Molecular Pathogenesis of Human Parechovirus 1 Infection

MOLECULAR PATHOGENESIS

HUMAN PARECHOVIRUS 1 INFECTION

CAMILLA KROGERUS

Department of Virology Haartman Institute Faculty of Medicine University of Helsinki Finland

ACADEMIC DISSERTATION

To be presented for public discussion, with the permission of the Faculty of Medicine of the University of Helsinki, in Lecture Hall 2, Meilahti Hospital, Haartmaninkatu 4, Helsinki, on March 9th 2007, at 12 o’clock noon.

HELSINKI 2007

SUPERVISED BY

Timo Hyypiä, M.D. Professor Department of Virology University of Turku Finland

REVIEWED BY

Maija Vihinen-Ranta, Ph.D. Docent Department of Biological and Environmental Science University of Jyväskylä Finland

and

Kristiina Mäkinen, Ph.D. Professor Department of Applied Chemistry and Microbiology University of Helsinki Finland

OFFICIAL OPPONENT

Leevi Kääriäinen, Ph.D. Professor emeritus Institute of Biotechnology University of Helsinki Finland

ISBN 978-952-92-1643-7 (paperback) ISBN 978-952-10-3737-5 (PDF) http://ethesis.helsinki.fi Yliopistopaino Helsinki 2007

To my Family

Table of Contents

TABLE OF CONTENTS TABLE OF CONTENTS ...... 4

LIST OF ORIGINAL PUBLICATIONS...... 7

ABBREVIATIONS...... 8

ABSTRACT...... 10

REVIEW OF THE LITERATURE ...... 12

1. PICORNAVIRUSES AND THEIR CLASSIFICATION...... 12 2. VIRION STRUCTURE AND GENOME ORGANIZATION OF PICORNAVIRUSES ...... 14 3. CELLULAR RECEPTORS AND ENTRY OF PICORNAVIRUSES ...... 16 3.1. Ig-like molecules as picornavirus receptors...... 17 3.2. Integrins as picornavirus receptors...... 18 3.3. Other molecules as picornavirus receptors ...... 19 4. TRANSLATION OF PICORNAVIRUS RNAS...... 20 5. POLYPROTEIN PROCESSING ...... 22 6. THE PICORNAVIRAL NON-STRUCTURAL PROTEINS...... 23 6.1. L ...... 23 6.2. 2A ...... 23 6.3. 2B ...... 24 6.4. 2BC and 2C...... 26 6.5. 3A, 3B and 3AB...... 28 6.6. 3C and 3CD ...... 29 6.7. 3D...... 29 7. REPLICATION OF PICORNAVIRUSES ...... 30 7.1. Cis-acting RNA elements ...... 30 7.2. Minus-strand RNA synthesis...... 31 7.3. Plus-strand RNA synthesis...... 32 7.4. Viral RNA replication is associated with cellular membranes...... 33 7.5. Membrane trafficking between the ER and the Golgi...... 34 7.6. Formation of the picornaviral replication complex...... 36 8. PARECHOVIRUSES ...... 38 AIMS OF THE STUDY...... 43

MATERIALS AND METHODS ...... 44

1. VIRUSES, CELLS AND TRANSFECTION (I, II, III, IV, V) ...... 44 2. PLASMIDS AND PROTEIN PRODUCTION (II, III, IV, V)...... 44 3. ANTIBODIES (I, II, III, IV, V) ...... 45 4. INFECTIVITY TITRATION AND BLOCKING OF INFECTION WITH CELL SURFACE ANTIBODIES (I)...... 45

Table of Contents

5. IMMUNOFLUORESCENCE (IF) LABELLING AND FLUORESCENCE MICROSCOPY (I, II, III, V)...... 46 6. PERCOLL-FRACTIONATION OF INFECTED HOMOGENATES (I) ...... 46 7. METABOLIC LABELLING OF VIRAL RNA (II, V) ...... 47 8. IN VITRO TRANSCRIPTION-TRANSLATION AND IMMUNOPRECIPITATION (II) ...... 47 9. FLUORESCENT IN SITU HYBRIDIZATION (II, III, V) ...... 47 10. FLOTATION ASSAY AND WESTERN BLOTTING (II)...... 48 11. ELECTRON MICROSCOPY (EM), IMMUNO-EM AND EM-IN SITU HYBRIDIZATION (II, V)...... 48 12. SEQUENCE ALIGNMENT AND COMPARISON (II)...... 49 13. IN VITRO SYNTHESIS OF RNA (III, IV) ...... 49 14. RNA-BINDING EXPERIMENTS (III, IV) ...... 49 15. ATP HYDROLYZATION AND CHROMATOGRAFY (IV)...... 50 RESULTS ...... 52

1. ENTRY OF HPEV1 (I)...... 52 1.1. Interactions with the cell surface...... 52 1.2. Entry...... 52 2. REPLICATION COMPLEX OF HPEV1 (II) ...... 53 2.1. Synthesis and localization of viral macromolecules in the infected cell ...... 54 2.2. Ultrastructural aspects of the infected cell and the site of viral RNA synthesis...... 55 3. BIOCHEMICAL CHARACTERIZATION OF THE HPEV1 2A PROTEIN (III)...... 56 3.1. Intracellular location...... 56 3.2. RNA-binding activity...... 57 3.3. Mutation analysis...... 59 4. ATP HYDROLYSIS AND AMP KINASE ACTIVITIES OF THE HPEV1 2C PROTEIN...... 60 4.1. ATP diphosphohydrolase activity ...... 60 4.2. Phosphoryltransfer activity and autophosphorylation ...... 61 5. INTRACELLULAR LOCALIZATION AND EFFECTS OF INDIVIDUALLY EXPRESSED HPEV1 NON-STRUCTURAL PROTEINS ...... 62 5.1. Localization of individually expressed HPEV1 non-structural proteins ...... 62 5.2. 2C protein can associate with viral RNA in infected cells...... 63 5.3. Effect of the HPEV1 infection and non-structural proteins on the secretory pathway...... 64 DISCUSSION ...... 66

1. ATTACHMENT AND ENTRY ...... 66 2. REPLICATION COMPLEX FORMATION...... 68 3. THE BIOCHEMICAL PROPERTIES OF THE 2A PROTEIN ...... 69 4. THE ATP DIPHOSPHOHYDROLASE AND AMP KINASE ACTIVITIES OF THE 2C PROTEIN...... 71 5. THE ROLE OF THE NON-STRUCTURAL PROTEINS IN RC FORMATION ...... 72 SUMMARY AND CONCLUDING REMARKS ...... 74

Table of Contents

SVENSKT SAMMANDRAG...... 76

ACKNOWLEDGEMENTS ...... 78

REFERENCES...... 80

ORIGINAL PUBLICATIONS ...... 105

List of Original Publications

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications, which are referred to in the text by the Roman numerals I-V

I. Joki-Korpela, P., Marjomäki, V., Krogerus, C., Heino, J. and Hyypiä, T. 2001. Entry of human parechovirus 1. Journal of Virology, 75: 1958-67

II. Krogerus, C., Egger D., Samuilova, O., Hyypiä, T. and Bienz, K. 2003. The replication complex of human parechovirus 1. Journal of Virology, 77: 8512-23

III. Samuilova, O., Krogerus C., Pöyry, T. and Hyypiä, T. 2004. Specific interaction between human parechovirus non-structural 2A protein and viral RNA. Journal of Biological Chemistry, 279: 37822-31

IV. Samuilova, O., Krogerus, C., Fabrichniy, I. and Hyypiä, T. 2006. ATP hydrolysis and AMP kinase activities of the non-structural protein 2C of human parechovirus 1. Journal of Virology, 80: 1053-58.

V. Krogerus, C., Samuilova, O., Pöyry, T., Jokitalo, E. and Hyypiä T. 2007. Intracellular localization and effects of individually expressed human parechovirus 1 non-structural proteins. Journal of General Virology, 88: 831-41

Published papers have been reprinted with the permission of copyright holders.

Abbreviations

ABBREVIATIONS aa amino acid Ab antibody AEV avian encephalomyelitis virus ATCC American Type Culture Collection AMP, ADP, ATP adenosine mono/di/triphosphate AMD actinomycin D BSA bovine serum albumin bus 3B-uridylylation site CAR coxsackievirus-adenovirus receptor CAV coxsackie A virus CBV coxsackie B virus cDNA complementary DNA CMC carboxymethylcellulose CNS central nervous system CPE cytopathic effect C-terminus carboxy-terminus DAF decay-accelerating factor E. coli Escherichia coli EGFP enhanced green fluorescent protein eIF eukaryotic initiation factor EM electron microscopy EM-ISH electron microscopy in situ hybridization EMCV encephalomyocarditis virus ER endoplasmic reticulum EV echovirus FCS fetal calf serum FISH fluorescence in situ hybridization FHV flock house virus FMDV foot-and-mouth disease virus FRET fluorescence resonance energy transfer GalT ß1,4 Galactosyltransferase GFP green fluorescent protein GST glutathione-S-transferase GTP guanosine triphosphate HA hemagglutinin HAV hepatitis A virus HAVcr-1 HAV cellular receptor 1 HBB 2-(a-hydroxybenzyl)- benzimidazole HCV hepatitis C virus HeLa human tumour-derived cell line HEV human enterovirus HPEV human parechovirus HRP horse radish peroxidase HRV human rhinovirus ICAM intercellular adhesion molecule IEM immuno electron microscopy IF immunofluorescence Ig immunoglobulin IRES internal ribosome entry site Kd dissociation constant kDa kilodalton LV Ljungan virus MAb monoclonal antibody

Abbreviations

MEM minimal essential medium MHC major histocompatibility complex mRNA messenger RNA m7Gpp 5’ 7-methylguanosine nt nucleotides N-terminus amino-terminus PAGE polyacrylamide gel electrophoresis PABP polyadenosine binding protein PBS phosphate-buffered saline PCBP poly-C binding protein PFU plack-forming units p.i. post-infection p.t. post-transfection PNS post-nuclear supernatant PV poliovirus PVR poliovirus receptor RC replication complex RdRp RNA-dependent RNA polymerase RGD arginine-glycine-aspartic acid tripeptide RI replicative intermediate RNP ribonucleoprotein RT room temperature SDS sodium dodecyl sulphate SF helicase superfamily SV40 Tag Simian virus 40 large tumor antigen TGN trans-Golgi network TLC thin-layer chromatography TMEV Theiler’s murine encephalomyocarditis virus tRNA transfer RNA UTR untranslated region VCAM-1 vascular cell adhesion molecule-1 VLDL-R very-low-density lipoprotein receptor VP viral protein VSV vesicular stomatitis virus wt/vol weight/volume wt/wt weight/weight 4E-BP 4E binding proteins

Abstract

ABSTRACT

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members.

We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3’UTR RNA. It was found to bind also duplex RNA containing 3’UTR(+)-3’UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1- infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic.

