract 7 Summary in Swedish–Populärvetenskaplig sammanfattning på svenska 9 List of papers 12 Abbreviations 13 Aims of the thesis 15 Introduction 16 History 16 Taxonomy and clinical/pathological aspects 17 Adenovirus 17 Picornavirus 20 Anatomy of the anterior segment of the eye 23 Description of EKC and AHC 25 Viral structure 28 Adenoviruses 28 Major capsid proteins 29 Minor capsid proteins 31 The adenovirus core 33 Non-structural proteins 34 Picornaviruses 35 Structural proteins 35 Non-structural proteins 36 Viral life cycle 38 Receptors 38 Immunoglobulin superfamily (IgSF) 41 Coxsackievirus and adenovirus receptor (CAR) 41 Intercellular Adhesion Molecule-1 (ICAM1) 42 Poliovirus receptor (PVR or CD155) 42 Regulators of complement activation (RCA) family 43 Membrane Cofactor Protein (MCP/CD46) 43 Decay Accelerating Factor (DAF or CD55) 44 Sialic acid 44 Gangliosides 45 Heparan sulfate proteoglycans (HSPGs) 46 Desmoglein-2 46 Integrins 47 Other receptors 47 Soluble components 49 The life cycle after binding to cells; adenoviruses 50 Internalization and trafficking 50 Structure of the genome 51

Gene expression and replication 52 Assembly and release 55 The life cycle after binding to cells; picornaviruses 56 Internalization and trafficking 56 Structure of the genome 58 Translation and replication 59 Assembly and release 59 Antivirals 61 Adenoviruses 61 Picornaviruses 64 Results and Discussion 66 Paper I 66 Paper II 69 Paper III 71 Paper IV 75 Concluding remarks 79 Acknowledgements 81 References 84

Abstract

Several viruses from different virus families are known to cause ocular infections, e.g. members of the Adenoviridae, Picornaviridae and the Herpesviridae families. These infections are spread by contact and in the case of adenoviruses (Ads) and picornaviruses they are also highly contagious. The ocular infections caused by Ads and picornaviruses are called epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC), respectively. Historically, EKC is caused mainly by three types of Ads from species D: Ad8, Ad19 and Ad37. The infection is characterized by keratitis and conjunctivitis but also involves pain, edema, lacrimation and blurred vision. AHC is caused mainly by two types of picornaviruses: coxsackievirus A24v (CVA24v) and enterovirus 70 (EV70), and is characterized by pain, redness, excessive tearing, swelling and subconjunctival hemorrhages. In addition, blurred vision, keratitis, malaise, myalgia, fever, headache and upper respiratory tract symptoms can also be experienced.

Both infections are problematic in many parts of the world, affecting millions of people every year. Ads are responsible for appriximately of 20-40 million cases of EKC every year, and the number of AHC-cases until 2002 have been estimated to be up to 100 million. EV70 and CVA24v are also known to cause pandemics and so far, three pandemics have occured. Despite the great need, the only treatment available today is supportive treatment; no antiviral drugs are available to combat these common viral infections.

Ad37 has previously been reported to use sialic acid (SA) as its cellular receptor. Since there is no antiviral treatment available against EKC we wanted to evaluate the inhibitory effect of SA-based antiviral compounds on Ad37 binding to and infection of ocular cells. We found that multivalent compounds consisting of SA linked to a carrier molecule, in this case human serum albumin, efficiently blocked Ad37 binding and infection at low concentrations. Further attempts were then made to improve the effect by

chemically modifying SA monosaccharides. However, no enhanced inhibitory effect was accomplished and the conclusion was that the best inhibitors are based on unmodified SA. We next hypothesized that development of efficient SA-based binding inhibitors may require detailed knowledge about the structure of the SA-containing receptor. Using a battery of biological and biochemical experiments, including glycan array, binding and infection assays, X-ray crystallography and surface plasmon resonance (SPR) we identified a specific glycan involved in the binding and infection of Ad37. This glycan turned out to be a branched, di-SA-containing motif corresponding to the glycan motif of the ganglioside GD1a. However, the ganglioside itself did not function as a cellular receptor, as shown by a number of binding and infection assays. Instead, the receptor consisted of one or more glycoproteins that contain the GD1a glycan motif. This glycan docked with both its SAs into the trimeric Ad37 knob resulting in a very strong interaction as compared to most other protein-glycan interactions. Hopefully, this finding will aid development of more potent inhibitors of Ad37 binding and infection.

The receptor for CVA24v, one of the main causative agents of AHC, has been unknown until now. We showed that this ocular virus, like Ad37, is also able to use SA as a receptor on corneal cells but not on conjunctival cells. This suggested that CVA24v may use two different receptors. As for Ad37, the receptor used by CVA24v on corneal cells also appears to be one or more sialic acid-containing glycoproteins. We believe that these findings may be a starting point for design and development of candidate drugs for topical treatment of AHC.

Summary in Swedish– Populärvetenskaplig sammanfattning på svenska

Virusinfektioner i ögat är ett stort problem i många delar av världen, speciellt i tätbefolkade områden. Varje år drabbas miljontals människor av dessa åkommor vilket i sin tur leder till stora samhällsekonomiska förluster. Ett antal virus från flera olika virusfamiljer har förmågan att infektera ögat, t.ex. medlemmar av adenovirus-, picornavirus- och herpesvirusfamiljerna. Från adenovirusfamiljen är det främst tre typer: 8, 19 och 37, som orsakar den allvarliga ögoninfektionen epidemisk keratokonjuktivit (EKC). Denna infektion karaktäriseras av inflammation i horn- och bindhinna men inkluderar även rinnande, svullna ögon, smärta, irritation och blödningar. Inflammationen i hornhinnan leder i många fall till synnedsättning. Infektionen går normalt sett över inom ett par veckor, men i vissa fall kan synnedsättningen dröja kvar i veckor eller månader. Även permanent synnedsättning har rapporterats.

Akut hemorrhagisk konjunktivit (AHC) orsakas framförallt av två medlemmar av picornavirusfamiljen: enterovirus 70 (EV70) och coxsackievirus A24 variant (CVA24v). Denna infektion karaktäriseras av inflammation i bindhinnan, rinnande, svullna ögon, smärta, irritation och blödningar. Man kan även drabbas av hornhinneinflammation, feber, huvudvärk och övre luftvägs-symptom. Dessa två virus, EV70 och CVA24v, orsakar epidemiska utbrott av AHC men även pandemier. Sedan upptäckten av EV70 och CVA24v 1969 så har dessa virus orsakat sammanlagt tre pandemier och under dessa pandemier har mer än 100 miljoner människor drabbats. I dagsläget finns ingen medicinsk behandling för ögoninfektioner som orsakats av adeno- eller picornavirus.

Kolhydraten sialinsyra har tidigare identifierats som den struktur på cellytan som adenovirus typ 37 (Ad37) binder till. Eftersom det inte finns någon

antiviral behandling av EKC så var målet med den första studien att utvärdera den hämmande effekten av sialinsyre-baserade molekyler vid bindnings och infektions experiment med Ad37. I denna studie så kopplades sialinsyra till en bärarmolekyl och detta resulterade i en multivalent molekyl (en molekyl med ett flertal sialinsyror kopplade till bäraren). Resultaten från denna studie visade tydligt att dessa molekyler är väl fungerande hämmare av Ad37 orsakade ögoninfektioner in vitro. Som en fortsättning på denna inledande studie så gjordes försök att genom små förändringar av sialinsyran förbättra den hämmande effekten. Ett litet bibliotek med tio stycken varianter av sialinsyra togs fram och utvärderades. Utifrån dessa försök kunde tydligt utläsas att den hämmande effekten hos de modifierade sialinsyre-varianterna inte uppnådde samma nivå som den ursprungliga sialinsyran och därmed drogs slutsatsen att ändringar i den ursprungliga strukturen inte bidrog till någon ökad effekt. När de sedan kopplades till en bärarmolekyl så visade det sig att de inte var lika bra hämmare som den ursprungliga föreningen där sialinsyra kopplats till en bärare.

Tidigare har man visat att även EV70 binder till sialinsyra på utsidan av ögonceller. Målet med den tredje studien var således att undersöka om så var fallet även för CVA24v, vars cellulära receptor dittills var helt okänd. Genom att använda ett antal olika metoder kunde det fastställas att sialinsyra fungerade som receptor även för detta virus på vissa celler, men det verkar även finnas ytterligare minst en receptor (på andra celler) som än så länge inte har identifierats.

Som nämnts ovan så använder Ad37 sialinsyra som sin cellulära receptor och i den fjärde studien var målet att försöka få fram en mer detaljerad bild av hur den sialinsyre-innehållande receptorn ser ut. Genom att studera bindningen av Ad37 till ett stort antal olika sockerstrukturer så identifierades en specifik sialinsyre-innehållande sockerstruktur som Ad37 band bra till. Denna struktur innehöll två sialinsyror som genererar en relativt stark bindning till viruset. Den kunskap som erhållits i denna studie

kan förhoppningsvis bidra till att bättre antiviraler kan tas fram, kanske baserade på en liknande struktur.

Sammanfattningsvis har jag identifierat kolhydraten sialinsyra som receptor för ytterligare ett ögonvirus (CVA24v) och i samarbete med andra, även arbetat fram en mer detaljerad bild av den struktur på cellytan som Ad37 binder till. Jag har också analyserat effekten av sialinsyre-baserade molekyler, vilket i sin tur kan leda till utveckling av bättre antiviraler för behandlingen av virus-orsakade ögoninfektioner.

List of papers

I Multivalent sialic acid conjugates inhibit adenovirus type 37 from binding to and infecting human corneal cells

Johansson SM*, Nilsson EC*, Elofsson M, Ahlskog N, Kihlberg J, Arnberg N. Antiviral Res. 2007. 73:92-100.

II Design, synthesis, and evaluation of N-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis.

Johansson S, Nilsson E, Qian W, Guilligay D, Crepin T, Cusack S, Arnberg N, Elofsson M. J Med Chem. 2009. 52:3666-78.

III Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential.

Nilsson EC, Jamshidi F, Johansson SM, Oberste MS, Arnberg N. J Virol. 2008. 82:3061-8.

IV The GD1a glycan is a cellular receptor for adenovirus that cause epidemic keratoconjunctivitis

Emma C. Nilsson, Rickard Storm, Johannes Bauer, Susanne M. C. Johansson, Aivar Lookene, Jonas Ångström, Mattias Hedenström, Lars Frängsmyr, Hugh Willison, Fatima Pedrosa Domelöf, Thilo Stehle and Niklas Arnberg. Nat Med. 2011. 17:105-9

Paper I-IV have been reprinted with permission from the publishers.

*Contributed equally to this work.

Abbreviations aa Amino acid Ad Adenovirus ADP Adenovirus death protein AHC Acute hemorrhagic conjunctivitis AIDS Acquired immunodeficiency syndrome ARD Acute respiratory disease CAR Coxsackievirus and adenovirus receptor CMV Cytomegalovirus CNS Central nervous system CsA Cyclosporine A CVA Coxsackievirus A CVB Coxsackievirus B DAF Decay accelerating factor DBP DNA binding protein DNA Deoxyribonucleic acid DSG-2 Desmoglein-2 ECM Extracellular matrix EKC Epidemic keratoconjunctivitis ER Endoplasmatic reticulum EV Enterovirus fk Fiber knob FMDV Foot and mouth disease virus GAGs Glucosaminoglycans GONs Groups of nine GRP78 Glucose-regulated protein 78 kDa HAVCR Hepatitis A virus cellular receptor HFMD Hand foot and mouth disease HIV Human immunodeficiency virus HRV Human rhinovirus HS Heparan sulfate HSPGs Heparan sulfate proteoglycans HVR Hypervariable region ICAM-1 Intercellular adhesion molecule-1 IFN Interferon IGDD Isoleucine-Glycine-Aspartic acid-Aspartic acid IgSF Immunoglobulin superfamily IRES Internal ribosome entry site ITR Inverted terminal repeat kDa Kilo dalton LDLR Low density lipoprotein receptor Lf Lactoferrin LFA-1 Leukocyte function-associated antigen-1 MCP Membrane cofactor protein MDa Mega dalton MHC-1 Major histocompatibility complex-1

mRNA Messenger ribonucleic acid Mw Molecular weight NCT N-chlorotaurine NES Nuclear export signal NLS Nuclear localization signal NPC Nulear pore complex NRTI Nucleoside reverse transcriptase inhibitor Pb Penton base PCF Pharyngoconjunctival fever PI3K Phosphatidylinositol-3-OH kinase PKR Protein kinase receptor PSGL-1 P-selectin glycoprotein ligand-1 PV Poliovirus PVP-I Povidone-iodine PVR Poliovirus receptor RCA Regulators of complement activation RdRP RNA-dependent RNA polymerase RGAD Arginine-Glycine-Alanine-Aspartic acid RGD Arginine-Glycine-Aspartic acid RISC RNA-induced silencing complex RNA Ribonucleic acid RSV Respiratory syncytium-forming virus SA Sialic acid SCARB2 Scavenger receptor B2 SCR Short consensus repeat siRNA Small interfering RNA STP Serine-threonine-proline SV40 Simian virus 40 TP Terminal protein UTR Untranslated region VA-RNA Virus-associated RNA VLA-2 Very late activation antigen-2 VLDLR Very low density lipoprotein receptor VPg Virus protein genome linked

Aims of the thesis

Overall aim The overall aim of this thesis was to identify and characterize the interactions between ocular viruses and their cellular receptors.

Specific aims

Aim 1: Evaluate the inhibitory effect of multivalent sialic acid-containing compounds on Ad37 binding and infection of ocular cells and to characterize the mechanism behind this inhibitory effect.

Aim 2: Increase the inhibitory effect previously observed with the multivalent compound by structural modification of sialic acid. If the binding between sialic acid and Ad37 could be increased then this would lead to a more potent antiviral compound.

Aim 3: Identify the so far unknown cellular receptor(s) for CVA24v.

Aim 4: Further characterize the cellular receptor for Ad37 with focus on the structure of the sialic acid-containing motif.