Abstract

Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Review of the Literature

REVIEW OF THE LITERATURE

1. Picornaviruses and their classification

The Picornaviridae family of viruses consists of small, non-enveloped particles with a single-stranded infectious RNA genome. The picornaviruses constitute one of the largest and most important families of human and animal pathogens. Currently the family is divided into nine genera: Enterovirus, Rhinovirus, Hepatovirus, Parechovirus, Cardiovirus, Aphthovirus, Erbovirus, Kobuvirus and Teschovirus (King, 2000). The genus Enterovirus contains several important human pathogens known to be responsible for a range of different clinical symptoms while the viruses belonging to the Rhinovirus genus are the most important causative agents of common cold. The hepatitis A viruses (HAV), which belong to the genus Hepatoviruses, are the most common causative agents of water- and food-borne hepatitis. The genus Parechoviruses consist of HPEV1 and HPEV2 (Stanway & Hyypia, 1999), and the recently identified serotypes HPEV3-5 (Abed & Boivin, 2005, Al-Sunaidi et al., 2007, Benschop et al., 2006a), as well as the Ljungan viruses (LVs), which have been isolated from rodents (Johansson et al., 2003, Johansson et al., 2002, Niklasson et al., 1999). The type member of the genus Kobuvirus, Aichi virus, was first recognised in 1989 as the cause of oyster-associated non-bacterial gastroenteritis in man (Yamashita et al., 1991). The genus Cardiovirus comprises of two rodent pathogens, encephalomyocarditis virus (EMCV) and Theiler’s murine encephalomyocarditis virus (TMEV). The (in)famous foot-and-mouth disease viruses (FMDV) belong to the genera Aphthoviruses. The members of the Erbo- (Bohm, 1964) and Teshovirus (Doherty et al., 1999) genera also infect only animals. The picorna-like viruses are distinguished by their RNA-dependent RNA polymerase (RdRp) sequence alignments and the conserved helicase-protease-replicase array (Koonin & Dolja, 1993) and are divided among the Picornaviridae, Caliciviridae, Sequiviridae, Comoviridae, Potyviridae and Dicistroviridae. These virus families comprise the "picornavirus superfamily" and are characterized by the production of an exclusive genomic RNA message, encoding one or two polyproteins which are post-translationally cleaved to generate the structural and non-structural viral proteins.

Review of the Literature

The recorded history of the picornaviruses goes back to 1400 B.C., with Egyptian temple records depicting a victim of poliomyelitis. One of the first viruses to be identified was FMDV; in 1897 Loeffler and Frosch demonstrated that the causative agent of foot-and- mouth disease in cattle, was filterable (Loeffler & Frosch, 1897). In 1908, Austrian physicians Karl Landsteiner and Erwin Popper showed that polio is a contagious disease spread by a virus and that initial infection confers immunity (Landsteiner & Popper, 1909). This finding prompted the development of vaccines against poliovirus (PV). Forty years later, the A and B subgroup coxsackieviruses (CAV and CBV), which characteristically could induce disease in newborn mice, were isolated from the feces of children with paralysis (Dalldorf & Sickles, 1948). Later, several viruses which shared physicochemical properties (solvent-resistant, acid-stable particles) with polio- and coxsackieviruses, but grew exclusively in cell culture, were isolated. These were called ECHO (enteric, cytopathogenic, human, orphan) viruses (Committee on the ECHO viruses., 1955). Since then, a number of picornavirus genera have been characterized and very recent additions suggest that more are yet to come.

Review of the Literature

Table 1. Classification of picornaviruses (Stanway et al., 2005) Genus Species (number of serotypes) Clinical symptoms in man Enterovirus Poliovirus (3) Poliomyelitis, gastroenteritis - Human polioviruses 1-3 Human enterovirus A (12 serotypes) Meningitis, encephalitis, paralysis, - Human coxsackieviruses A2-A8, A10-A14, A16 myocarditis, rash - Human enterovirus 71 Human enterovirus B (37) Meningitis, paralysis, encephalitis, - Human coxsackieviruses B1-B6 pleurodynia, myocarditis, - Human coxsackievirus A9 gastroenteritis, respiratory - Human echoviruses 1-7, 9, 11-21, 24-27, 29-33 infections - Human enteroviruses 69, 73 Human enterovirus C (11) Respiratory infections, - Human coxsackieviruses A1, A11, A13, A15, conjunctivitis A17-A22, A24 Human enterovirus D (2) Conjunctivitis - Human enteroviruses 68, 70 Bovine enterovirus (2) Porcine enterovirus A (1) Porcine enterovirus B (2)

Rhinovirus Human Rhinovirus A (74) Common cold, otitis Human Rhinovirus B (25) Common cold, otitis

Cardiovirus Encephalomyocarditis virus (1) Theiler’s murine encephalomyocarditis virus (2)

Aphthovirus Foot-and-mouth disease virus (7) Equine rhinitis A virus (1)

Hepatovirus Hepatitis A virus (1) Liver disease Avian encephalomyelitis virus (1)

Parechovirus Human parechovirus (5) Respiratory infections, Ljungan virus (2) gastroenteritis, CNS infections

Erbovirus Equine rhinitis B virus (2)

Kobuvirus Aichi virus (1) Gastroenteritis Bovine kobuvirus (1)

Teschovirus Porcine teschovirus (10)

2. Virion structure and genome organization of picornaviruses

Picornaviruses are non-enveloped particles of about 30 nm in diameter. The icosahedral capsid contains 12 pentagon-shaped pentamers consisting of 5 protomers. Each protomer is formed by one copy of the four structural proteins VP1 to VP4. The capsid surrounds a

Review of the Literature

single-stranded infectious RNA genome with a length of approximately 7500 nucleotides (nt). The atomic-resolution structures of several picornaviruses have been resolved by x- ray crystallography (Arnold & Rossmann, 1988, Hadfield et al., 1997, Hendry et al., 1999, Hogle et al., 1985, Lentz et al., 1997, Muckelbauer et al., 1995, Rossmann et al., 1985, Smyth et al., 1995) and they all share a remarkable degree of similarity. The major capsid proteins VP1 to VP3 are each folded into eight-stranded antiparallel β-sheets. Five copies of VP1 are located around the fivefold axis, while VP2 and VP3 alternate around the threefold axis. VP4 is much smaller than the other structural proteins and it has a less ordered structure. It is located on the inner surface of the capsid, facing the RNA. The amino-terminal (N-terminal) glycine of VP4 has a myristate covalently attached to it (Chow et al., 1987). In site-directed mutagenesis studies, myristoylation and myristate- protein contacts were found to be necessary for pentamer formation, RNA encapsidation (Moscufo et al., 1991) and for the stability of the virion (Moscufo & Chow, 1992). The VP4 protein has also been shown to be involved in uncoating and cell entry (Moscufo et al., 1993). In addition to protecting the viral genome, the viral capsid also mediates receptor recognition and determines the B-cell specific antigenicity of the virus. When the structure of human rhinovirus 14 (HRV14) was solved (Rossmann et al., 1985), it was proposed that the circular depression seen around each fivefold axis would be the receptor-binding site. This canyon hypothesis (Rossmann et al., 1985, Rossmann, 1989) suggested that one strategy to escape the immune surveillance of the host organism would be to hide the receptor attachment site in a surface depression. This would sterically protect the attachment site from antibodies, still allowing recognition by the smaller cell surface receptor. Indeed, it was shown that the receptor-binding site for the major group rhinoviruses was located within the canyon (Olson et al., 1993). However, the absence of the canyon formation in aphtho- and cardioviruses (Acharya et al., 1989, Luo et al., 1987) and the finding that the receptor binding site for the minor group rhinoviruses is located outside the canyon (Hewat et al., 2000), indicates that the canyon hypothesis cannot be extended to all picornaviruses. The picornavirus genome consists of a single molecule of infectious RNA. A small virus- encoded protein VPg, also called 3B, is covalently attached to the 5’ end of the molecule.

Review of the Literature

The open reading frame is flanked at both the 5’ and the 3’ end by an untranslated region (UTR). The 5’ UTR makes up approximately 10 % of the genome and is highly structured (Pilipenko et al., 1989, Skinner et al., 1989). The 3’ UTR is shorter, only 70- 100 nt in length, and followed by a poly-A tail. The 5’- and 3’-terminal structures of the genome have been designated as origins of replication at the left and right ends of the RNA, oriR and oriL. In PV, the 5’-terminal domain forms a cloverleaf-like structure (Andino et al., 1990, Andino et al., 1993). Another complex structure of the 5’ UTR, the internal ribosome entry site (IRES) (Jang et al., 1988, Pelletier & Sonenberg, 1988), is located upstream of the translation initiation site. The protein-coding region encodes a single polyprotein, which is proteolytically cleaved into precursor proteins P1, P2 and P3, and thereafter into the structural proteins VP1 to VP4 (P1), and seven non-structural proteins (P2 and P3). Many of the intermediate cleavage products are functional as well.

Figure 1. A schematic illustration of a picornavirus genome. The P1 region is processed into 3-4 capsid (structural) proteins, whereas the P2 and P3 regions are processed into non-structural proteins. Several precursors and alternative processing products are functional.

3. Cellular receptors and entry of picornaviruses

Initiation of viral infection begins with attachment to a cellular receptor molecule. Attachment to these molecules is specific and during evolution viruses have adapted to use a variety of receptors. Receptors do not only serve as attachment points, they can also induce conformational changes in the virus or induce signalling events which result in viral internalization. Enveloped viruses may be internalized by direct fusion at the plasma

Review of the Literature membrane, while non-enveloped viruses have to use different endocytic routes (Smith & Helenius, 2004). Picornavirus receptors include diverse cell surface molecules like members of the immunoglobulin (Ig) and integrin families. Most rhino- and enterovirus receptors are Ig- like molecules that attach to the viral capsid at the site of the canyon. Binding into the canyon destabilizes the virus and thus initiates the uncoating process. By contrast, non-Ig molecules, when used by picornaviruses as receptors, bind to regions outside the canyon and do not cause viral instability (Rieder & Wimmer, 2002).

3.1. Ig-like molecules as picornavirus receptors All three PV serotypes recognize the same cellular receptor molecule, poliovirus receptor (PVR) (Hogle & Racaniello, 2002, Mendelsohn et al., 1989). PVR is a transmembrane glycoprotein with three Ig-domains forming the extracellular component. The N-terminal domain D1 provides the virus-attachment surface which binds into the canyon of the PV capsid (He et al., 2000). After receptor interaction, conformational changes in the virus result in the formation of a functional intermediate (the A particle), in which the VP4 is absent and the N-terminus of VP1 is externalized (Fricks & Hogle, 1990). Subsequently, the hydrophobic N-terminus of VP1 and possibly the myristate group of VP4, presumably combined with changes in Ca2+ concentrations, allow membrane binding and pore-formation which leads to the internalization of the virus (Hogle, 2002). Whether the pore is formed in the plasma membrane or in the membrane of a cytoplasmic vesicle is still unknown (Bubeck et al., 2005a, Bubeck et al., 2005b, Danthi & Chow, 2004, DeTulleo & Kirchhausen, 1998, Kronenberger et al., 1998, Tuthill et al., 2006). Another Ig-like molecule, intercellular adhesion molecule 1 (ICAM-1), is used as a receptor by the major group rhinoviruses (Greve et al., 1989, Tomassini et al., 1989) as well as by CAV21 (Shafren et al., 1997, Xiao et al., 2001). Yet another receptor, coxsackievirus-adenovirus receptor (CAR), mediates attachment of CBVs (Milstone et al., 2005). The major group rhinoviruses are internalized via clathrin-coated pits and early endosomes into late endosomal compartments (Grunert et al., 1997). Recent evidence suggests that CBVs are internalized through tight junctions of epithelial cells, making use

Review of the Literature of decay accelerating factor (DAF or CD55, see below) on the apical side and CAR inside the junction (Coyne & Bergelson, 2006). Although the structure of HAV is not known, it is likely that it too uses the canyon for attachment to its receptor, the Ig-like molecule HAV cellular receptor 1 (HAVcr-1) (Silberstein et al., 2001).

3.2. Integrins as picornavirus receptors Integrins are cell-adhesion receptors, which bind many different ligands, including a large number of extracellular matrix proteins (e.g. collagens, laminins, fibronectin, vitronectin), counter receptors and plasma proteins (Hynes, 1992). Several different viruses exploit integrins as cell surface receptors (Bergelson et al., 1992, Berinstein et al., 1995, Ciarlet et al., 2002, Feire et al., 2004, Guerrero et al., 2000, Roivainen et al., 1994, Wickham et al., 1993). Most integrin ligands contain an arginine-glycine-aspartic acid

(RGD) tripeptide that binds to integrins such as α5β1, αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8 (Hynes, 1992, Hynes, 2002, Ruoslahti & Pierschbacher, 1987). Several picornaviruses have an RGD-motif in the capsid protein VP1, on the surface of the virus (Chang et al., 1992, Fox et al., 1989, Hyypia et al., 1992, Zimmermann et al., 1997). In contrast to many other picornavirus receptors, the integrins interacting with RGD-containing picornaviruses do not appear to bind into the virus canyon. Aphthoviruses possess an RGD-motif in the disordered GH loop of VP1 (Acharya et al., 1989, Fox et al., 1989), suggesting that this motif might be used for attachment to integrins. Indeed, most FMDV strains use αvβ3 integrin (Berinstein et al., 1995) or αvβ6- integrin (Jackson et al., 2000) as receptors. Other serotypes of FMDV can use heparin sulphate (Jackson et al., 1996) or oligosaccharides (Fry et al., 1999) as receptors. CAV9 was first shown to utilize αvβ3 integrin as a receptor (Roivainen et al., 1991, Roivainen et al., 1994), but later reports suggested that it also binds to other αv integrins, such as αvβ6 in an RGD-dependent manner (Williams et al., 2004). The RGD-motif is not, however, an absolute requirement for CAV9 infectivity (Hughes et al., 1995, Roivainen et al., 1991, Roivainen et al., 1996), suggesting that the virus can also use other receptors for cell entry. Indeed, an major histocompatibility complex (MHC) class I-associated protein, GRP78, has been implied in the CAV9 entry process (Triantafilou et al., 2002). EV1 has

Review of the Literature

been shown to interact with the α2I domain of α2β1 integrin (Bergelson et al., 1992), and the binding site is situated in the canyon (Xing et al., 2004). The viruses that bind to integrins are internalized by several different endocytic mechanisms. Lipid rafts have been implied in the entry of CAV9 (Triantafilou & Triantafilou, 2003), echovirus 1 (EV1) is internalized in mobile structures which contain caveolin-1 (Pietiainen et al., 2004) and FMDVs have been suggested to be internalized through the clathrin-mediated endocytosis pathway (O'Donnell et al., 2005).

3.3. Other molecules as picornavirus receptors Although capsids of enteroviruses and rhinoviruses all have a canyon and many of them tend to use Ig-like cell-surface molecules as their receptors, there are also examples of viruses in these genera which use non-Ig-like molecules that do not bind into the canyon, as receptors. The minor group rhinoviruses bind to the very-low-density-lipoprotein- receptor (VLDL-R) (Hofer et al., 1994) with a protrusion around the five-fold axes of the viral capsid (Hewat et al., 2000) whereas decay accelerating factor (DAF or CD55) is used by some EVs (Bergelson et al., 1994) and some CBVs (Bergelson et al., 1995) and it attaches to the viral capsid south of the canyon around the icosahedral two-fold axis (Hewat et al., 2000). Unlike the Ig-like receptors, these molecules do not cause viral instability upon binding (Hoover-Litty & Greve, 1993) and thus do not themselves trigger uncoating. VLDL-R binding leads to clathrin-mediated endocytosis of major group rhinoviruses, followed by a lowering of pH in endosomal vesicles and subsequent uncoating (Bayer et al., 2001). Viruses using the DAF molecule as a receptor are internalized in lipid rafts (Bergelson et al., 1994) or by caveolae-mediated endocytosis (Stuart et al., 2002). Cardioviruses have an apparent surface depression corresponding to the central portion of the canyon, identified as a ‘pit’ (Luo et al., 1987). There is evidence that the cellular receptor used by cardioviruses binds into the pit (Kim et al., 1990). The receptor might be sialic acid (Zhou et al., 1997) or vascular cell adhesion molecule-1 (VCAM-1) (Huber, 1994). Although the pit is in essentially the same site as part of the canyon, there is no evidence indicating that binding of sialic acid fragments to the capsid would cause viral instability.

Review of the Literature

Figure 2. Replicative cycle of a picornavirus. 1) Attachment and entry. 2) RNA uncoating. 3) Translation. 4) Polyprotein processing. 5) RNA replication. 6) Assembly. 7) Release. CP, capsid (structural) protein, NSP, non-structural protein, R, ribosome.

4. Translation of picornavirus RNAs

Successful amplification of the viral genomes requires that viral RNAs compete with cellular messenger-RNAs (mRNAs) for the host cell translation apparatus. Most cellular mRNAs begin with a 5’ 7-methylguanosine (m7Gpp) cap structure, which is the initial site of interaction between mRNAs and the cellular translation machinery. It is recognized by translation-initiation factor 4E (eIF4E), a component of the cap-binding complex eIF4F, which also includes eIF4A (an RNA helicase) and eIF4G, which binds several factors and bridges the mRNA and the 40S subunit of the ribosome. When the 40S subunit complex reaches the 5’-proximal AUG codon, where translation generally begins, initiation proteins are released allowing the 60S ribosomal subunit to associate with the 40S, forming the 80S initiation complex. A variety of additional cellular proteins

Review of the Literature aid in these processes (Dever, 2002, Pestova et al., 2001), among them the polyadenosine binding protein (PABP) which interacts with eIF4G (Sachs & Varani, 2000), resulting in translational enhancement. It has been known for a long time that infection of cultured cells with PV results in the translational inhibition of host mRNAs (Holland & Peterson, 1964). When the complete nucleotide sequence of PV was determined in 1981, it was found that the 5’-end was not capped, that the 5’-UTR was extremely long and highly structured and that numerous AUG codons were present but not utilized. Several lines of evidence suggested that the 5’-UTR was important for this unique and novel mechanism of translation initiation and experiments using bicistronic constructs engineered to encode two tandem protein sequences, separated by a viral 5’UTR, confirmed the presence of an internal ribosome entry site (IRES) in this part of the genome (Chen & Sarnow, 1995, Jang et al., 1988, Pelletier et al., 1988, Pelletier & Sonenberg, 1988). Later, it has been shown that IRES elements are not unique to picornaviruses since numerous other positive-stranded RNA viruses as well as some cellular mRNAs contain such elements (Martinez-Salas et al., 1996). The RNA sequences that constitute IRES elements extend through several hundred nucleotides and fold into complex, multidomain structures. Three types of picornavirus IRES structures have been defined: type I in entero- and rhinoviruses, type II in cardio-, aphtho- and parechoviruses and type III in hepatoviruses (Ehrenfeld & Teterina, 2002). It is the RNA structure, rather then the nucleotide sequence that is conserved within these groups. Translation initiation, mediated by an IRES element, requires the same set of initiation proteins as translation of mRNA by cap-dependant initiation, except for eIF4E (Ohlmann et al., 1996, Pause et al., 1994, Pestova et al., 1996a, Scheper et al., 1992). Also, the carboxy-terminal (C-terminal) cleavage product of eIF-4G, which harbours the binding sites for eIF3 and eIF4A, is sufficient for IRES-mediated translation (Pestova et al., 1996b). The function of the IRES is mediated and modulated by different cellular and viral proteins (Belsham & Sonenberg, 2000) and several studies indicate that the efficiency of the IRES can also be modulated by sequences outside the IRES (Bergamini et al., 2000, Blyn et al., 1997, Simoes & Sarnow, 1991).

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The inhibition of cellular protein synthesis in PV infected cells was long thought to depend solely on the fact that the virus-encoded protease 2A cleaved the eIF4G component at a specific site within the 176-kilodalton (kDa) protein (Etchison et al., 1982, Krausslich et al., 1987, Lamphear et al., 1993, Wyckoff et al., 1990, Wyckoff et al., 1992). However, eIF4G is only one of several eIFs that can be cleaved in picornavirus infected cells (Gradi et al., 1998, Joachims et al., 1999, Kerekatte et al., 1999, Svitkin et al., 1999). Picornaviruses can also inhibit cap-dependent translation by dephosphorylating 4E binding proteins (4E-BP), which in turn sequester eIF4E, lowering the abundance of the cap-binding protein complex through different mechanisms (Gingras et al., 1996).

5. Polyprotein processing

The entire coding region of the picornavirus RNA genome is translated into a single large polypeptide. Under normal conditions in the infected cell, this polyprotein is never observed because virally encoded proteinases initiate its cleavage cotranslationally. The proteinases first release themselves from the polyprotein by self-cleavage and then cleave the polyprotein precursor in trans to generate numerous intermediate and mature viral proteins that are required for a complete infectious cycle. In entero-and rhinoviruses the 2A proteins are proteinases which mediate the primary cleavage between the C-terminus of VP1 and their own N-terminus (Leong et al., 2002). The cardio- and aphthovirus 2A proteins are not proteinases, however, they mediate the primary cleavage between the C-terminus of 2A and the N-terminus of 2B, by a mechanism involving a ribosomal “skip”(Donnelly et al., 2001). Furthermore, in aphthoviruses, the leader protein is a protease which cleaves between its own C-terminus and the N-terminus of VP4 (Piccone et al., 1995, Strebel & Beck, 1986). The hepato- and parechoviruses encode only one proteolytic enzyme, 3C, which is thought to carry out both primary cleavage events, firstly between 2A and 2B and secondly between 2C and 3A (Jia et al., 1993, Schultheiss et al., 1995a, Schultheiss et al., 1995b). Of the picornaviruses studied, the majority of the secondary processing steps are performed by 3C (Leong et al., 2002). Uniquely, the 3C precursor, the 3CD protein, protein mediates two cleavages in the PV polyprotein (Jore et al., 1988, Ypma-Wong et

Review of the Literature al., 1988). Another secondary cleavage peculiar to PV and certain HRVs is a cleavage of the 3CD protein by 2A to give the alternate products 3D’ and 3C’ (Hanecak et al., 1982, McLean et al., 1976), the functions of which are not known. The secondary processing of HAV and HPEV has been poorly studied.

6. The picornaviral non-structural proteins

6.1. L The aphtho- and cardioviruses code for an L protein at the N-terminus of their polyproteins. The FMDV L protein is a papain-like cystein protease (Gorbalenya et al., 1991, Kleina & Grubman, 1992, Piccone et al., 1995) and it cleaves between its C- terminus and the N-terminus of VP4 (Piccone et al., 1995, Strebel & Beck, 1986). In addition, the proteinase is responsible for the proteolytic cleavage of eIF-4G, leading to host-cell protein shut-off in FMDV infected cells (Devaney et al., 1988). The L protein of FMDV is, however, not essential for FMDV replication (Piccone et al., 1995). The L protein of cardioviruses does not posses any proteolytic activity. Recent studies suggest a role for the protein in facilitating the bidirectional relocation of proteins between the nucleus and cytoplasm of infected cells (Lidsky et al., 2006).