Introduction

History

The first isolation of adenoviruses (Ads) was achieved in 1953 by Rowe et al. when searching for agents of acute respiratory infections in adenoid tissue [1]. Almost at the same time Hilleman and Werner identified an agent in respiratory samples from military recruits suffering from respiratory disease [2]. The first clinical description matching the clinical features of epidemic keratoconjunctivitis (EKC) was recognized already in 1889 in Austria and it was named keratitis punctuate superficialis [3-7]. The following years there were case reports of EKC from United Kingdom, Japan and the US [8-10]. In 1901 the first report regarding epidemic disease in Bombay, India was published [11]. At this time the epidemics were concentrated to the Indian subcontinent but in the 1930:s there was an epidemic outbreak in the US [12]. In 1941, there was a large outbreak in the marine shipyards of Pearl Harbor involving more than 10,000 individuals [13]. This coincided with a number of EKC outbreaks in shipyards along the west coast in the US, this is why the disease was named “shipyard eye” [14, 15]. Even though a filterable agent was identified at that time it took until 1955 to isolate the first EKC- causing Ad, which was Ad type 8 [16]. Many contributions to our understanding of viral and cellular gene expression and regulation, DNA replication, cell cycle control and cellular growth regulations have been made by studying Ad-infected cells. Probably, the biggest contribution to modern biology from the Ad research field was the discovery of messenger RNA (mRNA) splicing by Sharp and Roberts in 1993. For this discovery they were awarded the Nobel Prize in physiology/medicine.

The picornavirus family is one of the “oldest” known virus families. The first known animal virus discovered was the foot-and-mouth disease virus (FMDV) in 1898 and only 10 years later the well-known poliovirus was discovered [17, 18]. Picornaviruses have also, like Ads, played an important

role in the development of modern virology. Some of the advances where picornaviruses have contributed are: the discovery of the first RNA- dependent RNA polymerase, the demonstration of proteolytic processing of a polyprotein, and the propagation of virus in cell culture (viral replication) [19-21]. The more recent advances made by picornavirus researchers are e.g. infectious DNA clones and three-dimensional structures by X-ray crystallography [22-24]. In June 1969 a new type of picornavirus-caused conjunctivitis was reported and it was first named epidemic hemorrhagic conjunctivitis or “Apollo 11 disease” because it coincided with the moon landing of the Apollo 11 spaceship. Later it was re-named to acute hemorrhagic conjunctivitis (AHC) [25, 26].

Taxonomy and clinical/pathological aspects

Adenovirus

Taxonomy

The family of Adenoviridae contains five different genera according to the International Committee of Taxonomy of Viruses (ICTV): Mastadenovirus isolated from mammals, aviadenovirus isolated from birds, atadenovirus isolated from reptiles, birds, mammals and a marsupial, siadenovirus isolated from reptiles and birds, and the newest genus ichtadenovirus isolated from fish. The genus of mastadenovirus contains all the 51 human serotypes known today and they are further divided into species A-F. A number of new Ad types have been suggested: Ad53 and 54 that cause EKC will probably be classified as species D types and also Ad52 (species G), 55 and 56 have been suggested [27-31]. However, these have not been characterized with classical serology. The classification of new types is however under debate (manuscripts in press in J. Virol: Aoki et al. and Seto et al.)

Historically, the classification of Ads is based on the ability to agglutinate red blood cells, oncogenicity in rodents, DNA homology and tropism [32]. The classification of different serotypes is based on the resistance to neutralization by antisera of already known types [33]. The types are then separated into different species based on lack of cross-neutralization and a phylogenetic distance of more than 10%. If the phylogenetic distance is less than 5%, some of the other grouping criteria can be used to place the types in the same species even if they are isolated from different hosts [34]. To be identified as a new serotype the cross-reaction with previously known types must yield a titer greater than 16 [35].

Clinical and pathological aspects

Ads are common pathogens that infect a wide range of hosts in a highly species specific manner [36]. In many cases, Ad infections are asymptomatic but when disease occurs it is usually in the form of respiratory, ocular or gastrointestinal disease (see table 2) [37]. Infections of the urinary tract, lymphoid tissue, liver and other organs have also been reported. Only about half of the known Ad types are linked to illness. Respiratory disease in children is commonly caused by Ads from species C: Ad1, 2, 5 and 6, sometimes also Ad 3 and 7 from species B. In the adult population it is the other way around, Ad1, 2, 5 and 6 rarely cause disease but Ad 3, 4 and 7 do. Acute respiratory disease (ARD) has been a problem amongst the military recruits in the US. This population is probably more susceptible due to the crowded sleeping situation and the constant exhaustion that they experience. Historically, ARD is mainly caused by Ad 4 and 7, sometimes also Ad3 and more recently Ad14. The Ad-caused respiratory infections can be accompanied by ocular infections as well and the disease is then named pharyngoconjunctival fever (PCF). The most common PCF-causing Ads are Ad3 and 7. A more severe ocular infection called epidemic keratoconjunctivitis (EKC) is caused by Ad8, 19 and 37 from species D and also the new types Ad53 and 54. Since Ads are known to replicate in the intestine and regularly are isolated from stool they were believed to cause

diarrhea, but studies often showed the same amount of virus in the controls suggesting asymptomatic replication in the intestine [38]. Only the two Ads from species F, Ad40 and 41, are known to cause gastroenteritis to any larger extent, especially among young children [39].

Even though Ad infections are usually mild in the immunocompetent host they are severe and sometimes lethal in immunocompromized patients. The problem is increasing with the raising numbers of transplant recipients and AIDS patients. The first descriptions of fatal outcome of disseminated Ad infection were reported already in the 1960s [40]. Ad infections in the immunocompromized hosts can present very high fatality rates [41]. In many of the immunodeficient patients, Ads from species A, B and C are the most common. Ad31 from species A has been shown to be a main contributor to fatal disease [41].

Table 2. Classification and tropism of human adenoviruses.

Species Type Tropism

A 12, 18, 31 Intestine

B:1 3, 7, 16, 21 Respiratory tract, eye

B:2 11, 14, 34, 35, 50 Respiratory tract, urinary tract, eye

C 1, 2, 5, 6 Respiratory tract

D 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, Eye, intestine 36-39, 42-49, 51

E 4 Respiratory tract, eye

F 40, 41 Intestine

G 52 Intestine

Picornavirus

Taxonomy

The picornavirus family is divided into 12 different genera according to the International Committee of Taxonomy of Viruses (ICTV; table 3):

These genera are then further divided into different species, and the species are divided into types. The genus of enterovirus contain ten different species, seven of them contain human types (see table 3). The first classification of types was based on type of disease in humans and in newborn mice e.g. coxsackievirus A (CVA) caused acute flaccid paralysis in newborn mice and coxsackievirus B (CVB) caused spastic paralysis in newborn mice. Problems with this classification method lead to development of other criteria like: immunological response in humans, receptor usage and protection from disease. This type of classification also lead to problems since there was cross-reactivity between types and there was also antigenic variation within types [42]. Serological methods are still used for identification and classification, but thanks to the development of molecular methods it is easier to compare sequences. Criteria to fulfill to be classified within a species in the enterovirus genus are based on molecular methods [43]:

 Share greater than 70% aa identity in P1 (domain encoding the capsid proteins)  Share greater than 70% identity in the non-structural proteins 2C (ATPase)+3CD (proteinase)  Share a limited range of host receptors and a limited natural host range  Have a genome base composition (G+C) which varies by no more than 2.5%  Share a significant degree of compatibility in proteolytic processing, replication, encapsidation and genetic recombination.

Table 3. Classification and tropism of human enteroviruses.

Species Types Tropism

HEV-A Coxsackievirus A 2-8, 10, 12, 14, 16 CNS, skin and/or airways

Enterovirus 71, 76, 89, 90-92 Skin and/or GI tract

HEV-B Coxsackievirus B 1-6 Airways, heart, pancreas and/or CNS Coxsackievirus A 9, 23 CNS, skin and/or airways

Enterovirus 69, 73-75, 77-88, 93, 97, 98, Airways, skin and/or GI tract 100, 101, 106, 107

Echovirus 1-9, 11-21, 24-27, 29-33 Airways, GI tract and/or CNS

HEV-C Poliovirus 1-3 CNS

Coxsackievirus A 1, 11, 13, 17, 19-22, 24, CNS, skin, airways and/or eye 24v

Enterovirus 95, 96, 99, 102, 104, 105, 109 Airways, skin and/or GI tract

HEV-D Enterovirus 68*, 70, 94 Eye, airways and/or CNS

HRV-A Human rhinovirus 1, 2, 7-13, 15, 16, 18-25, Airways 28-34, 36, 38-41, 43-47, 49-51, 53-68, 71, 73-78, 80-82, 85, 88-90, 94- 96, 98, 100

HRV-B Human rhinovirus 3-6, 14, 17, 26, 27, 35, Airways 37, 42, 48, 52, 69, 70, 72, 79, 83, 84, 86, 91-93, 97, 99

HRV-C Serotypes not yet assigned Airways

*Previously identified as human rhinovirus 87 (HRV87).

Clinical and pathological aspects

Enterovirus (EV) infections are many times asymptomatic but a number of different symptoms are seen in the cases where they cause disease [44]. The diseases caused by enteroviruses includes severe symptoms such as poliomyelitis, meningitis, acute flaccid paralysis and fatal paralytic

poliomyelitis, and less severe symptoms such as ”common cold”, diarrhea, myalgia, conjunctivitis and skin rashes (see Table 3).

Poliovirus (PV) infections are in many cases asymptomatic but when symptoms occur it is usually in the form of a disease called abortive poliomyelitis. This is a mild febrile illness that sometimes includes gastrointestinal symptoms. About one of 200 symptomatic infections results in the severe paralytic disease known as poliomyelitis [44]. Other enteroviruses are also able to cause poliomyelitis e.g. EV70 and EV71 [45, 46]. Since the eradication period for polio started, EV71 has emerged as the major neurotropic EV. EV70, the major causative agent of acute hemorrhagic conjunctivitis (AHC) has also been shown to cause paralytic disease.

Aseptic meningitis is frequently caused by EVs as seen in the US were up to 95% of the cases are caused by EVs [47]. Typical symptoms of aseptic meningitis are fever, headache and meningeal signs that usually resolve within a week. The symptoms can be more severe and also lead to death, especially in neonates [48]. The EVs that are overrepresented as causative agents of aseptic meningitis are PV, EV70, 71, echovirus 7, 9, 11, 30 and CVB5. However, other types can also cause this disease [49].

Some of the EVs, in particular CVBs, have been associated with acute cardiac disease. A variety of different studies have detected EVs in heart tissue by immune assays, blotting and RT-PCR [50-52]. A link has also been suggested between EVs and dilated cardiomyopathy [50].

Respiratory disease is commonly caused by EVs, in particular by human rhinoviruses (HRV). These viruses usually infect the upper respiratory tract leading to “common cold”, but they also contribute to chronic respiratory diseases of the lower respiratory tract [53].

Acute hemorrhagic conjunctivitis (AHC) is a severe epidemic and sometimes pandemic ocular infection mainly caused by EV70 and coxsackievirus A24

variant (CVA24v) [54]. These viruses can also cause respiratory illness, gastrointestinal and neurological dysfunctions [44].

Herpangina and hand-foot-and-mouth disease (HFMD) are common EV infections among children [44]. Herpangina is characterized by lesions of the palate, tonsils and pharynx that usually disappear within a few days. The major causative agents of herpangina are: echoviruses, members of the CVAs and CVBs and also EV71 [55]. HFMD is characterized by blisters of the hands, feet and mouth and is caused mainly by CVA16 and EV71 [56].

Anatomy of the anterior segment of the eye

The sclera, “the white part of the eye”, is a fibrous tissue that protects the eye. The tissue consists of collagen bundles and elastic fibers. In the front part of the eye, the sclera proceeds into the cornea. The cornea is the clear outermost tissue of the eye. The cornea contains no blood vessels and receives its nourishment and protection from tears and the fluid in the anterior chamber behind it [57]. The corneal tissue is divided into five different layers (see figure 1):

(a) (b)

Figure 1. (a) Anatomy of the anterior segment of the human eye. (b) Cross- section of the human cornea showing the five different layers. Reprinted from [58] with permission from the publisher.

Epithelium: This is the outermost layer of the cornea; it is mounted on a basement membrane and is only five to six cell layers thick. The main functions are to protect the cornea from foreign material and to provide a smooth surface that absorbs nutrients and oxygen. The epithelium consists of: basal cells, wing cells and superficial cells [58]. The outermost cells have microvilli to increase the cell surface area contributing to a close contact with the tear film [59]. The cornea is highly sensitive to pain due to all the nerve endings that are present in the epithelium. In fact this is the tissue with the highest number of nerve fibers in the body. The corneal epithelium has the ability to regenerate due to the stem cells present in the corneal-sclera junction. This area is called the limbus.

Bowman´s Layer: This transparent layer anchors the epithelial cells and is composed of collagen, and if injured it can form scars when healing, which can affect the vision.

Stroma: The stroma corresponds to approximately 90% of the thickness of the cornea, and the main components are water, collagen and keratinocytes or fibroblasts, but it also contains glucosaminoglycans and proteoglycans [58]. It contributes to the strength and elasticity of the corneal tissue. Due to the fact that there are no blood vessels in the cornea, the stroma together with the endothelial cells serve as the circulatory system of the cornea.

Descemet´s Membrane: This is a thin, but strong tissue located just beneath the stroma. It is composed by collagen, which is produced by the endothelial cells beneath and serves as a barrier to protect the eye from pathogens and injuries. These cells are responsible for nutrient transportation and of maintaining the optimal hydration to prevent corneal edema [59].

Endothelium: The endothelium is the thin, innermost layer of the cornea. These cells regulate transport of fluid and other components in and out of the cornea. If it is injured the stroma swells up, becomes cloudy and in the end also opaque leading to visual damage. These cells do not regenerate.

The conjunctiva covers the inside of the eyelids and extends to the edge of the cornea which is called the limbus. It is composed of two layers, the stratified epithelia and the stroma. The thickness of the conjunctiva ranges between 2-10 cell layers depending on the part of the conjunctiva studied. The conjunctival epithelia contain goblet cells that have as their main function to produce mucins that lubricate the eye. In contrast to the cornea, the conjunctiva is highly vascularized. The conjunctiva is divided into three different areas: Palpebral conjunctiva, bulbar conjunctiva and the fornix. The palpebral conjunctiva covers the inside of the eyelids, the bulbar conjunctiva covers the sclera (until the limbus region) and the fornix is where the palpebral and bulbar conjunctiva meets.

The tear fluid layer continuously covers the surface of the eye and protects the cornea and conjunctiva from the external environment, and maintain the transparency of the cornea [60]. Other functions of the tear film are e.g. keeping the eye moist, creating a smooth surface, supplying oxygen and nutrients and protecting the eye against pathogens. The tear film consists of three layers: the lipid, the aqueous and the mucous layer [61]. The outermost lipid layer is present to prevent the evaporation of the aqueous layer beneath. The aqueous layer is responsible for taking the main nutrients and oxygen to the cornea and at the same time get rid of waste products. Since the epithelial plasma membrane of the corneal cells is hydrophobic, the hydrophilic mucin layer is needed to prevent the aqueous layer from rolling off. The mucous layer is produced by the goblet cells of the conjunctiva.

Description of EKC and AHC

Epidemic keratocconjunctivitis (EKC)

Epidemic keratoconjunctivitis is a severe ocular infection caused by viral infection of the cornea and conjunctiva. Ads are the main causative agents of the infection, especially Ad8, 19 and 37 but also Ad4 can cause a milder form

of the infection [62, 63]. More recently, two new types have been associated with EKC, Ad53 and 54 [27, 28]. However, EKC can also be caused by viruses from other families e.g. herpesviruses and picornaviruses. EKC is characterized by an acute and severe onset with swelling, lacrimation, discharge or photophobia [64, 65]. Follicular hypertrophy, edema, hyperemia and petechial hemorrhage are caused by viral replication in the palpebral and bulbar conjunctiva [65-70]. Replication in the cornea results in focal epithelial keratitis, which is seen as punctuate opacities and leads to a “foreign body” sensation [69, 71]. In severe cases chemosis, iritis and formation of pseudomembranes are also observed [65, 67, 68]. The infection normally lasts for 18-21 days, but the corneal opacities can last for months up to years and even be permanent [72-76]. The infection is usually bilateral, meaning that it starts in one eye and spreads to the other. Only about 25% of the EKC cases display a unilateral infection [77]. The infection is spread by contact and is therefore highly contagious causing outbreaks in densely populated areas of the world. In many cases the outbreaks starts in eye clinics, ophtalmologists´offices, schools or military bases [62]. Ad-induced EKC is endemic in Japan where half a million to one million individuals are infected every year, leading to substantial socioeconomical losses [63, 78].

Acute hemorrhagic conjunctivitis (AHC)

Acute hemorrhagic conjunctivitis is a highly contagious infection mainly caused by two members of the picornavirus family; CVA24v and EV70 [79]. The disease has a rapid onset with pain, redness, excessive tearing, swelling of the eyelid or photophobia within six to twelve hours after infection [80]. The infection usually spreads to the other eye within 24 hours. Another typical symptom of the infection is subconjunctival hemorrhages, usually beneath the bulbar conjunctiva covering the sclera (white part of the eye). In addition, blurred vision, malaise, myalgia, fever, headache, keratitis or upper respiratory tract symptoms may also appear [81]. There are some differences in the given symptoms depending on the virus type e.g. fever, headache and upper respiratory symptoms are more common in CVA24v infections and for

EV70 infections there are more pronounced subconjunctival hemorrhages [82-85]. There have also been reports about neurological symptoms like mild cranial nerve palsy or acute flaccid paralysis in EV70 infections [86]. The infection normally resolves within 7-10 days without any long-lasting or permanent problems [81, 85]. So far these viruses have caused three pandemics, 1969 to 1971 [87], 1980 to 1981 [88] and the most recent 2002 to 2004, which probably started in South Korea 2002 [89, 90] and thereafter spread to Malaysia [91], India [92, 93], Nepal [94-96], Tunisia [97], Congo and Marocco [98], Nicaragua, Honduras, Guatemala, El Salvador, Caribbean countries [99], French Guiana, the West Indies [100], Puerto Rico [101] and Brazil [102, 103]. Since the causative agents of AHC was identified it has been estimated that up to 100 million cases have occurred until 2002 [104].

Viral structure

Adenoviruses

Ads are nonenveloped double-stranded DNA viruses with an icosahedral capsid structure. The particle has a diameter of approximately 90 nm, a mass of 150 MDa and approximately 87 % is constituted of proteins. Eleven structural proteins have been identified by SDS-PAGE analysis and named II-XII after their decreasing molecular weight [105]. The capsid has 20 surfaces and 12 vertices with a fiber protein protruding from each vertice (see figure 2) [106].

Figure 2. A schematic picture of the Ad structure showing the major proteins (capsid proteins), the minor proteins and the core proteins. The picture has been reprinted from [107] with permission from the publisher.

Major capsid proteins

Hexon protein (pII)

The capsid contains 720 hexon monomers forming 240 trimers; this makes the hexon protein the main contributor to the Ad capsid (63 % of the total protein mass). The structure of the >900 amino acid hexon protein is complex: Each monomer consists of a base with two antiparallel β-barrels stabilized by an internal loop and three protruding loops on the external side [108]. The loops contain hypervariable regions (HVRs), which gives the type- specific epitopes (see figure 3a). This complex structure with a number of intermolecular contacts renders the trimeric hexon proteins extremely stable [105]. The hexons surrounding the pentons are called the peripentonal hexons and the hexons covering the centre of the 20 facets of the capsid are called “groups of nine” (GONs) [109, 110].

Penton base (pIII)

The penton base is a 340 kDa protein that consists of five monomers of polypeptide III and is located at the twelve vertices of the icosahedral capsid (see figure 2). The structure of the penton base monomer is similar to the hexon, consisting of a β-barrel domain and two loops in the distal end. The pentameric protein contains a central cavity that recognizes, and bind the N- terminal domain of the fiber protein [111]. Ad internalization into host cells is mediated by penton base binding to cellular integrins [112, 113]. The binding to cellular integrins leads to a clustering of the integrins, which in turn might lead to cell-signal induced endocytosis [114]. The integrin binding is mediated through the Arg-Gly-Asp (RGD) motif that is present in a loop on the penton base in all Ads except Ad40 and Ad41 (see figure 3b) [112, 115- 117]. These two viruses carry other motifs: Ad40 has an RGAD motif and Ad41 has an IGDD motif. Ad41 has been suggested to use another entry pathway than the typical clathrin mediated endocytosis that is used by other Ads [118].

Fiber protein (pIV)

The 182 kDa fiber protein is a trimer situated at the twelve vertices of the capsid in the center of the penton base. It consists of an N-terminal tail, a shaft and a C-terminal globular domain known as the knob, which is the receptor binding part of the virus (see figure 3 c & d). The N-terminal tail of the protein contains the highly conserved FNPVYPY sequence that anchors the protein to the center of the cavity in the penton base [111, 119]. The fiber shaft is built up by various numbers of repeating motifs of approximately 15 residues, rendering the difference in fiber length of different serotypes e.g. Ad3 has six repeats compared to Ad2 and 5 that have 22 repeats [120, 121]. Like the hexon protein and the penton base protein, the terminal knob domain contains β-barrels building up the core of the knob and loops that are exposed [122]. The three globular domains forming the knob of the virus give rise to a central cavity on the top of the knob, which was first thought to be the CAR-binding domain [123]. But, structural studies of the Ad12 knob in complex with an extracellular domain of CAR indicated that the binding site for CAR was surface loops at lateral subunit interfaces rather than the central cavity [124]. The same has been seen for the CD46-binding Ad11 where the binding also takes place on the outside of the knob, but, in contrast to CAR-binding, CD46-binding leads to a conformational change of the receptor [125]. The sialic acid-binding Ads from species D (Ad19 and Ad37) on the other hand interact with their receptor using the central cavity, which can bind three sialic acid saccharides [126]. Three of the serotypes, Ad40, Ad41 and Ad52 have a unique characteristic. They express two different fiber proteins on their capsid surface, one long, CAR-binding and one short with an as yet unknown receptor [127-129].

Figure 3. Structure of the capsomeres. (a) Ad5 hexon trimer model shown in side view and from the top with the hypervariable loops in colors. (b) Cryo-EM image of the Ad5 penton base shown as a side view with one of the RGD loops highlighted. (c) A ribbon representation of the fiber knob of Ad35 showing the three monomers in green, blue and pink. (d) Space-filling model of the Ad2 fiber based on the atomic structure, with some receptor- binding sites indicated. Reprinted from [107] with permission from the publisher.

Minor capsid proteins

The minor capsid proteins are far less studied than the major capsid proteins. They function as capsid cement managing the different requirements for accurate assembly of the virion, its fluctuating stability and its efficient disassembly [109].

Protein IIIa (pIIIa) pIIIa is a 63.5 kDa protein with a somewhat unknown function but it is believed to be important for correct encapsidation of the DNA since pIIIa mutants form empty capsids [130, 131]. The protein is phosphorylated to some extent, but the role of the phosphorylation is not known [132-134]. The location of this protein is on the interior side of the viral capsid where it binds the penton base to the peripentonal hexons and it also interacts with pVIII [135].

Protein VI (pVI) pVI is a 22 kDa protein located on the internal side of the capsid adjacent to the hexons suggesting it to be in contact with two peripentonal hexons [136, 137]. But it might also be situated in the central cavities of the hexons as suggested by cryo-EM studies [138, 139]. During the infection, pVI is involved in the endosomal escape by inducing a pH-independent disruption of the membrane [140]. Another important function of this protein is to facilitate the nuclear import of hexon proteins as determined by the inability of a pVI mutant to import hexons. The precursor of pVI acts as a shuttle, facilitating the hexon transfer into the nucleus. pVI contains two nuclear- localization signals (NLS) and two nuclear export signals (NES), which are proteolytically removed during maturation [141, 142].

Protein VIII (pVIII)

This protein is the least studied of the minor capsid proteins. It is a 15.3 kDa protein that forms dimers and interacts with nearby hexons facets. The location of this protein is on the inside of the capsid where it mediates interactions between GONs and their neighboring hexons and peripentonal hexons [138]. The protein has also a suggested role in the overall structural stability of the virion [143].

Protein IX (pIX) pIX is the smallest of the minor capsid proteins with a mass of only 14.3 kDa and it is also the only one of them that is unique to the mastadenoviruses, it is absent in all the other genera [144]. The 20 facets of the icosahedral capsid all contain twelve pIX molecules placed in the cavities between the peaks formed by the hexons [136, 137]. The pIX monomers are α-helical and form trimers due to its leucine-zipper domain, which allows the monomers to self- associate into a coiled-coil structure with three extended arms [105, 144]. Together with nine hexons, pIX has been termed the cement that stabilizes the GONs [145]. This is shown by Ad5 mutants lacking pIX, which can be propagated in the same way as wild type virions but they do not form GONs and they are heat-sensitive [146, 147]. Another potential role of this protein is to function as a transcriptional activator of the major late genes [148, 149]. pIX has also been proposed to play a role in the packaging of the virion; A virus defect in pIX can only carry a genome that is approximately 2 kb shorter than the normal genome, whereas others have demonstrated that pIX is not involved in the packaging but that lack of pIX renders the virus non-infectious [150, 151].

The adenovirus core

In contrast to the capsid, the core does not present a well-ordered symmetry or the coaxial coiled DNA as observed for bacteriophages [152, 153]. The genome of Ads is a linear double-stranded DNA of approximately 36 kb with a terminal protein (TP) attached to each 5´end, this protein is involved in initiation of replication [154, 155]. The core is associated with four other proteins aside from the TP named protein V, VII, µ and pIVa2. The most abundant of the core proteins is pVII with ~800 copies per virion. This protein is together with µ responsible for the packaging of the genome into nucleosome-like structures [110, 156, 157]. pV is only found in mammalian mastadenoviruses and interacts with pVI, pVII and/or DNA and thereby connects the core with the capsid [158, 159]. Like pVII and µ, which interacts

with the DNA, pIVa2 also interacts with the DNA but in a sequence specific way. pIVa2 is also known to activate the major late promoter and is responsible for the type specific packaging of DNA [160].

Non-structural proteins

There are about 30 non-structural Ad proteins identified. Most of these have a yet unknown function but are suggested to have catalytic or regulatory functions [34]. These proteins are produced in very small amounts and they are thereby difficult to study. However, two of the non-structural proteins have been structurally defined: the DNA binding protein (DBP) and the viral cysteine protease [134]. The DBP is a ~50 kDa phosphoprotein involved in DNA replication and other DNA metabolic processes [161]. It binds to single- stranded DNA and protects the DNA from digestion; DBP also destabilizes the DNA helix thus facilitating the replication of DNA [162, 163]. The adenovirus protease is a 25 kDa protein of which there are 10-30 molecules present in each virion [164]. Most of the minor capsid proteins and core proteins are cleaved by this protease and the cleavage product from pVI binds to the protease and increases its activity [165-167].

Picornaviruses

Picornaviruses are small (8.5 MDa) non-enveloped viruses with a positive sense RNA, which can act directly as an mRNA. They have an icosahedral symmetry and the size of the capsid ranges between 25-35 nm in diameter. Due to the lack of a lipid membrane these viruses are insensitive to organic solvents and the viruses with a tropism for the gastrointestinal tract are acid stable [168]. The picornavirus particles are composed of approximately 70 % protein and 30 % RNA [169].

Structural proteins

The icosahedral capsid is composed by 60 heteromeric protomers each consisting of four structural proteins: viral protein 1 (VP1), VP2, VP3 and VP4. Five protomers then assemble and form pentamers and twelve pentamers in turn assemble and form the capsid. Three of the proteins, VP1, VP2 and VP3 have a molecular weight (Mw) of approximately 30 kDa whereas the fourth protein VP4 is much smaller and has a Mw of 8 kDa [169]. The core structure of VP1, VP2 and VP3 is very similar, consisting of an eight-stranded antiparallel β-barrel and two α-helices. On the other hand, there is a high degree of diversity in the loops connecting the β-strands. The five-fold axis is formed by five copies of VP1, while the three-fold axis consists of a plateau formed by VP2 and VP3 (see figure 4) [22]. The N- terminal of VP4 and also its precursor VP0 is covalently linked to a myristic acid. Myristylation has been seen in several of the picornavirus genera, and this appears to be of importance during capsid assembly but has also been suggested to play a role in viral entry [170, 171]. An interesting finding in the structure of the first picornavirus studied by X- ray crystallography, human rhinovirus 14 (HRV-14), was a depression seen at the five-fold axis. This depression, or canyon, was believed to play a role in binding to receptors, since this location would be protected against neutralizing antibodies [172]. This hypothesis was later challenged when it

was discovered that neutralizing antibodies against HRV-14 could bind to residues within the canyon [173]. Below the canyon there is a “pocket” that contains a host-derived fatty acid. This “pocket-factor” is believed to stabilize the capsid [174]. Since the receptor-binding canyon and the pocket overlap it has been suggested that only one component at a time can be bound, this fact has been exploited when designing antivirals [175, 176]. However, more recent findings have questioned the role of “pocket-factors” for capsid stability, suggesting that it could be the cavity itself rather than the “pocket- factor” that is contributing to the stability [177].