6.2. 2A The protein encoded at the 2A locus differs dramatically among picornaviruses, and several distinct forms have been identified. The 2A proteins of entero- and rhinoviruses are chymotrypsin-like cysteine proteases that carry out the primary cleavage event between the C-terminus of the P1 region and the N-terminus of 2A (Palmenberg, 1990). The trans-cleavage activity of the protein is not essential for polyprotein processing. An important function of the 2A proteinase is the cleavage of cellular factors that are involved in cap-dependant translation. The proteinase cleaves the eIF-4G subunit of the cap-binding complex and, consequently, eliminates cap-binding activity (Etchison et al., 1982, Krausslich et al., 1987, Lamphear et al., 1993, Wyckoff et al., 1990, Wyckoff et al., 1992), which in turn, correlates with the selective inhibition of cellular protein synthesis in PV-infected cells (Gradi et al., 1998). The PV 2A protein is also known to target a

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variety of nuclear transcription factors and histones (Yalamanchili et al., 1997a, Yalamanchili et al., 1997b, Yalamanchili et al., 1997c). The cardiovirus 2A protein sequence (ca. 15kDa) exhibits no similarity to the 2A protein of the entero- and rhinoviruses and none of the characteristic proteinase sequence motifs can be found. The 2A protein has been found in the nucleus of EMCV infected cells (Aminev et al., 2003a, Aminev et al., 2003b) where it has been suggested to inhibit cap- dependent mRNA translation (Aminev et al., 2003b). The 2A proteins of aphtho-, tescho- and erboviruses are short, but highly similar to the C-terminal region of cardiovirus 2A. These proteins do not have proteolytic activities, however they exhibit a highly conserved NPGP motif at the 2A/2B boundary which has been linked to the “ribosomal skip” severing the tetrapeptide between the proline and glycine residue (Donnelly et al., 2001, Ryan et al., 2002). HAV 2A also lacks the consensus sequence of the putative catalytic site of the trypsin- like proteases (Lloyd et al., 1988) and peptides generated by in vitro translation of RNA transcripts encoding HAV 2A are apparently devoid of autocatalytic activity (Schultheiss et al., 1994). Moreover, deletion of 45 nt spanning positions 3155-3200 of HAV RNA did not affect infectivity of cDNA clones (Harmon et al., 1995), suggesting that, whatever the function(s) of HAV 2A is, the virus can dispense with it. However, recent data show that expression of an HAV-encoded peptide encompassing the putative 2A region inhibits cap-dependent gene expression, while internal initiation of translation is unaffected (Maltese et al., 2000). The nature of the cellular target of HAV 2A remains unclear. The 2A proteins of HPEVs and kobuviruses show homology to cellular proteins involved in control of cell growth (Hughes & Stanway, 2000). Ljungan virus has two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio-, erbo-, tescho- and aphthoviruses, and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses and avian encephalomyelitis virus (Johansson et al., 2002).

6.3. 2B The sequence of the 2B protein is poorly conserved among picornaviruses. Very little is known about the 2B protein of different picornaviruses, except for the enterovirus 2B which has been extensively studied, mainly through experiments with PV1 and CBV3.

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The 2B of picornaviruses is a small protein. However, its function seems to be critical at least for PV, as viruses with mutations in the 2B gene are defective in genome replication (Johnson & Sarnow, 1991). In PV-infected cells, the 2B protein has been localized at the rough ER membrane and the outer surface of the ER-derived membranous vesicles at which plus-strand RNA replication takes place (Bienz et al., 1987, Bienz et al., 1994). However, in COS-7 cells expressing low levels of PV 2B, antibodies against the protein stain the Golgi, suggesting a physical association of the viral protein with the Golgi complex, whereas expression of high levels of 2B provokes the disassembly of the Golgi complex (Sandoval & Carrasco, 1997). All picornavirus 2B proteins contain two hydrophobic regions, one of which is predicted to form a cationic amphipathic α-helix (van Kuppeveld et al., 1995, van Kuppeveld et al., 1996) and the other a potential transmembrane domain. The amphipathic α-helix displays characteristics typical for the group of membrane-lytic α-helical peptides that can build membrane-integral pores by forming multimeric transmembrane bundles (Segrest et al., 1990, Shai, 1999). Homomultimerization reactions of CBV3 2B proteins have been demonstrated by yeast and mammalian two-hybrid systems (Cuconati et al., 1998, de Jong et al., 2002), biochemical approaches (Agirre et al., 2002) and in living cells by using fluorescence resonance energy transfer (FRET) microscopy (van Kuppeveld et al., 2002). The CBV3 2B protein has been shown to modify both ER as well as plasma membrane permeability and facilitate virus release (van Kuppeveld et al., 1997). Recently, the 2B protein of CBV3 was shown to reduce the Ca2+ levels inside the ER and the Golgi and thus induce a rise of the Ca2+ concentration in the mitochondria as well as an increased influx of Ca2+ from the extracellular medium (van Kuppeveld et al., 2005). The 2B protein has also been found to suppress apoptosis induced by certain stimuli, such as actinomycin D and cycloheximide. Interestingly, 2B mutants that were unable to reduce the Ca2+ content of the stores failed to protect against apoptosis (Campanella et al., 2004). These data implicate the 2B protein in the enteroviral strategy to suppress premature abortion of the viral life cycle (Agol et al., 2000, Campanella et al., 2004, van Kuppeveld et al., 2005).

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Another activity identified for the 2B protein is the ability to interfere with protein trafficking through the vesicular system (Doedens & Kirkegaard, 1995). This function has been shown not only for PV 2B, but also for the corresponding protein in HAV (Jecht et al., 1998).

6.4. 2BC and 2C The sequence coding for the 2C protein is highly conserved within the picornavirus family (Argos et al., 1984). The 2C of PV has been extensively studied and has been found to be an exclusive part of the replication complex (Bienz et al., 1992) and involved in viral RNA replication. This was shown, for example, by the finding, that relevant mutations for both resistance to or dependence on the PV RNA replication inhibitors 2- (a-hydroxybenzyl)- benzimidazole (HBB) and guanidine, map within its coding region (Hadaschik et al., 1999, Klein et al., 2000, Pincus & Wimmer, 1986, Tolskaya et al., 1994). Subsequently, sequential and functional analysis of 2C revealed the presence of three highly conserved NTP-binding subdomains (Gorbalenya et al., 1990, Klein et al., 1999, Mirzayan & Wimmer, 1992). It has been proven that PV 2C can hydrolyse ATP and GTP (Klein et al., 1999, Mirzayan & Wimmer, 1992, Rodriguez & Carrasco, 1993) and that the ATPase activity is inhibited by 2 mM guanidine hydrochloride (Pfister & Wimmer, 1999). 2C is considered to be a putative RNA helicase (Gorbalenya et al., 1988, Kadare & Haenni, 1997), although the experimental evidence for this activity is still missing. The central domain of the 2C protein, which contains the NTP-binding and helicase motifs, is highly conserved among picornaviruses and other small RNA and DNA viruses (Gorbalenya et al., 1990). The 2C protein of PV has also been found to bind RNA (Rodriguez & Carrasco, 1993). Experiments with truncated 2C revealed that two regions (aa 21-45 and 312-319) are involved in RNA binding (Rodriguez & Carrasco, 1995). Specific binding of the protein to the 3’-terminal cloverleaf of the minus-strand RNA has also been reported (Banerjee et al., 1997). The exact role of 2C in the replication processes is not known. Studies with non-lethal 2C mutants suggest that the protein has at least two functions in RNA replication: a cis- acting guanidine-sensitive function required for initiation and a trans-acting function

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required for elongation (Wimmer et al., 1993). More recent results utilizing an in vitro translation/replication system (Barton et al., 1995, Molla et al., 1991), which produces viable PV, have shown that 2C is required prior to or during initiation of minus-strand RNA synthesis (Barton & Flanegan, 1997). 2C, 2B and 2BC are associated with the intracellular membranes of the host cell (Bienz et al 1987, Egger et al 1996). The membrane- targeting signal of 2C has been mapped to the N-terminal region (Echeverri & Dasgupta, 1995), which has been predicted to form an amphipathic helix, conserved among all picornaviruses studied (Paul et al., 1994). PV 2BC protein (Aldabe & Carrasco, 1995, Barco & Carrasco, 1995, Cho et al., 1994) as well as a fragment of 2C comprising the N-terminal 274 residues (Teterina et al., 1997b) are able to, in isolation, induce the formation of vesicular structures resembling the structures observed in PV-infected cells. The 2C protein of PV is indeed a multifunctional protein as it has also been implicated in the encapsidation of the virus (Vance et al., 1997) and recent evidence suggests that it is additionally capable of regulating virus-encoded proteases (Banerjee et al., 2004). Furthermore, yeast two-hybrid analysis has shown that the PV 2B, 2C and 2BC proteins interact with each other in all combinations except for 2C/2C (Cho et al., 1994). The precursor 2BC remains largely uncleaved in PV-infected cells. It exerts some of the functions of the mature 2B and 2C proteins (Wimmer et al., 1993) but also seems to be important as such for the replication of the virus (Molla et al., 1991, Wimmer et al., 1993). The PV 2BC has also been implicated in the induction of membrane proliferation and rearrangement of intracellular membranes (Aldabe & Carrasco, 1995, Aldabe et al., 1996, Barco & Carrasco, 1995, Cho et al., 1994). The 2C proteins of other picornaviruses have been poorly studied. HAV 2C and 2BC proteins, like their PV counterparts, can induce rearrangement of intracellular membranes and interact directly or indirectly with membranes (Teterina et al., 1997a). Like in PV 2C, the N-terminal amphipathic helix exerts this effect as well as the ability of the protein to bind RNA (Kusov et al., 1998). Recent findings indicate that the FMDV 2BC protein is responsible for the block in cellular protein secretion seen in infected cells (Moffat et al., 2005). The 2C protein is not only well conserved among picornaviruses, but common sequence elements, such as the N-terminal amphipathic helix, have also been found in the

Review of the Literature hepatitis C virus (HCV) NS5A protein, the putative viral RNA helicase (Teterina et al., 2006).