Non-structural proteins

The non-structural proteins of the picornaviruses are encoded from the 3´end of the genome. There are seven to ten known non-structural proteins: 2A, 2B, (2BC), 2C, 3A, (3AB), 3B, 3C, (3CD) and 3D. These proteins are much more conserved than the capsid proteins, probably due to the lack of immune pressure and because of their importance [178]. The 2A protein is known to differ in function between different genera of the picornaviruses, in enteroviruses it functions as a cysteine proteinase that cleaves its own N- terminus to release the capsid protein precursor and it also cleaves cellular proteins that affects viral growth [179]. Proteins 2B, 2C and its precursor 2BC are involved in the remodeling of intracellular membranes to support viral replication [180, 181]. The EV protein 2B has been called a viroporin, a protein that oligomerize and inserts itself into membranes to create pores or channels leading to an increased diffusion of small molecules [182]. The function of this pore formation is not clear. Protein 2C is one of the most conserved picornavirus proteins but the exact functions of the protein remains unclear. It is suggested to e.g. have ATPase activities and RNA- binding activities [183, 184]. Protein 3A has the highest degree of variability between the different genera, but they all contain a hydrophobic domain that anchors the protein to the membranes where the replication occurs. This protein has been suggested to play a role in the membrane remodeling and also to be able to disrupt the endoplasmatic reticulum (ER)-to-Golgi

secretory transport and disassemble the Golgi complex [185, 186]. The 3B polypeptide is also called VPg (viral protein genome-linked), and it is attached to the 5´end of the genome where it serves as a primer to initiate RNA synthesis [187, 188]. The 3AB precursor protein seems to be directly involved in the RNA synthesis, either by donating VPg to the initiation of the RNA chain elongation or as a cofactor for the polymerase [189, 190]. The 3C polypeptide encodes the proteinase responsible for the majority of the cleavage of both viral and cellular proteins. 3C and 3CD also contain an RNA-binding motif and may therefore play a role in the replication process [191-193]. The most important (and therefore the most studied) protein is 3D, which is an RNA-dependent RNA polymerase (RdRP). The RdRP is the main subunit of the replication complex, which is responsible for the synthesis of viral RNAs [191].

Figure 4. Structure of human rhinovirus 16 (HRV-16). (a) Subunit organization at 3 Å resolution showing capsid proteins VP1-3 (blue, green and red), VP4 is not exposed on the capsid surface, (b) Close-up of the star- shaped dome at the fivefold axis and the surrounding canyon and, (c) Arrangement of VP1–VP3 on the icosahedral lattice. Fivefold, threefold and twofold symmetry axes are indicated. Images are taken from the Virus Particle Explorer database (VIPERdb; http://viperdb.scripps.edu) [194].

Viral life cycle

Receptors

The initial interaction between the virus and the cell takes place through one or more cellular receptors. This interaction is the starting point of the infectious cycle. Most Ads bind to host cells via the most distal part, the “knob” of the fiber protein [195]. In the case of the picornaviruses, many of the receptor interactions take place through the canyon, which is formed by the VP1 protein. Canyon binding was first suggested for HRV14 even before the actual receptor had been identified [23]. The first receptors identified for picornaviruses were the poliovirus receptor (PVR/CD155) and the major group rhinovirus receptor ICAM1 [196, 197]. Some picornavirus receptor binding sites are localized outside the canyon as for FMDV that instead have an exposed flexible loop on its capsid surface [198]. A number of different receptors are known today both for the Ads and the picornaviruses, and even though they are not related in any way they share a great number of receptors (see figure 5, table 4 and 5).

Figure 5. Schematic picture of cellular receptors that Ads and picornaviruses have in common. SA (sialic acid), HSPGs (heparan sulphate proteoglycans), MHC-1 (major histocompatibility complex-1), integrins and CAR (coxsackievirus and adenovirus receptor).

Table 4. Cellular receptors of the human adenoviruses, classified in species.

Species Type Receptors*

A 12, 18, 31 CAR [129]

B:1 3, 7, 16, 21 Desmoglein 2 [199] CD80, CD86 [200] CD46 [201] HS [202]

B:2 11, 14, 34, 35, 50 CD46 [201, 203] Desmoglein 2 [199] CD80, CD86 [200]

C 1, 2, 5, 6 CAR [123, 129] Heparan sulfate [204] VCAM-1 [205] MHC-1 [206]

D 8, 9, 10, 13, 15, 17, 19, 20, 22-30, CAR [129] 32, 33, 36-39, 42-49, 51 Sialic acid [207] GD1a glycan [208] CD46 [209]

E 4 CAR [129]

F 40, 41 CAR [129]

G 52 N.D.*

*cellular receptors that mediate direct Ad binding. *N.D; not determined.

Table 5. Cellular receptors of the human enteroviruses, classified in species.

Species Types Receptors

HEV-A CVA 2-8, 10, 12, 14, 16 N.D.*

EV 71, 76, 89, 90-92 SCARB-2, sialylated glycans, PSGL-1 [210-212].

HEV-B CVB 1-6 CAR, DAF, HS [123, 213, 214]

CVA 9, 23 avβ3, avβ6 , MHC-1, GRP78 [215-217]

EV 69, 73-75, 77-88, 93, 97, N.D.* 98, 100, 101, 106, 107

E 1-9, 11-21, 24-27, 29-33 VLA-2 (a2β1), DAF, HS [218-221]

HEV-C PV 1-3 CD155 [196]

CVA 1, 11, 13, 17, 19-22, 24, 24v ICAM-1, DAF, sialic acid [222-224]

EV 95, 96, 99, 102, 104, 105, 109 N.D.*

HEV-D EV 68, 70, 94 DAF and/or sialic acid [104, 225, 226]

HRV-A HRV 1, 2, 7-13, 15, 16, 18-25, 28-34, ICAM-1 [197] 36, 38-41, 43-47, 49-51, 53-68, 71, 73-78, 80-82, 85, 88-90, 94- 96, 98, 100

HRV-B HRV 3-6, 14, 17, 26, 27, 35, 37, 42, LDLR [227] 48, 52, 69, 70, 72, 79, 83, 84, 86, 91-93, 97, 99

HRV-C Types not yet assigned N.D.*

* N.D; not determined.

Immunoglobulin superfamily (IgSF)

The immunoglobulin superfamily is a large family of cell surface proteins with diverse biological functions. These proteins are important for cell surface recognition and thereby they play a major role in the immune defense. Members of the IgSF are commonly used as viral receptors and all of the known interactions take place via the most membrane-distal domain D1 of the IgSF receptor [228].

Coxsackievirus and adenovirus receptor (CAR)

In 1976, it became evident that CVB3 and Ad2 competed for the same receptor and that this receptor was different from the already known poliovirus receptor PVR/CD155 [229]. It was not until 1997 that the receptor was identified and given its name CAR [123, 230, 231]. CAR belongs to the immunoglobulin family and it consists of an extracellular domain, an intracellular domain and a single membrane-spanning domain and it has a Mw of 46 kDa. The extracellular part has two Ig-like domains, D1 and D2. The outermost D1 is the domain that binds both CVBs and Ads but they do not bind to the same part of the domain [124, 232]. CAR function as a receptor for all CVBs within the picornavirus family and for selected Ad types in species A, C-F [129, 230, 233, 234]. However, the Ad knob-CAR interaction function in vitro, which does not necessarily mean that it is a functional receptor in vivo. This was first indicated in 2001 when CAR was discovered to be located in tight junctions of polarized epithelial cells were it associates with ZO-1 [235]. These studies showed that in polarized epithelia, CAR is not accessible from the apical side and viruses might need a disruption of the epithelial surface to be able to enter the cells. Recently, an isoform of CAR (CAREx8) was discovered on the apical side of polarized airway epithelial cells and this isoform was enough to support viral attachment [236]. This indicates that CAR could be expressed on the apical side and thus, accessible as a receptor for Ads. Basolaterally and laterally expressed CAR has also been suggested to facilitate spread instead of entry,

since free Ad fibers can disrupt intercellular CAR-CAR-mediated junctions and thereby permit intercellular virus transport [237]. The distribution of CAR is not completely known, but it is present in heart, brain, pancreas, intestine and also in lung, liver and kidney [230, 238].

Intercellular Adhesion Molecule-1 (ICAM1)

ICAM-1 is present on many cells of the immune system and is involved in leukocyte adhesion to endothelial cells at sites of infection. ICAM-1 can be expressed on a wide variety of cells but normally, ICAM-1 expression is localized to a few cells since the expression have to be induced by the inflammatory response [239]. The natural receptor for ICAM-1 is a member of the integrin family called LFA-1 (leukocyte function-associated antigen-1) [240]. ICAM-1 is highly glycosylated and consists of five extracellular Ig-like domains, a transmembrane domain and a cytoplasmic domain [241]. ICAM- 1 is one of the major receptors for picornaviruses e.g. the major group of the rhinoviruses as well as CVA13, 18 and 21 but it is also a receptor for plasmodium falciparum [197, 223, 242-244].

Poliovirus receptor (PVR or CD155)

The PVR is the receptor for probably the most extensively studied picornavirus, PV. The receptor is a long, highly glycosylated member of the IgSF. It is composed of a C-terminal cytoplasmic part, a transmembrane part and an extracellular part consisting of three Ig-like domains D1, D2 and D3 [196, 245]. PVR is known to activate natural killer cells (NK cells) and it has a suggested role in cell motility and invasion by tumour cells [246-248]. The PVR gene is expressed as four different splice variants of which two lack the cytoplasmic part and therefore are released after expression [249]. The other two are functional PV receptors. The domain responsible for the interaction between PVR and the PV canyon is domain D1 [245, 250, 251]. The N- glycosylation of the D1 domain of the receptor is not involved in viral binding and PV interacts directly with the protein [252]. This receptor is not known to function as a receptor for any other virus. The tropism of PV for CNS

cannot be explained solely by the expression of the receptor since PVR is expressed in tissues other than those infected by PV, meaning that other factors are probably of importance also [196, 253].

Regulators of complement activation (RCA) family

The RCA family has the important role to distinguish “self” from “non-self” molecules via a number of proteins. They control the activity of the complement system and thereby prevent the destruction of host tissue and direct the complement degradation to e.g. harmful microorganisms [254]. These proteins have a common structure consisting of short consensus repeats (SCRs). Some members of the family are: membrane cofactor protein (MCP/CD46), factor H, decay accelerating factor (DAF/CD55), C4b-binding protein and complement receptor types 1 and 2 [255].

Membrane Cofactor Protein (MCP/CD46)

Membrane cofactor protein (MCP/CD46) belongs to the RCA family and its function is to protect healthy cells from complement-mediated destruction by binding C3b and C4b to facilitate their degradation by serum factor I. The protein consists of a cytoplasmic tail, a transmembrane region, an O- glycosylated STP (serine-threonine-proline) domain and four SCRs (short consensus repeats). The SCRs are characteristic for proteins of the RCA family [256]. Several Ads from species B are known to use CD46 as a receptor: Ad11, Ad16, Ad21, Ad35 and Ad50. From species D, Ad37 have been reported to recognize CD46 [201, 203, 209, 257, 258]. CD46 is expressed on all cells except for erythrocytes [259], and in opposite to CAR, CD46 is available on the apical surface of polarized epithelial cells and not constricted to the basolateral side or the tight junction [260]. Many pathogens have been shown to bind to CD46 e.g. Measles virus, bovine viral diarrhea virus, human herpes virus-6, Streptococcus spp. and Neisseria spp. [261-265].

Decay Accelerating Factor (DAF or CD55)

Like CD46, DAF also protects the cell from autologous lysis by complement. The protein consists of four SCRs, a heavily O-glycosylated STP region, but unlike CD46, DAF lacks a cytoplasmic region, and is instead linked to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Several EVs have been reported to use DAF as a receptor either for binding only or for both binding and internalization. However, there is no uniform binding between these viruses in that they bind to different SCR domains. CVA21 and EV70 both interact with SCR1; CVB interact with SCR2 (CVB3-RD (adapted to growth in RD cells)) or SCR3 (CVB1-3 and 6) [224, 266-269]. The only EV that can use DAF for both binding and internalization is echovirus 7 (E7), many or most of the other EVs also need another receptor as co-receptor for internalization [270].

Sialic acid

N-acetyl neuraminic acid (Neu5Ac), which was first discovered in the 1930s by Klenk and Blix [271], belongs to the family of sialic acids (SA) that contains more than 50 saccharides with a large diversity. Unlike most of the vertebrate monosaccharides which have five- or six-carbon backbones, SAs have a nine-carbon backbone [272]. A characteristic feature of SA is the carboxyl group at position 1 that gives a negative charge under physiological conditions, and the amino group at carbon number 5. The SAs are mainly present on the outermost part of glycan chains of glycoproteins or glycolipids and thereby easily accessible for interactions with other cells or with pathogens. They are linked via glycosidic bonds from carbon 2 to either carbon 3 or carbon 6 on the neighboring galactose or to carbon 8 on a neighboring SA [273].

The main function of SA is linked to cellular and molecular recognition such as binding and transport of positively charged molecules (e.g. Ca2+), protection of glycoproteins or entire cells from degradation by e.g. proteases

or macrophages, helping the immune system to distinguish self from non- self structures, and they are also involved in leukocyte “rolling” [273, 274]. A number of different microorganisms (virus, bacteria and protozoa) take advantage of this and use SA as an attachment receptor [272]. Some examples are; Influenza A virus [275, 276], JC and BK virus [277, 278], Theiler´s murine encephalitis virus [279], Haemophilus influenza [280] and Helicobacter pylori [281].

The three EKC-causing types, Ad8, Ad19 and Ad37 belonging to species D use SA as a receptor [207]. The positive charge of the fiber knobs of these viruses (9.0-9.1) together with the negative charge of sialic acid (pKa = 2,6) results in a charge-dependent interaction [282]. The AHC-causing EV70 from the picornavirus family is another virus with ocular tropism that uses SA as its attachment receptor, but this is only on specific cells [283].

Gangliosides

Gangliosides are glycosphingolipids containing one or more sialic acid residue linked to a glycan backbone, which is further attached to a ceramide base [284]. Gangliosides were first discovered in the 1930s by Ernst Klenck, and they were named due to their association with brain gray matter (“Ganglionzellen”) [285]. They are present in all vertebrate tissues, which imply that they play a crucial role in the cell [286, 287], however they are more concentrated in tissues of neuronal origin [288]. Several different gangliosides have been reported to play a role during infection, either as a receptor for a toxin or as a receptor for a microbe. For example, the cholera toxin from Vibrio cholera binds to the ganglioside GM1 a ganglioside that is also used as a receptor by the SV40 virus [289, 290]. Members of the polyomaviruses have been shown to interact with gangliosides: the murine polyomavirus binds GD1a and Gt1b, BK virus binds the gangliosides GD1b and

GT1b and JC virus recognizes GM3, GD2, GD3, GD1b, GT1b, GQ1b and GD1a to some extent [289, 291, 292].