6.5. 3A, 3B and 3AB The homology between different picornaviruses in the region coding for the 3A protein is quite low. The 3A proteins also show wide differences in length: the HRV 3A proteins are around 77 aa, whereas the FMDV 3A protein is 153 aa. Several studies have shown that mutations in the enterovirus 3A give rise to defects in viral RNA synthesis (Giachetti et al., 1992, Hope et al., 1997, Xiang et al., 1995). The protein contains a C-terminal hydrophobic anchor which is responsible for its membrane association (Towner et al., 1996). When individually expressed, 3A induces swelling of the ER (Egger et al., 2000), interferes with ER-to-Golgi transport (Doedens & Kirkegaard, 1995, Doedens et al., 1997, Wessels et al., 2005, Wessels et al., 2006a, Wessels et al., 2006b) and modifies the antiviral response of the infected cell (Deitz et al., 2000, Dodd et al., 2001, Neznanov et al., 2001). HAV and avian encephalitis virus (AEV) 3A proteins have also been shown to interact with cellular membranes and interfere with cellular protein secretion (Beneduce et al., 1997, Liu et al., 2004, Pisani et al., 1995). The 3A protein of FMDV, on the other hand, does not have the ability to interfere with cellular secretion (Moffat et al., 2005). The 3B protein of picornaviruses is usually referred to as VPg, a small peptide covalently linked via a completely conserved tyrosine to the 5' terminus of all full-length and nascent viral plus- and minus-strand RNAs (Flanegan et al., 1977). Uniquely, FMDV RNA encodes three functionally equivalent copies of the 3B peptide (King et al., 1980). VPg is removed by a cellular unlinking enzyme (leaving 5' pU) from those viral RNAs destined to become mRNAs, suggesting that 5'-linked VPg may serve as an encapsidation signal, leaving the mRNAs free for translation without obstruction from the replication machinery (Paul, 2002). Different approaches have shown that VPg plays a role in the initiation of viral RNA synthesis (Paul, 2002). The initial modification of VPg to VPgpU or VPgpUpU is achieved using the 3B-uridylylation site or “bus” (previously called cis replicative element or “cre”) as a template (Gerber et al., 2001, Paul et al., 2000, Paul et al., 2003b, Tiley et al., 2003). Free uridylylated VPg peptides are found in the cytoplasm

Review of the Literature of infected cells and act as primers for the initiation of viral RNA synthesis, explaining why VPg is present at the 5’-terminus of both positive- and negative-sense RNA transcripts (Paul et al., 2000, Paul et al., 2003a, Paul et al., 2003b, Yang et al., 2002, Yin et al., 2003). The membrane-bound precursor 3AB is most likely the donor of VPg to the membranous replication complex (Paul et al., 1998, Porter, 1993). 3AB of PV has also been shown to serve as a cofactor for the binding of 3CD to the 5’-and 3’-termini of the RNA genome (Harris et al., 1994), for the polymerase activity of 3Dpol (Lama et al., 1994, Paul et al., 1994) and for the autocatalytic processing of 3CDpro to 3Cpro and 3Dpol (Molla et al., 1994).

6.6. 3C and 3CD The 3C regions of all picornaviruses code for a chymotrypsin-like serine protease (Cheah et al., 1990, Hammerle et al., 1991, Jia et al., 1991, Skern et al., 2002). This protease is responsible for the majority of the cleavages in the precursor polyprotein (Leong et al., 2002). Proteolysis by 3C occurs in a complex and incompletely understood cascade of cis- and trans-cleavages. In PV, but not in other picornaviruses, two cleavages are carried out by the precursor of 3C, 3CD. These are between the capsid proteins VP0 and VP3 and between VP3 and VP1 (Jore et al., 1988, Ypma-Wong et al., 1988). The 3C proteins of several different picornaviruses have also been implicated in the modification of cellular proteins (Belsham et al., 2000, Falk et al., 1990). The cardiovirus precursor 3BCD (Aminev et al., 2003a) as well as the rhinovirus 3CD’ and/or 3CD proteins (Amineva et al., 2004) and the PV 3CD (Sharma et al., 2004) have been localized to the nucleus of infected cells where they supposedly regulate cellular mRNA and tRNA transcription.

6.7. 3D The 3D regions of all picornaviruses code for an RNA-dependent RNA polymerase which has regions of homology with all known DNA and RNA polymerases (Cameron et al., 2002). In vitro, the oligo(U)- or host factor-dependent PV RNA syntheses will accept any polyadenylated RNA as a template, indicating that one or more host (or viral)

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components which confer specificity on PV 3D for initiating viral RNA replication in vivo are missing from the in vitro reaction (Paul, 2002). The solution of crystal structure of the PV 3D protein (Hansen et al., 1997) greatly increased the understanding of the function of the protein. The protein can be compared to a cupped right hand with “fingers”, “palm” and “thumb”. The fingers and thumb subdomains are thought to be involved in nucleic acid binding while the palm subdomain is likely involved in nucleic acid and nucleotide binding along with catalysis (Cameron et al., 2002). The crystal structure of 3D also revealed two potential oligomerization domains. The protein has been proposed to form a lattice that might act as a scaffold for RNA replication (Hobson et al., 2001). In HAV the precursor 3ABC is also a stable intermediate which binds specifically to the 5’ and 3’UTRs of the HAV genome (Kusov et al., 1996, Kusov et al., 1997).

7. Replication of picornaviruses

7.1. Cis-acting RNA elements The cis-acting elements are signals in the picornaviral genome which are recognized by the replicative proteins and which are thought to confer template specificity in vivo. Most studies have concentrated on the 5’- and 3’-terminal structures, termed OriL and OriR (Paul, 2002). Recently, however, the importance of sequences in the coding region of the polyprotein have also become clear (Gerber et al., 2001, Goodfellow et al., 2000, Lobert et al., 1999, McKnight & Lemon, 1998). All picornaviruses contain 5’-terminal nucleotide sequences that form complex secondary structures. Entero- and rhinoviruses have 5’-terminal structures resembling cloverleaves, whereas the corresponding regions of other picornaviruses have less defined structures (Agol, 2002). In PV, the 3AB and 3CD proteins interact with each other (Molla et al., 1994, Xiang et al., 1998) and a cellular protein called poly-C binding protein (PCBP) (Blyn et al., 1996) to form an ribonucleoprotein (RNP) complex with the 5’ cloverleaf. This RNP has an essential role in PV RNA replication (Harris et al., 1994, Xiang et al., 1995). The 3’UTR and the poly(A) tail form the origin of replication, OriR, for minus-strand synthesis (Pilipenko et al., 1996). However, the 3’ UTRs of picornaviruses studied are

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not crucial for replication (Meredith et al., 1999, Todd et al., 1997). In PV the 3AB/3CD complex was found to have affinity for the 3’UTR (Harris et al., 1994), whereas in EMCV the interaction has been found between 3D and the 3’UTR (Cui et al., 1993) and in HAV between 3AB and 3ABC and the 3’ UTR (Kusov et al., 1996). The poly(A) tail of picornaviruses is also highly variable in length; it is shortest in cardioviruses (35 nt) and longest in aphthoviruses (100nt) (Agol, 2002). The poly(A) tail of PV is genetically encoded (Dorsch-Hasler et al., 1975) and its length is crucial for minus-strand synthesis (Herold & Andino, 2001). Amongst the various picornaviruses studied, internal cis-acting elements called bus- structures, have been identified in different locations in the genome. For example, the HRV-14 bus, which was the first one to be identified, is located within the VP1-coding region (McKnight & Lemon, 1998) and the PV bus is located within the 2C region (Goodfellow et al., 2000). The FMDV bus on the other hand is located in the 5’-UTR (Mason et al., 2002). Although these elements have been found in different parts of the genome and differ in sequence and structure, they all seem to be involved in the uridylylation of VPg (Paul et al., 2000). The bus-structure is essential for plus-strand RNA synthesis, but not for minus-strand synthesis (Goodfellow et al., 2003, Morasco et al., 2003, Murray & Barton, 2003). The IRES may also influence RNA replication (Borman et al., 1994, Ishii et al., 1999) and replicative proteins of PV also bind to the cloverleaf at the 3’ end of the minus strand RNA (Banerjee et al., 1997). Undoubtedly many other, hitherto unidentified cis-acting RNA elements, exist.

7.2. Minus-strand RNA synthesis The first event in the replicative process of picornaviruses is initiation of a complementary strand synthesis using the viral RNA as a template. As with other positive stranded viruses, the genome of picornaviruses must first be translated before it can be used as a template. Why there is a coupling between translation and replication is not known. It is possible that the passage of ribosomes alters the secondary structure of the RNA or then the nascent proteins must be delivered in cis to the site of RNA replication (Novak & Kirkegaard, 1994). The switch from translation to minus strand RNA synthesis

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is proposed to happen when a critical concentration of the 3CD protein accumulates in the infected cell (Gamarnik & Andino, 1996). The 3CD protein binds to the 5’ clover-leaf and sequesters a cellular protein, PCBP2, which is needed for viral translation. The observation that another cellular protein, PABP, interacts with on the other hand the PCBP2 and 3CD proteins and the 5’ cloverleaf and on the other hand the poly(A) tail, suggests that the genome circularizes prior to or during minus-strand synthesis (Herold & Andino, 2001). The 3CD protein cleaves the membrane bound 3AB to yield VPg and 3A. It has been proposed that the 3D, 3CD and VPg proteins then form a complex with the bus RNA hairpin and the polymerase synthesizes VPgpU and VPgpUpU using the AACA sequence in the bus as a template (Paul et al., 2000). The protein complex is then transferred to the 3’ end of the poly(A) tail where VPg-linked precursors serve as primers for the polymerase during the elongation step. Recent reports, however, have showed that a structurally disrupted CRE mutant retained the capacity to induce minus-strand RNA synthesis in a cell-free translation/ replication system, suggesting that the CRE is only required for plus-strand RNA synthesis (Goodfellow et al., 2003, Morasco et al., 2003, Murray & Barton, 2003). It is suggested that the tyrosine hydroxyl of VPg primes minus- strand RNA synthesis without the synthesis of stable VPgpUpU intermediates (Murray & Barton, 2003). The elongation step most likely involves a structural change in the 3D polymerase, which might involve a dissociation of 3D oligomers into monomers (Hansen et al., 1997, Pata et al., 1995). The end product of minus-strand synthesis is the replicative form (RF) which is a viral double-stranded RNA, continuously formed in low amounts in the infected cell (Agol et al., 1999).

7.3. Plus-strand RNA synthesis Picornaviral plus-strand synthesis starts with the double-stranded RF. It is not known how the end of the double stranded molecule is unwound. The 2C protein has ATPase activity and has been predicted to be the helicase (Gorbalenya et al., 1990, Pfister & Wimmer, 1999, Rodriguez & Carrasco, 1993). Alternatively, the end of the RF is destabilized by the binding of PCBP2/3CD and 3AB/3CD to the plus strand and of 2C to the minus-strand of the 5’ cloverleaf (Agol et al., 1999, Andino et al., 1993, Banerjee et

Review of the Literature al., 1997, Gamarnik & Andino, 2000, Harris et al., 1994, Parsley et al., 1997, Roehl & Semler, 1995, Roehl et al., 1997). CRE-dependent VPg uridylylation is required for plus-strand RNA synthesis (Goodfellow et al., 2003, Morasco et al., 2003, Murray & Barton, 2003). It has been proposed that accumulated uridylylated VPg primes the initiation of plus-strand RNA synthesis and that each uridylylated VPgp molecule may remain associated with the 3D protein involved in its formation (Murray & Barton, 2003). Accumulated uridyylated VPg-3D-complexes may then prime plus-strand RNA synthesis via the complementarity of VPg with the 3’-terminal adenosine residues of minus-strand RNA templates. The polymerase elongates the precursors into plus-strands, forming a replicative intermediate (RI) which is multistranded and consists of a genome-length minus-strand and a varying number of complementary plus-strands of different length (Agol et al., 1999).

7.4. Viral RNA replication is associated with cellular membranes All positive-stranded viruses studied so far replicate on cellular membranes. The RNA replication complex (RC) of many virus families is associated with the ER, but Golgi membranes, endosomes, lysosomes, peroxisomes and mitochondria are also used as sites for RNA replication (Salonen et al., 2004). Studies of individual non-structural proteins have indicated that the association of the viral RNA replication to the membranes is mediated by these proteins rather than by the viral RNA. In the case of alphaviruses, for example, the non-structural proteins nsP1 and nsP3 seem to jointly target the replicative machinery to the endosomal membranes, the site of viral RNA synthesis (Salonen et al., 2003). However, it has been shown that membrane-bound complexes containing all the individual non-structural proteins and mimicking the RC seen during alphavirus infection, can only form following expression of the polyprotein precursor protein and not upon coexpression of all four non-structural proteins individually (Salonen et al., 2003). This suggests that the membrane association of the complex is mediated through a polyprotein intermediate. The modes of membrane binding and targeting to specific intracellular organelles of different viral proteins are so far poorly understood. Many non-structural proteins have hydrophobic sequences and may transverse the membrane like polytopic integral

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membrane proteins, whereas others interact with membranes monotopically. Some viral proteins have classical targeting signals; the flock house virus (FHV) A protein contains a mitochondrial targeting signal which localizes viral replication to the outer mitochondrial membrane (Miller et al., 2001, Miller & Ahlquist, 2002). The association of the alphavirus nsP1 protein with certain membranes may be dependant on their lipid composition (Ahola et al., 1999) and the HCV 5A protein might on the other hand guide the replicative machinery to the right place through interaction with caveolin II (Shi et al., 2003). Concomitantly with viral RC formation, the viral non-structural proteins not only attach the viral replicative machinery to intracellular membranes, but they also modify the membranes. Not much is known about how different viruses cause these fundamental structural changes in the membranes, but non-structural proteins of several viruses, when expressed in isolation, seem to be sufficient to induce the membrane modifications seen in the infected cells (Egger & Bienz, 2002, Egger et al., 2002b, Salonen et al., 2003, Snijder et al., 2001). The membrane association is thought to provide a structural framework for replication and to fix viral RNA replication to a spatially confined place, increasing the local concentration of necessary components and offering protection for the viral RNA molecules against host defense mechanisms (Salonen et al., 2004). It is also possible that the membrane lipids provide active components for the replication complex, binding the viral replicative machinery, thereby changing the conformation of the proteins and activating them (Ahola et al., 1999).

7.5. Membrane trafficking between the ER and the Golgi An understanding of the membrane traffic between the ER and the Golgi is important for the analysis of the intracellular changes seen in picornavirus infected cells. The ER contains protein-manufacturing ribosomes and transports proteins destined for membranes and secretion. The ER is connected to the nuclear envelope as well as linked to the cis cisternae of the Golgi complex by vesicles that shuttle between the two compartments. Export from the ER, the anterograde transport, is mediated by a protein complex called COPII, which coates the vesicles. Fusion of these anterograde vesicles

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depends on proteins called anterograde v-SNAREs on the vesicles and t-SNAREs on the cis Golgi. Anterograde transport continues on the trans side of the Golgi with clathrin- coated vesicles. Retrograde transport within the Golgi and from the Golgi to the ER depends on COPI coats and retrograde v- and t-SNAREs (Bannykh & Balch, 1997). The fungal macrocyclic lactone brefeldin A (BFA) has proven to be of great value as an inhibitor of protein trafficking in the endomembrane system of mammalian cells (Sciaky et al., 1997). Lately new insights have been obtained in how BFA achieves these effects (Nebenfuhr et al., 2002). The target of BFA is a subset of Sec7-type GTPexchange factors (GEFs) that catalyze the activation of a small GTPase called ARF1 (Jackson & Casanova, 2000). ARF1, in turn, is responsible for the recruitment of COPI proteins as well as clathrin via the adaptor complex AP-1 to membranes, resulting in the formation of transport vesicles (Scales et al., 2000). Therefore, in BFA-treated cells, all vesiculation at the Golgi stops due to the inhibition of ARF1. As a result, retrograde v-SNAREs remain exposed on the Golgi and the cisternae fuse directly with the ER. Cisternal maturation continues in the presence of BFA so that early Golgi compartments assume a more trans-like morphology. At the same time, the later cisternae and the TGN are lost to the cytoplasm and, eventually, to the BFA compartment. COPII vesicle formation at the ER is initially not inhibited by BFA, but anterograde vesicles may no longer be able to fuse with the maturing cis cisterna, thus effectively blocking ER-to-Golgi transport (Nebenfuhr et al., 2002).

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Figure 3. Membrane traffic between the ER and the Golgi. Modified from Nebenführ et al 2002.

7.6. Formation of the picornaviral replication complex The association of PV with its membranous replication complex has been well studied. The PV RCs consists of clusters of vesicles of 40-200 nm in diameter, which after isolation are associated as large “rosette-like” structures of numerous vesicles interconnected with tubular extensions. The rosettes can dissociate reversibly into tubular vesicles, which carry the PV non-structural proteins on their surface and synthesize viral RNA in vitro (Egger et al., 2002a). The immunoisolated vesicles contain cellular markers for the ER, lysosomes and the trans-Golgi network, suggesting a complex biogenesis (Schlegel et al., 1996). Involvement of the secretory route in the biogenesis of PV RCs has been suggested based on the early finding that BFA inhibits PV replication both in vivo and in vitro (Egger et al., 2002a). Furthermore, PV-infection is known to inhibit the transport of secretory and plasma membrane proteins (Doedens & Kirkegaard, 1995). Experiments showing COPII coat components colocalizing with PV non-structural proteins on vesicles budding from the ER, strongly suggested that the ER is indeed the primary source of PV RCs. Resident ER proteins were found to be excluded from the induced vesicles, and they were found

Review of the Literature not to fuse with the Golgi complex, like ordinary COP II vesicles, but rather accumulated in the cytoplasm (Rust et al., 2001). Thus it seems that in PV-infected cells, a continuous proliferation and loss of ER membranes takes place, which could explain why virus replication is so sensitive to lipid synthesis inhibitors, such as cerulenin (Fogg et al., 2003, Pfister & Wimmer, 1999). An autophagocytosis-like process has also been implied in the formation of the PV RC formation (Suhy et al 2000). Recently, more information has been obtained about the mechanism of PV BFA sensitivity. It has been shown that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of PV RNA. The accumulation of ARFs on membranes correlates with active replication of PV RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of PV 3A or 3CD proteins, and mutations in these proteins may abolish this activity (Belov et al., 2005). The process of RC formation requires all non-structural proteins (Wimmer et al., 1993). When the PV 2C protein is individually expressed it is localized to the ER, causing its extension into tubular structures (Suhy et al., 2000). On the other hand, the precursor 2BC, induces vesicles similar to those seen in PV infected cells, when individually expressed (Teterina et al., 1997a). Coexpression of 2BC with 3A resulted in vesicles both morphologically and biochemically identical to those seen in PV-infected cells (Suhy et al., 2000). However, vesicles formed in cells expressing individual non-structural proteins could not be recruited to support the replication of superinfecting PV RNA, suggesting that functional RCs are formed only in cis by the incoming RNA (Egger et al., 2000). Assuming that the newly synthesized plus-strands create in turn new RCs by a similar in cis mechanism, the rosette-like structures have been suggested to consist of closely packed assemblies of a parent replication complex and its numerous offspring, which are loosely bound to each other (Egger et al., 2002a). Until recently, not much has been known about the RCs of other picornaviruses. Experiments using BFA have revealed that different viruses within the Picornaviridae family have different susceptibility to BFA (Gazina et al., 2002, Maynell et al., 1992, O'Donnell et al., 2001) and may use different host membrane components. PV and EV 11

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are extremely sensitive to BFA treatment (Gazina et al., 2002, Maynell et al., 1992), whereas FMDV and EMCV are resistant to BFA effects (Gazina et al., 2002, O'Donnell et al., 2001), and HPEV1 displays partial sensitivity (Gazina et al., 2002). Based on these results and colocalization studies, it has been suggested that the COPI complex is directly involved in the formation of replication complexes of enteroviruses, but not cardioviruses or parechoviruses (Gazina et al., 2002). Studies with ARF1 in PV-infected cells, supports these findings (Belov et al., 2005). Interestingly, mutations within 2C and 3A have been observed to render PV resistant to BFA, indicating that other mechanisms might also be used (Crotty et al., 2004). The FMDV RC has been pinpointed to a site close to the Golgi complex (Knox et al., 2005). However, the FMDV RC did not to contain any marker proteins of the Golgi, the ER, ER intermediate compartment, endosomes or lysosomes, which suggests that the membranes generated at FMDV assembly sites are able to exclude organelle-specific marker proteins, or that FMDV uses an alternative source of membranes as a platform for assembly and replication (Knox et al., 2005). A study on the intracellular changes seen in FMDV-infected cells revealed a fragmented ER, almost totally devoid of ribosomes as well as dispersed Golgi stacks (Monaghan et al., 2004). Sensitivity to cerulenin, an antifungal antibiotic that inhibits sterol and fatty acid biosynthesis, seems to be a common property in the picornavirus superfamily (Carette et al., 2000, Ritzenthaler et al., 2002). Many plant viruses in the superfamily appear to replicate on ER-derived membranes, but in contrast to enterovirus-infected cells, the Golgi complex remains normal (Salonen et al., 2004).

8. Parechoviruses

In 1956, during studies of summer diarrhea, Wigand and Sabin (Wigand & Sabin, 1961) isolated previously unrecognized viruses from rectal swabs of infants. Two of these viruses, originally classified as EV 22 (Harris strain) and EV 23 (Williamson strain), have been designated as prototypes of HPEV1 and HPEV2, respectively. Already during their original characterization, these viruses were found to exhibit growth properties distinct from those of other enteroviruses. These included difficulty in passage and adaptation to cultures of monkey kidney cells and the restriction of cytopathogenic effect

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to peripheral parts of the cell monolayer (Shaver et al., 1961). It was also found that these two viruses differed from other enteroviruses by not being inhibited during replication by guanidine hydrochloride (Tamm & Eggers, 1962) and by lacking the host cell protein synthesis shut-off seen in cells infected with typical enteroviruses (Coller et al., 1990, Stanway et al., 1994). Later, it has been shown that cleavage of eIF4G, the principal mechanism involved in the shut-off in enterovirus-infected cells, does not to occur in HPEV1-infected cells (Coller et al., 1991). Evidence of exceptional secondary structure in the HPEV1 genome compared to PV RNA was also reported (Seal & Jamison, 1984, Seal & Jamison, 1990). When nucleic acid hybridization assays were developed for identification of enteroviruses, it was observed that the HPEVs were not recognized by cDNA probes originating from members of the enterovirus subgroups (Auvinen et al., 1989, Auvinen & Hyypia, 1990, Chapman et al., 1990, Hyypia et al., 1987). These findings led to more detailed molecular analysis of HPEV1, and subsequently HPEV2, including determination of the genomic sequences (Ghazi et al., 1998, Hyypia et al., 1992). The sequences revealed a number of unusual features of the HPEVs and showed that in any region of the genome, with the exception of the 5’UTR, HPEVs clearly constitute a separate molecular entity among picornaviruses. The availability of the genomic sequence made it possible to compare the predicted viral proteins of HPEV1 with those of other representatives of the picornavirus family (Hyypia et al., 1992, Stanway et al., 1994). Similarities in the primary structures between HPEV1 and picornaviruses whose three-dimensional structure was known suggested that the common overall architecture of the major capsid proteins VP1 to VP3 is also found in HPEV1. Interestingly, it was observed that in HPEV1-infected cells, most of the VP0 molecules do not undergo the processing to VP2 and VP4 seen in other picornaviruses during the final maturation cleavage (Stanway et al., 1994). Many of the HPEV1 and 2 non-structural proteins could also be identified using sequence alignments (Ghazi et al., 1998, Hyypia et al., 1992). The 3D, 3C and 2C coding regions are relatively well conserved between HPEV1 and 2 and other representatives of the picornavirus family, while the regions coding for 2A, 2B and 3A exhibit only limited identity with those of other picornaviruses.