Heparan sulfate proteoglycans (HSPGs)

Proteoglycans are built by long chains of glucosaminoglycans (GAGs) linked to a core protein, often membrane proteins such as glypicans or syndecans. GAGs are linear polysaccharides composed of disaccharide building blocks of either GlcNAc or GalNAc and uronic acid (glucuronic or iduronic). HSPGs function as co-receptors by binding via heparan sulfate (HS) to either insoluble ligands in the extracellular matrix (ECM) or to cells, or to soluble ligands such as growth factors or cytokines and thereby promoting interactions and subsequent signaling. HSPGs can also function directly as internalization receptors for soluble ligands e.g. enzymes [293]. A number of different bacteria, protozoa and viruses have been shown to interact with HS for infection, including members of the Borrelia burgdorferi, Chlamydia trachomatis, cytomegalovirus (CMV), dengue virus and herpes simplex virus [294-298]. HSPGs have been reported to function as receptors to some extent for Ad types 2 and 5 and as a probable co-receptor for Ad3 and Ad35 [202, 204]. Also, members of the picornaviruses interact with HSPGs; echoviruses, coxsackievirus B3 and FMDV [214, 220, 299].

Desmoglein-2

Desmoglein 2 (DSG-2), a member of the cadherin cell adhesion molecule superfamily, was recently reported as the previously unknown receptor “X” for species B Ad3, Ad7, Ad11 and Ad14 [199, 202]. DSG-2 is, like CAR, a member of the intercellular junctions. By binding to DSG-2, species B Ads open the junctions to access other junctional proteins. Species B Ads are known to produce large amounts of dodecahedral particles (PtDds) during infection and these incomplete particles are capable of interacting with DSG- 2 and thereby open the junctions. This overproduction of incomplete particles may be a way for corresponding virions to spread from or within the epithelial cell layer [199].

Integrins

Integrins are part of the immune system where they play a crucial role in e.g. leukocyte adhesion and cell polarization. They are heterodimeric molecules composed of a α and a β subunit. So far, 18 α and 8 β subunits form the 24 αβ pairs known for vertebrates [300]. Several integrins are functioning as co-receptors for Ads e.g. αvβ1/3/5, αMβ2, α5β1 and α3β1 [301-308]. One of the known binding motifs of integrins is the RGD (Arg-Gly-Asp) motif, which is exposed on the Ad penton base protein. Pb-mediated clustering of integrins leads to cell signaling and subsequent internalization [112, 309]. Several different Ad Pb proteins interact with integrins [117, 158]. Ad40 and Ad41 however lacks the RGD- motif leading to a delayed uptake [117].

Also picornaviruses are known to utilize integrins as their receptors e.g. echovirus 1 binds to α2β1 (also known as VLA-2; very late activation antigen-

2), CVA9 use αvβ3 or αvβ6 and FMDV use αvβ1/3/6/8 or α5β1 [218, 310-316]. CVA9 and FMDV both contain RGD-motifs and are dependent on this motif for binding to integrins on the cell surface as blocking of the RGD-motif protect the cells from infection [216, 317]. However, the binding between echovirus and α2β1 takes place in an RGD-independent fashion since α2β1 does not recognize this motif [218].

Other receptors

Two molecules from the IgSF, CD80/CD86 have been reported to function as receptors for Ads from species B [200]. CD80 is expressed on monocytes and B cells and CD86 is present on antigen-presenting cells. However, CD80 and 86 have been questioned by others about their role as functional receptors for species B Ads [318].

Scavenger receptor B2 (SCARB2) was recently identified as a receptor for EV71 and also for CVA16 [211]. Both these viruses cause the common hand, foot and mouth disease (HFMD) in children, however, EV71 infection can also lead to neurological complications and death. SCARB2 is a

transmembrane protein usually located in the endosomal-lysosomal compartment, and is involved in e.g. transportation across the endosomal- lysosomal membranes [319]. This protein has been reported to be important for internalization of Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes but also as a receptor for high-density lipoprotein [320-322].

P-selectin glycoprotein ligand-1 (PSGL-1) has also recently been reported to function as a receptor for HFMD-causing EV71 and CVA16 [210]. This protein is expressed on cells of hematopoietic origin, on dendritic cells in lymph nodes and on macrophages in the intestinal mucosa, and play an important role for recruitment of leukocytes during inflammation (stimulated by infection) [323-325].

Low density lipoprotein receptor (LDLR) and very low density lipoprotein receptor (VLDLR) have been reported to be receptors for the minor group rhinoviruses [227, 326]. Cryo-EM reconstruction of HRV2 and VLDLR revealed that the receptor interaction takes place on the capsid surface and not within the canyon [327]. Virus binding to LDLR does not trigger a conformational change in the capsid or RNA release, instead, the uncoating is dependent on acidification and therefore takes place in the endosomal compartment [328-330].

T-cell immunoglobulin and mucin-1 (TIM-1 or HAVCR) have been identified as a receptor for Hepatitis A virus (HAV) [331]. This is a class I integral membrane glycoprotein that is expressed in every human organ including the liver, small intestine, colon, spleen, kidney and testis. The natural function of this molecule is not known. Recently, HAVCR was shown to co- localize with the tight junction protein ZO-1, suggesting that HAVCR is located to and forms a part of the tight junctions [332].

Major histocompatibility complex-1 (MHC-1) has been suggested to function as a receptor for the fibers of Ad2 and 5 [206]. An increase of fiber binding

and gene transfer was seen when MHC-I was expressed on lymphoid cells as compared to cells lacking MHC-I expression. Later studies have not been able to confirm the role of MHC-I as an Ad receptor [333, 334]. From the picornavirus family, CVA9 has been shown to also interact with MHC-I via a co-receptor called glucose-regulated protein 78 kDa (GRP78) [215]. In their work they show that MHC-I molecules interact with GRP78 on the cell surface and that CVA9 in addition to αvβ3 uses GRP78 as a receptor molecule. αvβ3 and GRP78 are used for binding, but for uptake MHC-I is needed.

Vascular cell adhesion molecule-1 (VCAM-1) is a member of the IgSF. An increase in Ad transduction of NIH T3T cells was observed when VCAM-1 was expressed on the cell surface [205]. The D7 domain of VCAM-1 shares homology with the D1 domain of CAR thus, possibly explaining the interaction between Ads and VCAM-1.

Soluble components

Other, alternative pathways have been suggested where Ads use components of blood or other body fluids to bind and infect cells in a CAR-independent fashion [335-338]. Lactoferrin (Lf) is a body fluid component known to enhance binding and infection of species C Ads [336]. By interacting with Lf, Ads can take advantage of Lf receptors. Lf thereby functions as a bridging factor. From blood, coagulation factors IX, X and also complement factor C4bp have been reported to function as bridging factors for Ad5 on epithelial cells and hepatocytes [337, 338]. This interaction takes place via the cell surface HSPGs. Ad31 from species A is able to interact with factor IX although if this interaction takes place via HSPGs is not clear [337]. Dipalmitoyl phosphatidylcholine (DPPC) is a major contributor to the lipid pool of surfactants. Surfactants are used in the treatment of respiratory distress symptoms in premature children however; these surfactants have been shown to promote Ad gene delivery [339, 340]. DPPC interacts with the hexon protein of Ads [341].

The life cycle after binding to cells; adenoviruses

Internalization and trafficking

The main mode of entry for Ads is by receptor-induced endocytosis. The two most studied types, Ad2 and Ad5, enter cells via clathrin-coated pits, but this mechanism also triggers a second endocytic process, macropinocytosis [342- 344]. After the initial attachment of the Ad-fiber to its receptor, the RGD- motif in the Pb interacts with integrins (αvβ1/3/5, α5β1), which mediates viral entry [301, 303-306, 308]. This is confirmed for Ad2, Ad3, Ad4 and Ad5, but most Ad types display an RGD motif in their penton base, indicating similar interactions [301]. Integrins that are unable to recognize the RGD-motif have also been shown to interact with Ads, αMβ2 and α3β1 [304, 307]. Integrin-clustering caused by Ad binding in turn leads to activation of phosphatidylinositol-3-OH kinase (PI3K) and Rho GTPases which induces cytoskeletal rearrangements leading to clathrin-mediated uptake [345b, 346a]. Approximately 15 min after uptake, the fiberless virus escapes from the endosome into the cytoplasm through a process that involves αvβ5 integrins rather than αvβ3 [347]. It is also known that the virus needs the acidification of the endosome to lyse the endosomal membrane [348]. The capsid is dismantled step by step in a highly ordered fashion that starts by release of protein IIIa and the fiber leading to release of the Pb, a crucial step needed for endosomal escape. Protein VI has also been suggested to have a membrane lytic activity, thus, potentially aiding the endosomal escape [140]. The partially dismantled particle interacts with dynein and is thereby transported on microtubules to the nucleus and the NPC (nuclear pore complex) [349-351]. After viral binding to the NPC, the viral protease cleaves pVI and thereby completeing the capsid dismantling. Cellular proteins suggested to play a role in NPC binding are the nuclear pore protein CAN/Nup214, the chaperone Hsc70 (heat shock cognate protein) and also histone H1 [352, 353].

Figure 6. Infectious entry pathway for Ad2 and 5. Reprinted from [354] with permission from the publisher.

Structure of the genome

The genome of Ads is a linear double-stranded DNA that ranges between 34- 36 kbp in size and it encodes approximately 40 different proteins (see figure 7). It is characterized by the presence of 36 to 200 bp inverted terminal repeats (ITRs) at its ends and a TP linked to the 5´end [158]. The genome organization is highly conserved between the genera, and is divided into early transcription units (E1A, E1B, E2, E3 and E4), delayed early transcription units (IX, IVa2 and E2 late) and the late transcription unit (major late) [158, 355]. The major late transcript is further processed into five mRNAs (L1 to L5) [356]. All are transcribed by the cellular RNA polymerase II. The genome also encodes one or two virus associated RNAs (VA RNAs) depending on the serotype. These are transcribed by the cellular RNA polymerase III [357].

Figure 7. Transcription of the Ad genome. The early genes expressed are coloured green and the late are blue. The VA RNAs and the major late promoter (MLP) are also outlined in brown and black. Reprinted from [358] with permission from the publisher.

Gene expression and replication

Early gene expression

The three main functions of the early proteins are: a) to trigger S-phase entry of the host cells, b) to protect the infected cell from different types of antiviral defense mechanisms and c) to synthesize the gene products needed for the viral replication [355]. The E1A proteins are so called trans- activators, meaning that they are able to activate transcription of both viral and cellular genes. The E1A proteins can also interfere with complexes formed by members of the pRb family and the E2F family and thereby push the cells into S-phase [359, 360], which creates an optimal environment for viral replication. E1B encodes two proteins that block apoptosis and the function of both the E1B proteins is needed for inhibition of the E1A-induced apoptotic program. E2 encodes three proteins that are all involved in DNA replication, the polymerase (AdPol), the DBP and the TP. E3 proteins are

involved in modulation of host responses to the infection. The E4 proteins have a number of different functions, including transcriptional and translational regulation, nuclear export of viral mRNA and inhibition of apoptosis [355]. The Ad genome encodes one or two VA RNA:s, these small abundant RNAs inhibits the interferon (IFN) response of the innate immunity by binding to protein kinase receptors (PKR) [357, 361].

Replication

In cell culture, the replication of the Ad genome starts approximately five to eight hours after infection and is triggered by the accumulation of E2 gene products. The replication takes place in two steps (see figure 8) [362]: Initiation takes place at both ends of the genome and thereafter proceeds continuously giving rise to one double-stranded complex and one single- strand of DNA (Type I replication). In the second step, complementary strands to the single-strands are produced. The single-stranded DNA circularizes by annealing its self-complementary termini to form a “panhandle”-structure. This panhandle has the same structure as the double- stranded genome and is therefore recognized by the same machinery that function in the first step, resulting in the production of a complementary strand (Type II replication) [355].

The terminal ITRs of the viral chromosome function as replication origins. By associating with two E2-encoded proteins; the TP and the AdPol, viral replication is initiated. The TP functions as a protein primer [363]. However, for efficient replication a number of cellular proteins are also needed e.g. nuclear factor I &II (NF-I, NF-II) and Oct-1 [364-366]. The role of the viral DNA binding protein (DBP) for replication is believed to be to displace the non-replicated strand during the elongation [367]. The viral proteins can themselves initiate replication but not as efficient as when the cellular factors are present. In that case, the replication initiation is approximately 200 times more efficient [368].

Figure 8. The replication of the Ad genome, shown in the two steps: type I replication and type II replication. Reprinted from [362] with permission from the publisher.

Delayed early gene expression

Expression of the delayed genes takes place immediately after onset of DNA replication but before expression of the late genes and they encode pIX and pIVa2. pIX is a capsid component but it also activates transcription. The IVa2 gene product has two functions, one is to activate transcription from the MLP (major late promoter) and the other is to facilitate packaging of the genome into the capsids [355].

Late gene expression

The late genes encode the capsid components, which are expressed after the onset of DNA replication. These genes encode one single transcript of about 28 kb, which is spliced into twenty different mRNAs. These mRNAs are grouped into five families, L1 to L5. The expression of these genes is controlled by the major late promoter (MLP) [355].

Assembly and release

The accumulation of large amounts of structural proteins and new genome copies results in the Ad capsid assembly. Hexon monomers assemble into trimers in the cytoplasm with the help of the L4-encoded 100K protein. Pb and fiber monomers assemble independently of each other in the cytoplasm but later join to form pentons [369, 370]. After this initial assembly, the hexons and penton capsomers are transported into the nucleus where the final assembly takes place. Import of hexon to the nucleus relies on the capsid stabilizing protein VI, which shuttles between nucleus and cytoplasm [371]. The DNA is packaged from the 5´end of the genome were the 200 bp packaging sequence is situated [372]. The DNA packaging is promoted by protein IVa2-, 52/55K- (L1) and 22 kd protein (L4) binding to the packaging sequence [373, 374]. The complete maturation of the provirion into an infectious virion requires enzymatic processing of proteins VI, VII, VIII, µ and TP by the Ad protease [373]. There are at least three different viral systems for release of progeny virions and escape from the site of infection: a) Interfering with the cellular intermediate filaments by proteolytic cleavage of the cellular cytokeratin K18 by the viral L3 protease, which renders the cell more susceptible to lysis [375], b) Accumulation of the adenovirus death protein (ADP) late during infection leads to cellular lysis [376], c) As previously mentioned, extensive production and release of Ad fibers during infection interferes with CAR at the tight junctions and facilitates the transport of progeny virions from the infected cell [237].

The life cycle after binding to cells; picornaviruses

Internalization and trafficking

Previously, picornaviruses were believed to undergo uncoating already at the cell surface but now it appears as if they are internalized into vesicles first [329, 377, 378]. The picornaviruses use a number of different entry pathways: a) FMDV use integrins as their receptors and are known to enter cells by clathrin-mediated endocytosis [379, 380]. This is a pH-dependent pathway where the acidification of the endosome leads to capsid disassembly [168]. Cell culture adapted FMDV use instead HS as receptors and enter cells in a caveolin- and lipid raft-dependent way [299, 381]. b) CVB3 use DAF and CAR as their receptors and have been shown to enter cells in a complex process [382]. The entry mechanism of these viruses is dependent of caveolin-1 and therefore it was suggested that they enter via caveoli. However, a more recent study showed that entry over the tight junction leads to internalization of occludin via macropinocytosis [383]. Therefore CVB3 seems to somehow combine caveolin-mediated and macropinocytic uptake. c) PV uptake was recently shown to depend on actin, energy and an unidentified tyrosine kinase [384]. The uptake was not due to clathrin- mediated endocytosis, caveoli or macropinocytosis. The release of RNA takes place just beneath the plasma membrane (100-200 nm) (see figure 9).

For PV, HRV and EV, the virus-receptor interaction triggers a conformational change in the capsid leading to the formation of A-particles (135S particles) [385, 386]. In the A-particles, the myristoyl-VP4 and the N- terminal of VP1 are exposed on the capsid surface. The N-terminal part of

VP1 has been suggested to penetrate the plasma membrane and thereby either disrupting the membrane or forming a pore for RNA injection into the cytoplasm [387]. However, more recent data have suggested a central role of VP4, as shown by mutational analysis disrupting RNA release and pore- formation. Also, bacterially expressed myristoylated VP4 from HRV-14 can by itself form pores in liposomes [388, 389].

Figure 9. A schematic picture of the entry mechanism of poliovirus. After viral binding to PVR, the virion is internalized through a clathrin- and caveolin-independent, but actin- and tyrosine kinase-dependent mechanism. The release of RNA takes place close to the cell membrane (100-200 nm). After RNA release the empty (80S) capsids are transported via microtubule towards the cell center perhaps for degradation in the endosomal or lysosomal compartments. Reprinted from [384].

Structure of the genome

The picornavirus genome consists of a single positive-stranded RNA molecule that by itself is infectious since it encodes all the proteins needed for viral replication. A feature of the picornavirus mRNA is the VPg protein that is attached to the 5énd [390, 391]. The genome size varies from 7209 to 8450 bases and contains one single open reading frame (ORF). Both the 3énd and the 5énd of the genome are flanked by untranslated regions (UTRs). The long 5ÚTR of the genome contain the internal ribosome entry site (IRES) that bind to the ribosomes to initiate translation [392]. The short 3ÚTR contain a poly A tail and a secondary nucleic acid structure called a pseudoknot, these are involved in controlling viral RNA synthesis [393, 394]. The picornaviral genome is divided into three protein encoding domains: P1, P2 and P3. The P1 region encodes the capsid proteins (structural proteins), the P2 region mainly encodes proteins involved in structural membrane rearrangements and the P3 region encodes proteins involved in RNA synthesis (see figure 10) [168].

Figure 10. The picornavirus genome is a plus stranded RNA and can thereby be translated directly into proteins. The three protein encoding domains P1-P3 are outlined, and the products and their functions are visualized. Reprinted from [395].

Translation and replication

Picornaviruses do not carry any proteins into the host cells and since the RNA cannot be copied by cellular RNA polymerases it must first be translated into proteins. Although the cap-dependent cellular mRNA translation system is shut down by picornaviruses the cap-independent synthesis of viral proteins is allowed due to the existence of an IRES sequence in the viral genome [392, 396]. The 40S ribosomal subunit bind to the IRES sequence by the help of a number of different viral translation initiation proteins (depending on the picornavirus type), and the mRNA is translated into a polyprotein. The polyprotein is then processed by the viral proteases, 2A and 3C or 3CD.

Picornavirus infection triggers a rearrangement of intracellular membranes, filling the cytoplasm with double-membraned vesicles [397]. The cytoplasmic surface of these vesicles is the place for viral replication [398]. The positive-sense RNA is then copied by the 3D polymerase, using VPg as its primer, into negative-sense RNA that carries VPg at its 5´end. This negative-sense RNA then function as a template for production of new positive-sense RNAs of which some in turn loses their VPg and are translated into viral proteins whereas others are packaged into capsids during assembly (see figure 11).

Assembly and release

Very little is known about the encapsidation of picornaviruses. The VPg protein has been suggested to play a role in the encapsidation of the genome since VPg-lacking RNAs are not packaged correctly [399, 400]. If this actually result directly in defect packaging, or if there is a link between RNA synthesis and packaging is not yet known. Moreover, each of the capsid proteins interacts with the RNA suggesting that encapsidation takes place at the pentamer level and that packaging of empty capsids does not occur [401, 402]. The final step of the assembly is the maturation of the capsid by cleavage of VP0 into VP2 and VP4, which stabilizes the capsid. The

mechanism behind the maturation is not yet known [403]. The newly produced virions normally leave the cells by lysis [404]. However, it has also been suggested that picornaviruses are able to cause non-lytic, persistent infections [405-407]. More recently, one of the innate responses to microbial infections, autophagy, was suggested as a way for non-lytic release of picornaviruses [408].

Figure 11. The poliovirus life cycle divided into different steps: 1) viral attachment to the PVR, 2) uncoating of the viral RNA, 3) cleavage of the VPg and cap-independent translation of the RNA, 4) proteolytic processing of the polyprotein, 5) the positive-sense RNA serves as template for complementary negative-strand synthesis, 6) initiation of many positive strands from a single negative strand, 7) newly synthesized positive-sense RNAs either lose their VPg and serve as templates for translation or, 8) associate with capsid precursors to undergo encapsidation, 9) release of progeny virions. Reprinted from [409].

Antivirals

Adenoviruses

Since the infections caused by Ads in otherwise healthy (immunocompetent) individuals, are self-limiting and rather mild, the development of antivirals have not been of great interest. Previously, the only type of infection where antivirals were thought to be needed was for treatment of EKC. Later, an increased knowledge about the severity of Ad infections in immunocompr0mized patients has led to a boost in the development of anti- adenoviral drugs [78, 410, 411]. However, no antivirals have yet been approved for treatment of Ad infections.

The antivirals that are available today against double-stranded DNA viruses all target the viral polymerase, and some of them have been evaluated for treatment of Ad infections also [412]. One of these, cidofovir, which is an acyclic nucleoside phosphonate seems to be the most effective [413]. The effect of cidofovir has been tested against ocular infections in New Zeeland rabbits caused by a number of Ads, but mainly from species C (Ad1, Ad5 & Ad6) [414]. Unfortunately, the EKC-causing types (Ad8, Ad19 & Ad37) do not infect these rabbits or any other animal model, which makes it difficult to test the in vivo effect of anti-Ad drug candidates. Nevertheless, the positive results from this and subsequent studies lead to a large clinical trial in the US for the evaluation of cidofovir for treatment of ocular Ad infections. Unpublished results from this study showed a significant efficacy both in the clearance of the infection and protection from spread to the other eye [78]. However, later usage in Europe and Hawaii was associated with rare cases of lachrimal canalicular blockade. Thus, the narrow efficacy-to- toxicity ratio lead to the discontinuance of this treatment [78].

CMX-001 is a lipid conjugate of cidofovir that is orally administered [415]. Normally, cidofovir is given intravenously due to the low bioavailability when administered orally. By adding a lipid moiety the bioavailability of

orally administered cidofovir was improved. CMX-001 was shown in cell culture to be a potent antiviral against all tested DNA viruses and animal studies showed protection against orthopoxvirus, CMV and Ads [412]. Clinical studies are ongoing.

Zalcitabine, a nucleoside reverse transcriptase inhibitor (NRTI) used in the treatment of human immunodeficiency virus (HIV) infections have been reported to have an anti-Ad effect both in vitro and in vivo [416, 417]. The effect of zalcitabine against ocular Ads indicated a more potent antiviral effect than cidofovir and clinical evaluation of the ocular toxicity gave similar result, indicating that zalcitabine would be safer to use than cidofovir [418].

A sialylated lipid called NMSO3 have been suggested to have anti-Ad activity [419]. This compound is a non-toxic broad-spectrum compound with an activity against a number of different viruses; respiratory syncytial virus (RSV), HIV, influenza A virus and rotavirus [420-422]. It was shown to have an effect against all the tested Ad types (Ad2, Ad4, Ad8 and Ad37), and compared to cidofovir and zalcitabine, NMSO3 showed almost no cellular toxicity. The effect was due to the interaction of NMSO3 with the virus and not the cells, and most probably a charge dependent interaction with regard to the negative charge of the compound. The charge dependent interaction is the most likely in the case of Ad8 and Ad37, which normally bind to cellular sialic acid, but the effect seen on the species C Ads (2 and 4) that normally bind to CAR is not clear. Somehow the Ad-NMSO3 interaction blocks the ability of the virus to interact with its receptor [419].

N-chlorotaurine (NCT) is the N-chloro derivative of the amino acid taurine. This amino acid has previously been shown to have microbiocidal effects against bacteria, yeast and mold [423, 424]. NCT has also been shown to have an anti-Ad activity against types from species B, C, D and E (1, 2, 3, 4, 5, 7a, 8, 19 and 37) [425]. The anti-Ad effect in the Ad5/NZW rabbit ocular model indicated a promising in vivo activity. Clinical trials have been conducted with positive results, reduction of symptoms was seen especially

in the more severe infections but NCT could not prevent the formation of subepithelial infiltrates [426, 427]. However, larger trials are needed to fully evaluate the efficiency of NCT as an anti-Ad compound.

The immunosuppressant cyclosporine A (CsA) inhibits the transcription of interleukine-2 (IL-2) and is used during organ transplantation to prevent rejection [428, 429]. CsA has been evaluated as a treatment for ocular infections caused by Ads [430, 431]. Topical treatment with 1% CsA reduced the regular symptoms but also the corneal opacities and topical treatment with 2% CsA reduced the subepithelial infiltrates in chronic cases [430, 432].

Povidone-iodine (PVP-I) is a common antiseptic used in general surgery and ophthalmology. PVP-I is known to be toxic for viruses, parasites, fungi and bacteria [433]. Dexamethasone is a steroid commonly used as an ophthalmic anti-inflammatory agent [434]. By combining this steroid and the antiseptic PVP-I a good inhibitory effect was seen on ocular Ad infections both in a small clinical trial and in the Ad5/NZW rabbit ocular model [435, 436].

Picornaviruses

The development of antivirals against picornaviruses started during the 70s and received a boost in the 80s when the first crystal structure was determined for HRV-14, a member of the enterovirus genus, and the capsid canyon was discovered [23]. This canyon was subject for targeting of different compounds that disrupted viral binding or uncoating [175, 437]. These first compounds were later named WIN compounds, referring to the Sterling-Winthrop company that developed the compounds [438, 439].

Modifications to increase the potency of these WIN-compounds led to the development of a substance called Pleconaril [440]. This substance was found to have a broad-spectrum activity against the prototype strains of EVs and also against the most commonly isolated EV types and in 1996 the drug was used in patients with life-threatening enterovirus infections [441, 442]. However, from 2002 the drug was disapproved by the Food and Drug Administration because of its unconvincing safety profile [443].

A number of replication inhibitors have also been developed, targeting protein 2C, 3A and 3D. The protein 2C inhibitor guanidine hydro- chloride was one of the first reported picornavirus inhibitors [444]. The effect of this compound is due to inhibition of negative strand RNA synthesis by disruption of the 2C/2BC association with the cellular membrane during replication [445, 446].

Two other replication inhibitors called EnvirX (Enviroxime) and EnvirD (Enviradone) have been evaluated for the treatment of AHC-causing viruses (EV70 and CVA24v) [447]. These compounds gave a significant block of viral replication and cytopathic effect (CPE) at concentrations that were at least 10 times lower than the cytotoxic levels. However, none of the replication inhibitors have been very successful since the effect seen in vitro is not obtained in vivo.

Two different protease inhibitors have also been evaluated; Rupintrivir and “Compound 1” [448, 449]. Although both of them showed promising effects in initial studies the clinical trials were unsatisfying and the clinical development of the drugs was terminated [450].

Another way of interfering with viral infections is by blocking the receptor with antibodies to hinder viral attachment, or by adding soluble receptor analogues, thus competing out the viral binding. Antibodies against the major rhinovirus receptor ICAM-1 has been evaluated but the avidity of these antibodies was judged to be insufficient [451, 452]. Further development of these antibodies into recombinant high-avidity multivalent antibodies is an ongoing and apparently promising project [453].

A new promising strategy of inhibiting virus replication is by means of small interfering RNAs (siRNAs), which is a strategy used in the eukaryotic cells to inactivate translation of specific proteins. The mechanism behind this is as follows; cellular double-stranded RNA is used to produce small interfering RNAs (siRNAs), these siRNAs are bound by RISC (RNA-induced silencing complex) and directs the degradation of mRNAs containing sequences complementary to one of the strands of the siRNA [454]. The development of siRNAs to target the AHC-causing viruses (EV70 and CVA24v) have shown some promising results [455]. The target for their siRNA is the homologous regions within the viral polymerase (3D) gene.

Results and Discussion

Paper I

Multivalent sialic acid conjugates inhibit adenovirus type 37 from binding to and infecting human corneal epithelial cells.

Johansson SM, Nilsson EC, Elofsson M, Ahlskog N, Kihlberg J, Arnberg N. Antiviral Res. 2007. 73:92-100.

This study was a continuation of a previous study by Johansson et al. 2005 where the effect of multivalent 3´SLA (3´sialyl lactose) on Ad37 binding to and infection of human corneal epithelial cells (HCE) was evaluated [456]. In that study the conclusions were that Ad binding and infection was inhibited up to 1000 times more efficient with multivalent SA compounds as compared to corresponding SA monosaccharide. At approximately the same time, the Ad37 fk was crystallized together with 3´SLA and one intriguing finding was that the only part of 3´SLA that interacted with the fk was the SA monosaccharide [126]. No interactions were seen between the fk and the neighboring galactose or glucose saccharides of the compound. These two previous studies were the starting point for paper I.