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The 2A protein of HPEVs showed none of the characteristics of other picornavirus 2A proteins, and was found to lack proteolytic activity (Schultheiss et al., 1995a). The 2A proteins of HPEVs are homologous to the corresponding region of the polyprotein of the hepato-like virus AEV and, to a lesser extent, to the 2A protein of Aichi virus (Marvil et al., 1999, Yamashita et al., 1998). It was shown that these proteins are also related to recently identified cellular proteins, H-rev107, Tazarotene-induced gene protein 3 (TIG3) and lecithin retinol acyltransferase (LRAT) (Hughes & Stanway, 2000). These protein sequences are relatively diverse, but all have a conserved H-box (HWA(I/L) in human and rat H-rev107, TIG3 and Aichi virus 2A; and H(Y/F)G(I/V) in HPEV1/2, AEV 2A and LRAT), an NC-motif, and a long, hydrophobic domain. (DiSepio et al., 1998, Hajnal et al., 1994, Husmann et al., 1998, Ruiz et al., 1999, Sers et al., 1997). It is known that H-rev107 is down-regulated in a number of tumour cells and that its overexpression leads to inhibition of cell proliferation and resistance to transformation by the ras oncogene homologue (Hajnal et al., 1994, Husmann et al., 1998, Sers et al., 1997). This suggests that H-rev107 may act as a negative regulator of oncogenic ras signals, possibly by binding to and inhibiting an effector molecule (Husmann et al., 1998). Expression of TIG3, which is induced by retinoic acid, likewise reduces cell proliferation (DiSepio et al., 1998) and may contribute to the known inhibition of cell growth by retinoic acid. Both Hrev107 and TIG3 have been described as class II tumour suppressors. These are proteins that are expressed at very low levels in cell lines or tumours, permitting cell proliferation, even though their genes are intact (Husmann et al., 1998). Like that of other picornaviruses, the HPEV1 5’UTR is long (712 nt) and contains several AUGs which are probably not functional. Analysis of the secondary structure indicates the presence of 12 stem-loops (A to L), several of which are similar to the structural domains seen in cardio- and aphthoviruses (Ghazi et al., 1998, Nateri et al., 2000, Stanway & Hyypia, 1999). The HPEV1 5’-proximal structure consists of a long stem- loop (A), together with a short stem-loop (B) whose loop appears to participate in a pseudoknot structure (C). Downstream, there is a variable AC-rich tract (Ghazi et al., 1998). Studies with replicons have revealed that these two terminal stem-loops, A and B, together with the pseudoknot element, form the cis-acting signals which are required for

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HPEV1 replication (Nateri et al., 2002). Recently, a putative bus has been identified in the VP0 region of the HPEV genome (Al-Sunaidi et al., 2007). Interestingly, the sequence of HPEV1 also revealed that it carries an RGD motif in its VP1 capsid protein (Hyypia et al., 1992). The RGD motif was shown to be functional by blocking experiments with RGD-containing synthetic peptides (Stanway et al., 1994), and it was shown that HPEV1 competes for cell surface binding with CAV9, known to

recognize the vitronectin receptor (αvβ3 integrin) on the cell surface (Roivainen et al., 1994). Affinity selection of virus-binding peptides from a phage display library showed that integrins were involved in the cellular entry of HPEV1 (Pulli et al., 1997). A separate

study indicated that both integrin αvβ3 and integrin αvβ1 would be directly involved in HPEV1 attachment (Triantafilou et al., 2000). Infections caused by HPEV1 have been reported from all around the world. Most HPEV1 infections occur in children under the age of one and almost all infections occur before the age of 15 (Ehrnst & Eriksson, 1993, Grist et al., 1978, Joki-Korpela & Hyypia, 1998, Nakao et al., 1970, Sato et al., 1972). In a Finnish study, 90% of 1-year-olds had antibodies against HPEV1 and among adults the seroprevalence of HPEV1 antibodies exceeded 95% (Joki-Korpela & Hyypia, 1998). Infections are often asymptomatic, but the predominant symptoms caused by HPEV1 are diarrhea and respiratory illness (Birenbaum et al., 1997, Ehrnst & Eriksson, 1993, Grist et al., 1978, Nakao et al., 1970). Symptoms associated with the CNS (Figueroa et al., 1989, Koskiniemi et al., 1989) or the myocardium (Maller et al., 1967, Russell & Bell, 1970) have also been described. During a search for an infectious agent linked to myocarditis in humans, a new virus, Ljungan virus, was isolated from Swedish bank voles (Clethrionomys glareolus) (Niklasson et al., 1999). The partial sequences of the prototype strain indicated that LV is most closely related to HPEVs (Niklasson et al., 1999). However, recent studies have suggested unique polyprotein processing, revealed unusual sequence characteristics of the capsid proteins, and identified two different 2A proteins (Johansson et al., 2002). Very recently, several new parechovirus serotypes have been characterized (Abed & Boivin, 2005, Al-Sunaidi et al., 2007, Benschop et al., 2006a, Ito et al., 2004). HPEV3 was isolated from a stool specimen of a 1-year-old Japanese girl with transient paralysis (Ito et al., 2004), whereas one new serotype was isolated from a neonate with high fever

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(Benschop et al., 2006a) and two new serotypes were found in an analysis of Californian isolates (Al-Sunaidi et al., 2007). Intriguingly, infection with HPEV3 has been connected to severe sepsis-like illness and central nervous system (CNS) involvement in neonates (Abed & Boivin, 2006, Benschop et al., 2006b, Boivin et al., 2005).

Aims of the Study

AIMS OF THE STUDY

The Parechovirus genus has several unique features when compared to other picornaviruses. However, little is known of the intracellular pathology of infection. The aims of this study were:

1. To study cell surface receptor interactions and the entry route of HPEV1 into the host cell

2. To characterize the membranous replication complex formed in HPEV1-infected cells and to study intracellular changes accompanying infection

3. To biochemically characterize the non-structural 2A protein to gain information about its function in the viral lifecycle

4. To characterize functions of the non-structural 2C protein

5. To study the intracellular effects following expression of individual HPEV1 non- structural proteins

Materials and Methods

MATERIALS AND METHODS

1. Viruses, cells and transfection (I, II, III, IV, V)

The HPEV1 virus stock was obtained by transfection of cells with infectious genomic RNA, obtained by in vitro transcription of clone pHPEV1 (Harris strain, kindly provided by G. Stanway, University of Essex, Colchester, United Kingdom). CAV9 (Griggs strain) and CBV3 (Nancy strain) were originally obtained from the American Type Culture Collection (ATCC). The A549 cells (human lung carcinoma) as well as the HeLa cell lines were obtained from the ATCC. Transfections with Fugene 6 (Roche) or Lipofectamin-2000 (Invitrogen) were carried out according to the manufacturers’ protocols.

2. Plasmids and protein production (II, III, IV, V)

pHPEV1 and pCBV3 (Nancy strain, kindly provided by R. Kandolf, University Hospital of Tubingen, Tubingen, Germany) were used as parental plasmids for cloning experiments. Plasmid construction and propagation were done in E. Coli DH-5α cells according to standard protocols. To clone and subsequently express 2A and 2C proteins, pGEX 4T-1 (Amersham Biosciences) or pQE-30 (Qiagen) were used. A hemagglutinin (HA) tag and restriction sites were added to the pC1-neo vector (Promega) and used to express non-structural proteins as HA-tagged fusion proteins. The pEGFP vector (Clontech) was used to contruct enhanced green fluorescent protein (EGFP) fusion proteins. All the constructs were verified by sequencing. The amplified viral UTR sequences were cloned into pGEM3Z vector (Promega). The resulting constructs allowed synthesis of positive- and negative- strand RNA transcripts under control of SP6 or T7 RNA polymerases. For production of 2A and 2C proteins, the E. coli strain BL21 (DE3) was transformed with 2ApGEX4T-1 or 2CpGEX4T-1, and strain M15 [pREP4] was transformed with 2ApQE-30 or its derivatives. The proteins were purified under native conditions in glutathione-Sepharose 4B columns (Amersham Biosciences/Pharmacia) in the case of

Materials and Methods

GST-2A and GST-2C or on Ni-NTA-agarose (Qiagen) in the case of His-2A according to the manufacturers’ protocols. The plasmid pVSVG3-GFP (Toomre et al., 1999), which encodes the ts045-VSVG protein fused to GFP at its C-terminus, was provided by F. van Kuppeveld (Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands), with the permission of P. Keller (Max Planck Institute of Molecular Biology and Genetics, Dresden, Germany).

3. Antibodies (I, II, III, IV, V)

HPEV1 antisera were obtained by immunizing rabbits and mice with sucrose gradient- purified viruses (Abraham & Colonno, 1984). Rabbits were immunized with three sequential 20 to 100 mg doses injected at 2- to 4-week intervals with Freund’s complete adjuvant included in the first dose. The sera were collected 2 weeks after the last injection. The mice were immunized subcutaneously with three sequential doses of purified virus at 2-week intervals, and Freund’s complete adjuvant was used in the first dose. Polyclonal antisera against the HPEV1 2A and 2C proteins were produced by immunization of rabbits with GST-fusion proteins as described above. A variety of antibodies against different surface molecules as well as organelle specific markers were used for virus entry studies, characterization of the viral replication complex and localization of individually expressed non-structural proteins. Bromodeoxyuridine (BrdU)-labelled nascent viral RNA was visualized by an anti-BrdU mouse MAb (Biocell Consult). A mAb against HA (Babco) was used to visualize the HA-tagged proteins and a rabbit Ab against the GFP-tag (Molecular Probes) was used to detect the EGFP-fusion proteins in EM and to intensify the signal prior to hybridization with RNA probes.

4. Infectivity titration and blocking of infection with cell surface antibodies (I)

For infectivity titration, samples were diluted from 101 to 106, added to the A549 cells, and incubated for 30 min at 37°C. The virus dilutions were then replaced by 0.5% carboxymethyl cellulose (CMC) in the culture medium (minimal essential medium,

Materials and Methods

MEM, Gibco BRL, supplemented with 0.2% bovine serum albumin, 1% FCS and 20 mM HEPES, pH 7.4), and the incubation was continued for 48 h at 37°C. The cells were stained using crystal violet solution prior to counting the number of viral plaques. Blocking experiments were performed on confluent A549 cells. The cells were washed once and Ab dilutions were added and incubated at room temperature (RT) for 45 min. Subsequently, approximately 100 PFU of purified HPEV1, CAV9, or CBV3 was added on to the cells and incubated at RT for 15 min. The virus solution was then replaced by 0.5% CMC in culture medium, and the number of plaques was determined as described above.