Thus, in this study we wanted to see if the same effect could be accomplished with the much simpler multivalent compound that was based on SA monosaccharides coupled to HSA (human serum albumine) instead of 3´SLA-HSA. Initially, a number of SA-containing monovalent compounds were tested and the results showed that mono-, di-, tri-SA as well as 3´SLA had a similar effect on Ad37 binding to corneal cells. The IC50 values (50% inhibition of binding compared to the control) of all compounds were between 1 to 5 mM. Next, the multivalent SA-HSA compounds were synthesized in a process similar to the one used for producing the multivalent 3´SLA-HSA compounds. One major difference however, was the number of steps in the synthesis, 3´SLA-HSA was produced in eleven steps

and SA-HSA synthesis only involved four steps. The synthesis was designed to result in three compounds with varying multivalency: 3-, 8- and 13- SA saccharides incorporated per HSA, which were further evaluated. The inhibitory effect of the compounds was first evaluated in a binding assay using radiolabeled virions. The results showed that SA-HSA were competent inhibitors of Ad37 binding to HCE cells. The IC50 values (with respect to SA monosaccharide) for the different compounds were: 5 mM for monovalent SA, 0,2 mM for 3-valent, 10 µM for 8-valent and approximately 5 µM for the

13-valent compound. The IC50 values for mono- and 13-valent SA were comparable with the corresponding results seen for mono- and 19-valent 3´SLA-HSA in the previous study. The effect of these new compounds was then tested on Ad37 infection of corneal cells and as expected, the results were similar to the ones from the binding assay. The IC50 value for the 8- and 13-valent SA-HSA was approximately 3 µM whereas monovalent SA however did not reach 50% inhibition at concentrations up to 5 mM. The 3-valent compound was not included in this experiment. The results from both the binding and the infectivity assay showed a clear valency-dependent inhibition. Based on the previously suggested hypothesis that the effect seen of 3´SLA-HSA on Ad37 binding and infection was due to the ability of the compound to aggregate virions we set up a new experimental method to try to confirm and also measure the level of aggregation. The setup was to incubate virions with or without (control) compound and then measure the amount of radioactively labeled virions in the supernatant (top 90 µl) and in the pellet (bottom 10 µl) after centrifugation at different speeds (g). In samples with compound, centrifugation at increasing g lead to increased amounts of virions in the pellet, and to decreased amounts of virions in the supernatant. This was also seen in the controls (w/o compound), but to a much smaller extent. For example, at the highest g the majority of virions in the control were in the supernatant, whereas in samples with compound, the majority of virions were found in the pellet. Thus, this simple assay confirmed our hypothesis that the effect seen on Ad37 binding and infection was in fact due to the aggregating ability of the compound.

From these data we concluded that SA-HSA is as efficient as the previously studied 3´SLA-HSA in inhibiting Ad37 binding and infection. The concentration needed to reach the IC50 was similar for both compounds and was approximately 1000 times lower than the concentration required to reach IC50 with SA monosaccharides. There was a clear correlation between the order of valency and the effect seen. Using the aggregation assay we could also conclude that the effect seen on viral binding and infection is because of the ability of the compound to bind to several viral particles at the same time and thereby aggregating them, which more efficiently prevent binding of virions to cells. SA-HSA and 3´SLA-HSA were equally efficient inhibitors, but since SA-HSA is synthesized in only four steps this molecule would be easier and cheaper to produce and would therefore be a better antiviral candidate than 3´SLA-HSA. This smaller SA-containing compound may also be easier to systematically modify at different positions in order to increase the affinity for the Ad37 fk as compared to the multivalent 3´SLA, which is a more complex structure, and would therefore be more difficult to modify since e.g. there are more groups to protect. Multivalent SA has been shown to be an effective inhibitor of both viruses and bacteria in vitro [457, 458]. The problem with many of these drugs is that the drug is delivered to a large organ that is difficult to reach. We present a candidate drug that targets viruses that infect the eye, which is a small and accessible organ for topical administration. In conclusion, we present here a new drug candidate type for treatment of Ad-caused epidemic keratoconjunctivitis (EKC).

Paper II

Design, synthesis, and evaluation of N-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis.

Johansson S, Nilsson E, Qian W, Guilligay D, Crepin T, Cusack S, Arnberg N, Elofsson M. J Med Chem. 2009. 52:3666-78.

In this paper we set out to evaluate the effect of modified SA on Ad37 binding to and infection of human corneal epithelial cells. The aim was to see if the binding between the Ad37 fk and modified SA could be enhanced as compared to native SA. If so, such results could be used for design and development of more efficient antiviral drug candidates for treatment of EKC. Structure-based design and docking studies (based on the known crystal structure of Ad37 fk in complex with 3´SLA) were used to design a small library of ten different SA-derivatives. In these derivatives, the N- acetyl group was replaced by a number of different groups. According to the structural data of the Ad37 fk-3´SLA complex, the N-acetyl group of SA was directed into a deep hydrophobic pocket, and thus we hypothesized that the inhibitory effect of SA could be enhanced by substituting this group with a larger, more hydrophobic group.

The inhibitory effect of the compounds was evaluated in biological assays. Three of the compounds inhibited binding of Ad37 virions to corneal cells. These three compounds were 17a, which was predicted to be a good inhibitor according to docking data, and 17e and 17g. 17a was equally efficient as SA and the other two were slightly less efficient. All of the other SA-derivatives with large N-acetyl substituents (17b, 17c, 17d, 17h and 18) were unable to inhibit Ad37 binding. Five of the compounds, 17a, 17b, 17c, 17e and 17g were then evaluated by their ability to inhibit Ad37 infection (17b and c were

included as negative controls). Once again, compounds 17a and 17g showed a similar activity as SA, but compound 17e on the other hand had almost no effect at all. Compounds 17b and 17c had no effect either in the binding or infectivity assays. Next, compounds 17a-c was coupled to HSA in a similar way as in the previous studies with SA-HSA, with a varying valency (14-17- valent). These multivalent compounds were then evaluated for their ability to block Ad37 infection and the results showed that the N-acyl modified SAs all behaved as their monovalent counterparts. The only multivalent compound that actively blocked Ad37 infection was compound 22a (which was based on compound 17a) however, the inhibitory effect was not as good as the effect seen with SA-HSA. The final step was then to co-crystallize the fk together with the three active compounds 17a, 17e and 17g to get a detailed picture of the exact interaction, and then compare this interaction with that of SA-Ad37 fk. In the docking studies compound 17e was suggested to interact with the fk in a different way as compared to SA. This was confirmed by X-ray crystallography data, showing that 17e was not detected in the known SA binding site or anywhere else in the fk protein. The effect seen by this compound is most likely due to unspecific binding, or, due to binding to another capsid protein. The structural data showed that compound 17a and 17g interacted with the fk in a similar way as SA. However, no additional interactions were obtained due to the substitutions made.

In this study we evaluated the possibility to improve the binding between SA and the Ad37 fk by substituting the N-acyl group of SA. Of the ten initial compounds, three were shown to be as effective as SA in inhibiting binding of Ad37 virions to HCE cells. Of these three compounds, only two had an inhibitory effect comparable to SA in the infectivity assay. The corresponding, multivalent conjugates showed a valency-dependent inhibition of Ad37 infection, although not as efficient as SA-HSA. Finally, co- crystallization data confirmed that the modified compounds that displayed an inhibitory effect interacted with the fk in the same way as unmodified SA. The conclusions were however, that the most promising anti-Ad compound was the previously tested SA-HSA. In order to obtain a more efficient

inhibitor of Ad37, we conclude that more extensive structure-activity studies are required. An alternative strategy for development of successful inhibitors might be needed due to the complicated synthesis of modifying SA. A possible strategy could be the use of non-carbohydrate sialyl mimetics, which would require less complicated synthesis and a greater possibility of varying the functional groups, or, a trivalent SA with three spacers designed to fit into the three known SA binding sites.

Paper III

Sialic acid is a cellular receptor for coxsackievirus A24 variant, an emerging virus with pandemic potential.

Nilsson EC, Jamshidi F, Johansson SM, Oberste MS, Arnberg N. J Virol. 2008. 82:3061-8.

Acute hemorrhagic conjunctivitis (AHC) is a severe ocular infection caused primarily by two members of the picornavirus family: Coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70). These viruses have since they were first discovered in 1969 caused three pandemics affecting probably more than 100 million people [25, 104]. Previously, EV70 was the main causative agent but during the past 10 to 15 years most of the outbreaks have been caused by CVA24v. Two receptors for EV70 have previously been identified, a) SA on human corneal epithelial cells and on human monocytes (U937) and b) CD55/DAF on Hela cells [104, 225]. In this study we set out to identify the so far unknown receptor for the other AHC-causing picornavirus, CVA24v. First we evaluated the ability of CVA24v to bind and infect different CHO cells that express specific, known picornavirus and Ad receptors. CVA24v bound almost equally well to all CHO-cells expressing CAR, CD55/DAF, CD46/MCP, ICAM-1 and also GAG deficient (pgsB-618) cells. Lec2 cells however, which are deficient in SA expression showed a

much weaker binding of CVA24v. When SA was enzymatically removed from the CHO cells, a strong reduction in binding was observed for all cells, except for Lec2 where only a 10 % reduction of the originally low binding was observed. An infectivity assay was then performed to investigate if SA was important also for the infection and again, CVA24v infected all CHO-cells to an equal extent except for the Lec2 cells, which were more or less resistant to infection. These initial results suggested that SA play a role in CVA24v binding and infection.

To find suitable cell lines for further tests we first evaluated a number of epithelial cells that had been used previously for isolation of CVA24v, for their susceptibility to virus binding and entry and also expression of known picornavirus receptors: Hep2, Hela, A549 and green monkey kidney cells (GMK). Also, two cell lines that represent the ocular tropism of this virus were included: human corneal epithelial cells (HCE) and normal human conjunctival cells (NHC). Here we found that the cells most susceptible to infection were the NHC and HCE cells together with the larynx epithelial cell line Hep2. CVA24v also showed the highest binding to two of these, NHC and Hep2. Expression of cell surface receptors by flow cytometry showed that NHC cells had, in comparison to the other epithelial cells, the highest expression of the following known picornavirus receptors: SA, CAR, CD55/DAF, CD46/MCP and ICAM-1. The ocular cell lines NHC and HCE had SA expression levels approximately three to four times higher than the other cell lines. The receptor identified for other Coxsackie A viruses, ICAM- 1, was almost only expressed on NHC cells. Based on these findings HCE, NHC and Hep2 cells, representing the ocular and respiratory tropism of CVA24v, were used in further studies.

To test if SA was a functional attachment receptor for CVA24v, NHC, HCE and Hep2 cells were treated with neuraminidase to remove cell surface SA, and then incubated with isotope-labeled CVA24v virions. A substantial reduction in virus binding was seen on CVA24v binding to HCE cells (85%) and Hep2 cells (58%) but only a modest decrease was seen with the NHC

cells (28%). Further, incubation of cells with the SA-binding lectin wheat germ agglutinin (WGA) resulted in a strong reduction in CVA24v binding to HCE (93%), but not to NHC or Hep2 cells (46% and 30% respectively). The effect of neuraminidase and WGA was also evaluated in an infectivity assay showing a strong inhibitory effect with both neuraminidase and WGA using HCE cells (90% in both cases), but only minor inhibitory effects were observed with the other two cell types. The previously discussed 13-valent SA-HSA, which is a very potent inhibitor of Ad37 binding to and infection of HCE cells, was also evaluated for its ability to inhibit CVA24v binding to ocular cells. The binding to NHC cells was not affected at all but the binding to HCE cells was reduced by more than 50% at the highest concentration (0,5 mM with respect to SA). This effect was however not at all in the same range as the effect seen for Ad37 binding where the IC50 was at a concentration of approximately 5 µM. Thus, SA-HSA was thereby considered not to be a suitable candidate for treatment of CVA24v-caused AHC. A possible explanation for the lack of effect of SA-HSA on CVA24v binding could be that SA, when coupled to HSA, is less able to access the binding site on the viral capsid.

Since other viruses with an ocular tropism have been shown to have a preference for SA linked via α2,3 glycosidic bonds to neighboring galactose (SAα2,3Gal) [459], we set out to investigate if this was also the case for CVA24v. We first used two types of lectins, maackia amurensis lectin type II (MALII), which recognizes SAα2,3Gal-containing glycans, and the sambucus nigra (SNA) lectin, which recognizes SAα2,6Gal-containing glycans. SAα2,3Gal was the dominant type present on the surface of NHC and HCE cells, according to flow cytometry analysis. When trying to inhibit viral binding to these cells, MAA only displayed a small inhibitory effect on binding to HCE cells and the opposite was seen for the NHC cells, were only SNA exerted a small inhibitory effect. A pronounced inhibitory effect on CVA24v binding was however seen when the two lectins were mixed: 67% reduction for NHC and 66% for HCE. Finally, in order to test whether the SA-containing receptor(s) were linked to either a protein or to a lipid, we

first treated cells with proteases prior to the addition of isotope-labeled virions. The use of three proteases resulted in a significant reduction of CVA24v binding, thus suggesting that CVA24v uses a sialylated protein rather than a lipid as a cellular receptor.

In this work we investigated whether any of the previously known picornavirus or Ad receptors were used by CVA24v. Using receptor- expressing CHO cells we found that CVA24v interacted with SA and not with any of the other known receptors. From experiments where we i) enzymatically removed cell surface SA, ii) blocked cell surface SA with SA- binding lectins, or iii) pre-incubated virions with soluble SA-containing molecules, we concluded that the major receptor for CVA24v on CHO cells and on human corneal epithelial cells contain SA. Regarding cells originating from conjunctiva or larynx, SA contributes to some extent to binding and infection but on these cells SA does not seem to be a main receptor. From experiments where cells were treated with proteases, we also concluded that the receptor used by CVA24v on corneal and conjunctival cells most likely contain one or more protein component(s), and gangliosides are less likely to be used as receptors for CVA24v. It has been demonstrated that SA-binding viruses have a tendency to use either SAα2,3Gal or SAα2,6Gal as cellular receptors, and the general idea has been that respiratory viruses like human influenza A uses SAα2,6Gal whereas ocular viruses such as EV70 and Ad37 prefer SAα2,3Gal. The distribution of SA can to some extent explain the tropism for different SA-binding viruses since it is known that the most frequent SA type found on corneal and conjunctival cells is SAα2,3Gal, and in the respiratory tract the most frequently found SA type is SAα2,6Gal [460]. For CVA24v a pronounced linkage could not be established since the virus seems to be able to interact with both types of SA, which we found a bit puzzling in the beginning. However, whereas the other AHC-causing virus, EV70, is not associated with respiratory disease, CVA24v is more often coupled to respiratory symptoms [81, 83, 84, 461]. Therefore, our results indicating a possible interaction with both SA2,3Gal and SA2,6Gal actually matches the tropism of CVA24v.

Paper IV

The GD1a glycan is a cellular receptor for adenoviruses that cause epidemic keratoconjunctivitis

Ad37 is one of the main causative agents of the ocular infection called epidemic keratoconjunctivitis (EKC) and has previously been reported to interact with two different receptors: SA and CD46 [207, 209]. However, so far only SA has been confirmed by others to be a functional receptor [462- 464]. The main goal with this study was to identify the exact structure of the SA-containing glycan that function as a receptor for Ad37. The starting point was a glycan array performed by the Consortium for Functional Glycomics (CFG), where Ad37 fk interaction with a library of 260 different glycans was investigated. The array resulted in a number of hits, which represented sialylated plasma proteins (that were not investigated further), sulfated glycans (that were found to not prevent Ad37 binding) and one interesting hit corresponding to the disialylated glycan found in ganglioside GD1a. To test if the GD1a glycan was a functional receptor for Ad37, a number of binding and infection assays were performed. We found that soluble GD1a glycans and monoclonal antibodies against GD1a (clone EM9) each inhibited viral binding to and infection of HCE cells. Soluble GD1a glycans also blocked Ad37 fk binding to cells and in situ staining of corneal tissue showed binding of both Ad37 fk and EM9. These results together suggested that Ad37 binds to cell surface molecules that contained the GD1a glycan motif.

To find out if the receptor actually was the GD1a ganglioside we performed a number of assays: a) a combinatorial glycolipid array was performed to see if

there was any interaction between the Ad37 fk and GD1a or any other gangliosides; b) ELISA with immobilized GD1a and soluble fk; c) viral binding to Lec2-cells pre-incubated with or w/o GD1a ganglioside; d) viral binding to HCE cells in which the de novo ganglioside synthesis was inhibited; e) pre-incubation of virions with GD1a-containing liposomes prior to binding experiments. However, none of the above experiments indicated that ganglioside GD1a would be a cellular receptor for Ad37. On the other hand, protease treatment of cells as well as cellular inhibition of de novo O- linked glycosylation significantly reduced viral binding, leading to the conclusion that the receptor used by Ad37 is not the ganglioside GD1a but rather one or more proteins that carry the GD1a glycan motif, or, a very similar motif.

To better understand the Ad37 fk-GD1a interaction we first modeled the glycan onto the X-ray crystallography-based Ad37 fk model obtained previously [126]. This led to a preliminary model indicating that both SAs of the GD1a glycan interacted with two out of the three known SA binding sites in the Ad37 fk. To further confirm this model, a saturation transfer difference (STD) NMR was performed. Large methyl peaks were seen, corresponding to the acetamide groups of the two SAs (Neu5AcA and Neu5AcB), which were not seen with fk alone or with fk incubated with galactose, thus indicating a close contact between both SAs in GD1a and Ad37 fk. This was also confirmed by resolving the protons H3eq and H3ax for Neu5AcA and Neu5AcB. The large methyl peak probably resulted from an overlap of the two Neu5Ac peaks. Thus, all peaks in the STD spectrum of the Ad37 fk-GD1a were directly related to an interaction between these molecules.

To verify the results from the modeling and the STD-NMR we also solved the crystal structure for Ad37 fk in complex with GD1a at 1.65 Å resolution. This confirmed our previous results that the fk engages one single GD1a glycan and that both SAs bind similarly to two of the three known binding sites. The amino acids directly involved in the binding are: Tyr312, Pro317

and Lys345. In silico substitutions suggested that Lys345 played a major role in the interaction between GD1a and the Ad37 fk protein. This was later confirmed by flow cytometry analysis with wt Ad37 fk and Lys345Ala mutants. By substituting Lys345 with Ala the SA-dependent binding was lost.

The SA-Ad37 fk interaction between the highly positively charged fk (pI:9,1) and the negatively charged SA (pKa:2,6) is charge-dependent [282]. As described previously, this interaction is quite weak, with a Kd of 5 mM, which can be compared to the affinities known for the interactions between CAR and many other Ad fk:s (nM range) [465]. The affinity between the Ad37 fk and the GD1a glycan was analyzed by surface plasmon resonance (SPR). The results showed that the association rate constant was different at high and low glycan concentrations, one interaction was of high affinity (19 µM: measured at low glycan concentrations) and one interaction was of low affinity (265 µM: measured at high glycan concentrations), suggesting that the GD1a glycan binds to two different parts of the fk protein. The fact that the high affinity binding was more than 250-fold higher than the SA-Ad37 fk interaction is probably due to the fact that both GD1a SAs interact with the fk at the same time. The low affinity binding on the other hand needs to be investigated further. The fact that only one GD1a molecule interacts with one fk at a time (1:1; according to structural data) suggested that the internalization of Ad37 is not dependent on receptor clustering as could be the case for CAR- and CD46-binding Ads, which are able to interact with three receptor molecules/fk (3:1). GD1a binding capacity seems to be a conserved feature among the species D Ads as shown by flow cytometry analysis with fk proteins from Ad8, 9, 19p, 19a and 37. Soluble GD1a prevented all these fk:s but not species C Ad5 fk from binding to HCE cells. However, Ad9 and 19p are not associated with EKC, indicating that the ability to bind GD1a is not a determinant of the ability to cause EKC. To confirm this, the ability of GD1a to inhibit infection of these species D types would need to be evaluated.

In this work we have identified the GD1a glycan motif as a receptor for Ad37 using a number of different methods. We concluded that the cellular receptor for Ad37 is not the ganglioside GD1a but rather one or more glycoproteins carrying the GD1a motif, or, a highly similar motif. We could also confirm that both SAs of the glycan interacted with the knob, leading to a relatively high affinity binding (µM range). The detailed structural and functional analysis of the interaction presented here might be useful for the design and development of antiviral drugs for treatment of EKC. A small (drug-like) compound occupying all of the three SA binding sites could perhaps be an even more potent inhibitor than the previously discussed SA- HSA. A smaller compound may also be easier to synthesize and therefore cheaper to produce than the large and complex SA-HSA.

Concluding remarks

Virus-caused ocular infections are problematic in many parts of the world, especially in densely populated areas. EKC and AHC are highly contagious and severe ocular infections caused by members of the adenovirus and picornavirus families, respectively. Millions of people fall ill every year due to these viruses, resulting in substantial economic losses. Despite this, there are no antiviral drugs available to treat these infections.

I have evaluated the inhibitory effect of multivalent sialic acid-containing compounds on Ad37 binding and infection and could conclude that these are potent inhibitors. The effect seen of these compounds were highly dependent on the order of valency of sialic acid. We also concluded that the mechanism of action of these compounds were due to aggregation of virions.

By structural modifications of native sialic acid we tried to improve the binding capacity between Ad37 and sialic acid. Ten different compounds were biologically evaluated but only two of them had an inhibitory effect on Ad37 infection similar to the effect seen with native sialic acid. These compounds were then coupled to human serum albumin in a multivalent fashion but none of them were as potent inhibitor as the previously evaluated multivalent compound based on native sialic acid.

The picornavirus member coxsackievirus A24v (CVA24v) is one of the main causative agents of AHC. We identified sialic acid as a cellular receptor for this ocular virus and, also found that CVA24v is, unlike many of the other sialic acid-binding viruses, able to use both SAα2,3Gal- and SAα2,6Gal- containing glycans as receptors. This ability is in agreement with the tissue distribution of these glycans as well as the tropism of CVA24v.

Finally, we have in detail characterized the sialic acid-containing receptor used by Ad37. We show that the branched, di-sialic acid containing glycan motif of ganglioside GD1a is a functional receptor for Ad37 in vitro. By using

a number of methods we concluded that the receptor is not the GD1a ganglioside, but rather one or more proteins containing the GD1a glycan motif, or, a highly similar motif. Based on structural analysis, STD-NMR and X-ray crystallography we also showed that both sialic acids of the GD1a glycan dock into the binding cavity of the Ad37 fk, thus occupying two of the three known sialic acid-binding sites. This would also explain the relatively high affinity binding, for being a protein-carbohydrate interaction, that we see (19 µM).

These results show that there seem to be a common receptor for these EKC- and AHC-causing viruses, namely, sialic acid. By studying the sialic acid- virus interaction in detail we can gain knowledge that will help to design and develop antivirals for treatment of ocular infections.

Acknowledgements

Tack till alla underbara människor som jag har haft möjligheten att lära känna under resans gång samt alla nära och kära som har stöttat mig.

Först av allt ett stort tack till min fantastiska handledare Niklas. Din positiva anda och ditt brinnande intresse för forskningen har smittat av sig. Jag är otroligt imponerad över allt som du har åstadkommit. Jag är glad och tacksam för de möjligheter som du har gett mig samt allt det som jag har fått uppleva under alla år på virologen. Tack!

Göran, det var du som väckte intresset för virus hos mig genom någon av dina inlevelsefulla föreläsningar. Få personer har den omfattande kunskap om virus, i synnerhet adenovirus, som du. Tack för dina uppmuntrande ord, kluriga frågor och tänkvärda kommentarer.

Mina biträdande handledare, Magnus du finns alltid där för att svara på frågor om det mesta. Tack för att du uppmärksammar mig på lämpliga kurser att gå. Johan, du brinner verkligen för forskningen och vi visste nog redan för länge sedan att du skulle lyckas med något stort.

Ett stort tack till dig Katrine för allt du ordnar, det är papper som ska fyllas i, saker som ska bokas och inte minst svar på alla frågor som man har. Du gör dessutom alltid detta med ett leende på läpparna.

Kickan och Karin för att ni alltid finns där och svarar på de frågor som man kommer med. Ni vet allt som man behöver veta om vad som ska göras på lab. En extra kram till dig Kickan, som har blivit en del av vår numera stora grupp, för allt jobb du har gjort när det gäller Nature-peket.

Mina fantastiska vänner, Cissi och Emma, ni har varit saknade under vintern även om vi har träffats lite då och då. Ni har bidragit till att jag alltid har haft roligt på jobbet, även om själva ”jobbet” inte alltid har gått som man har velat. Cissi, jag tror aldrig att jag har träffat på en sådan glädjespridare som du. Det gör att du kommer att lyckas med allt du tar dig för och du kommer att bli en mycket omtyckt läkare. Jag bokar in mig redan nu  Emma, en mer träningstokig och påhittig tjej får man nog leta efter. Du har en sådan energi och härlig inställning till det mesta. Jag funderar fortfarande ibland på vad han tänkte när det dök upp två små tjejer (Emma och Emma) med släpvagn (som vi dessutom inte kunde köra med ) för att hämta det

ganska så tunga skåpet. Jag hoppas verkligen att du snart hittar tillbaka till ditt rätta element, virus!

Carin, Annasara, Maria och Anna, mitt ”nya” kaffe-gäng. Stort tack till er, ni förgyller min vardag. Annasara, för alla pratstunder under den ”gemensamma” graviditeten samt trevligt resesällskap och barnlek. Carin, för ditt glada humör och för att du ställer upp vid alla tillfällen. Maria, för att du orkar med allt prat om barn och vabbande, och Anna för att du har fört in lite ”nytt blod” här på virologen.

Rickard, vilken tur att du dök upp när du gjorde, precis när jag skulle vara mammaledig. Du tog över där jag lämnade och drev projektet framåt fastän jag inte var där. Fantastiskt att Ad37 projektet får fortsätta några år till nu när jag slutar.

Nitte, det är tur att någon har tagit hand om mitt kära coxsakievirus så att det lever vidare ett tag till. Tack också för att du emellanåt förgyller vår vardag med dina härligt spydiga mail.

Fariba, för all den tid och arbete som du lade ner på coxsackievirus- projektet. Stort tack även till Ya-Fang, Fredrik, Mari, Therese, Lars, Marko, Sharvani, Johanna, Jim, Mårten, Jonas, Karin, Janne, Lisa och Dan för den trevliga stämning som ni alla bidrar till.

Ett stort tack till alla på landstinget, trevliga luncher, vinlotteri med vinprovningar och framförallt, det efterlängtade fredagsfikat. Tack även till alla de studenter som har hjälpt mig under åren.

Tack Sussi och Mikael Elofsson för allt som har med kemi att göra. Extra stort tack till dig Sussi för allt samarbete, jag insåg att ditt namn finns med på alla mina publikationer 

Soffe och Hasse, vi ses alldeles för sällan men ändå så känns det som igår när vi ses. Soffe, du är min äldsta och bästa vän. Minns du alla våra omvägar hem från skolan, eller vår gemensamma partygarderob på Nydala . Tänk att även vi har blivit vuxna, det trodde jag nog aldrig. Hasse, jag har nog aldrig träffat någon som är så härligt tankspridd som du. Ni är underbara!

Nicke och Anneli, tack för att ni har varit vårt trevliga rese-sällskap under alla år. Vi börjar ha gjort några resor tillsammans, även om det nu är mer än två år sen senast. Det lilla uppehållet kan ju ha berott på att det har tillkommit två underbara och fantastiska små killar.

Elisabeth, Rebecca, Lars, Andreas, Elisabeth, Vickan och Ida min fina ”extra familj”. Jag minns fortfarande den varma och välkomnande känslan som infann sig första gången jag hälsade på hos er för snart 13 år sedan. Rebecca, min vän du är saknad här i Umeå. Tack för alla trevliga middagar, pratstunder och träningspass. Kom bara ihåg, du är underbar och unik och kommer att lyckas med allt du vill här i livet.

Många kramar och tack till mormor Margit och farmor Gunnel. Ni har under åren snällt frågat vad det är jag håller på med och förklarat för mig hur viktigt det är för forskningen det just jag håller på med. För att göra min vardag lite lättare så har ni slitit i köket för att skicka påsar med alla sorters kakor och även världens godaste palt.

Petra, min kära syster. Jag sörjer att vi bor så långt ifrån varandra, tänk så mycket shopping vi skulle kunna göra om vi sågs lite oftare, och tänk vad roligt det hade varit för våra finaste pojkar att kunna leka med varandra. Jag skulle önska att jag hade fått en gnutta av din energi och spontanitet, du är underbar! Glöm aldrig det!

Kerstin och Michael, mina älskade föräldrar, tack för allt ert stöd. Utan er hade detta inte varit möjligt. Ni har under alla år gjort allt för mig, mer än vad jag någonsin förväntat mig. Det är en fantastisk trygghet att veta att vad som än händer så finns ni där och ställer upp för mig och min lilla familj.

Min lilla glädjespridare Hjalmar, du är meningen med livet. Hur trött jag än är så glömmer jag det när jag ser ditt leende och den kärlek som du utstrålar. Mitt livs kärlek David, du är underbar, fantastisk, stöttande och uppmuntrande, du är min bästa vän. Jag har dig att tacka för allt. Du är min trygghet här i livet. Jag älskar dig! Kaan Naan!

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