5. Immunofluorescence (IF) labelling and fluorescence microscopy (I, II, III, V)

Cells grown on coverslips were either fixed with methanol for 6 min at -20°C (I) or fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Sigma) (II, III, V). Indirect IF was performed using Abs diluted in phosphate-buffered saline (PBS) with or without 3% bovine serum albumin. For visualization, an appropriate fluorochrome- conjugated secondary Ab was used. Subsequently, the cells were mounted in glycerol containing 1% N-propyl gallate (Sigma) or Mowiol (Sigma). The following chemicals were used to study the viral entry procedures (I): nocodazole (20 mM, Sigma) for depolymerization of microtubules, chlorpromazine (25 and 50 mM, Sigma) to inhibit the formation of clathrin-coated pits, and cycloheximide (100 mg/ml, Sigma) to prevent de novo protein synthesis in the cells. Lipid droplets were visualized with Oil Red-O solution (Sigma) (V). Conventional light microscopy was performed using a Nikon E800 or Zeiss Axioplan 2 microscope, and confocal microscopy was carried out with a laser scanning microscope (Zeiss LSM510, Leica TCS4D or Leica SP2 Confocal Microscope). Pictures were corrected for contrast and intensity using the Adobe Photoshop software (Adobe).

6. Percoll-fractionation of infected homogenates (I)

Percoll fractionation was carried out to enrich the early endosomal fraction from the infected cells. HPEV1 infected A549 cells were incubated with horse radish peroxidase

Materials and Methods

(HRP) (Type II; 2 mg/ml in DMEM, Sigma). The cells were then homogenized, the post- nuclear supernatant (PNS) was collected and Percoll gradients (20%) prepared as described (Punnonen et al., 1994). HRP activities were determined in order to identify early endosomes whereas HPEV1 proteins were identified by immunoblotting.

7. Metabolic labelling of viral RNA (II, V)

To determine the kinetics of viral RNA replication, A549 cells infected with HPEV1 were treated with actinomycin D (AMD) to block cellular RNA synthesis 30 min before the cells were pulse-labeled for 10 min at different times p.i. with [5,6-3H]uridine (Amersham). RNA was acid precipitated, and radioactivity was quantified by liquid scintillation counting. To label nascent viral RNA for in situ detection, HPEV1-infected cells were incubated with 5 µg of AMD/ml for 30 min, washed twice with serum-free medium and incubated for 1 h with Lipofectin (Invitrogen) containing 5-bromo- UTP (Br-UTP, Sigma) and AMD. The cells were fixed and bromo-RNA (Br-RNA) was detected by indirect IF.

8. In vitro transcription-translation and immunoprecipitation (II)

To confirm that the anti-2C Ab produced, recognized both 2C as well as the precursor 2BC, in vitro transcription-translation was performed with plasmids expressing 2C and 2BC in the presence of [35S]methionine with the TnT quick-coupled transcription- translation system (Promega). For immunoprecipitation, 2C Ab was bound to protein A Sepharose (Amersham) overnight at 4°C. The radioactive samples of in vitro translated 2C and 2BC proteins were added and incubated overnight. Immunoprecipitated proteins were separated by sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) in 12% gels. Radioactive proteins were visualized by exposing the gels to X-ray film (Kodak).

9. Fluorescent in situ hybridization (II, III, V)

For the detection of viral genomic RNA, a riboprobe of minus polarity, covering the entire viral sequence, was prepared from plasmid pHPEV1 or pCBV3 by transcription with T3 or SP6 RNA polymerase in the presence of FITC- (Roche) or Alexa 546- conjugated (Molecular Probes) UTP. The probe was subjected to alkaline hydrolysis to

Materials and Methods

generate fragments of approximately 100 nt in length and hybridized to the fixed cells at 42°C overnight (Bolten et al., 1998, Egger et al., 1999). The specimens were mounted in glycerol containing 2.5% (wt/vol) DABCO (Sigma) as an antifade reagent.

10. Flotation assay and western blotting (II)

To study the membrane-binding properties of the 2C protein a flotation assay was performed. HPEV1-infected and uninfected A549 cells were homogenized and a PNS was obtained by centrifugation. The PNS was divided into two equal samples; one was treated with 1% Triton X-100 for 30 min on ice and the other one was left untreated. Sucrose was added to both samples to a final concentration of 60% (wt/wt). The samples (2 ml) were layered on a 1 ml 67% (wt/wt) sucrose cushion and overlaid by 8 ml of 50% sucrose and 1 ml of 6% sucrose. The sucrose gradients were centrifuged at 35 000 rpm for 18 h at 4°C in a Beckman SW 41 rotor. One ml fractions were collected and trichloroacetic acid precipitated. PNS of infected and uninfected cells as well as flotation samples were subsequently separated by SDS-PAGE in 12% gels and electrotransferred to a nitrocellulose membrane (Schleicher and Schuell). 2C protein and BAP31 were visualized by indirect immunodetection with secondary Abs, conjugated to HRP (DAKO). The filters were developed with an enhanced chemiluminescence detection system (Amersham).

11. Electron microscopy (EM), immuno-EM and EM-in situ hybridization (II, V)

For conventional EM, cells were trypsinized, centrifuged, fixed with 2.5% glutaraldehyde and 2% osmium, and embedded in Poly/Bed 812 (Polysciences) according to standard procedures. For EM-in situ hybridization (EM-ISH), the specimens were fixed with 2% paraformaldehyde and embedded in LRGold (London Resin Co.) (Bienz et al., 1987). A DIG-UTP (Roche) labeled riboprobe was prepared as described above, and visualized by indirect immunogold labelling with a DIG Ab (Aurion). The sections for conventional EM were post-stained with Reynolds lead citrate, and the LRGold sections were post- stained with 4% uranyl acetate and Millonig’s lead hydroxide stain. The preparations were viewed in a Philips CM100 EM.

Materials and Methods

For IEM, A549 cells grown on coverslips were fixed with PLP fixative (2% paraformaldehyde, 10 mM periodate, and 75 mM lysine-HCl in 75 mM phosphate buffer, pH 7.4) for 2 h. The cells were permeabilized with 0.01% saponin (Sigma) and immunolabeled with anti-2C Ab and Nanogold, which was subsequently silver enhanced with an HQ Silver kit (Nanoprobes) and gold-toned with 0.05% gold chloride. After washing, the cells were dehydrated in an alcohol series and processed for Epon embedding (Seemann et al., 2000b). Sections were post-stained with uranyl acetate and lead citrate and examined with a Jeol JEM-1200EX II EM.

12. Sequence alignment and comparison (II)

For sequence comparisons, the HPEV1 2C protein was aligned with PV1 2C and HAV 2C. The Wisconsin Package, version 10.2 (Genetics Computer Group), was used for sequence entry and analysis, and the phylogenetic tree was calculated by using PHYLIP with 500 bootstrap replicates.

13. In vitro synthesis of RNA (III, IV)

Non-radioactive RNA was transcribed in reactions containing 10 µg of linearized plasmid, and 30 units of the appropriate RNA polymerase (Promega). [33P]-Labeled RNAs were synthesized in the presence of 50 µCi of [α-33P]UTP (Amersham) and 0.5 mM of unlabeled UTP. The transcripts were extracted with phenol/chloroform and filtered through a Sephadex G-50 (Amersham) spin column. The synthesized RNA products were analyzed by gel electrophoresis, in the case of radio-labeled RNA probes, the gel was dried and autoradiographed. The transcripts were quantified by measuring the absorbance at 260 nm and, in the case of the radio-labeled transcripts, also by Cerenkov counting. Full-length transcripts of the viral genome were synthesized using the pHPEV1 plasmid as a template whereas plasmid pGEM3Z was used to synthesize control transcripts.

14. RNA-binding experiments (III, IV)

In a north-western assay approximately 1 µg of protein was run in a 12.5% SDS-PAGE and then transferred to a nitrocellulose membrane (Schleicher & Schuell). After incubation in denaturation buffer, the membrane was renatured at RT and probed with

Materials and Methods

radio-labeled RNA corresponding to the full-length positive-stranded HPEV1 RNA. The membrane was subsequently washed and subjected to autoradiography. In a UV cross-linking assay the radio-labeled RNA probe was incubated with the protein in binding buffer at RT for 45 min. The mixtures were placed as drops on a microwell plate and irradiated on ice with UV light using Stratalinker 2400 (Stratagene). Non- protected RNA was digested by addition of RNase A (Promega). The protein-RNA complexes were separated in 12.5% SDS-PAGE, and the gels were dried and subjected to PhosphorImager analysis (Fuji) or autoradiography. In competition binding experiments, radio-labeled pGEM3Z RNA was incubated with the protein with or without single-stranded or double-stranded unlabeled RNA competitors in various concentrations. The double-stranded (ds) competitors were generated by annealing of the (+) and (-) polarity strands of 5’UTR and 3’UTR RNA in hybridization buffer at 90 °C for 5 min and cooled to RT. In the gel retardation assay, the radio-labeled RNA transcript was incubated with the protein in binding buffer for 45 min at RT. In control experiments the protein-RNA complexes and free RNA were subsequently incubated in buffer SB, containing 1% SDS and 100 mM β-mercaptoethanol. Some samples were additionally treated with 1 µg of RNase A prior to incubation in SB buffer. The reaction products were analyzed by electrophoresis and the gels were dried and subjected to analysis using PhosphorImager or autoradiography. The experimental binding curve was statistically fitted to the data using the Sigma Plot program (SSPS Inc.).

15. ATP hydrolyzation and chromatografy (IV)

33 32 Hydrolysis of [γ- P] and [α- P]ATP by GST-2C or the mutant GST-2CK146A was investigated by thin-layer chromatographic separation (TLC) of the resulted products on PEI-cellulose F sheets (Merck) along with known non-radioactive standards. Non-linear least-square fitting of the kinetic data was performed using SCIENTIST version 2.01 (Micromath). ATP-binding activity of GST-2C was assayed in a reaction containing. [α- 32P] ATP, in the absence or in the presence of a 40-fold excess of unlabeled NTP:s. The

Materials and Methods

phosphoryltransfer activities of the protein were studied in a reaction containing [γ-33P] ATP or [32P] and unlabeled ATP, ADP or AMP as competitors. To study the autophosphorylation of GST-2C, the protein was incubated with [γ-33P] ATP. After the reaction was stopped, half of the probe was subsequently treated with λ- protein phosphatase for 30 min while the other half was left untreated. The probes were analyzed by SDS-PAGE and autoradiography.

Results

RESULTS

1. Entry of HPEV1 (I)

Previous reports have implied the RGD-motif found at the C-terminus of the VP1 capsid protein of HPEV1 in cell surface interactions of the virus (Stanway et al., 1994) and preliminary evidence (Pulli et al., 1997, Triantafilou et al., 2000) suggested that HPEV1

recognizes αv integrins as cellular receptors. In this study we wanted to clarify the mechanisms of virus attachment and entry in HPEV1 infection.

1.1. Interactions with the cell surface To learn more about the cell surface interactions of HPEV1, we studied the ability of Abs against selected receptor candidates to prevent HPEV1 infection in A549 cells. Abs

against β1, αv and β3 integrin subunits were found to block HPEV1 infection most

efficiently (39% , 42% and 74% inhibition, respectively). When Abs against αv and β3 were used in combination, the infection was almost completely inhibited (97%). The other antibodies tested had no significant blocking effect.

Next, we studied the cocalization of HPEV1 capsid proteins and integrin subunits (α2, αv,

β1 and β3) during the early steps of infection using confocal microscopy. HPEV1 was

found to partly colocalize with β3 integrin after virus attachment, but 5 min p.i., when HPEV1 was internalized into early endosomes, no colocalization or significant redistribution was observed with any of the integrin subunits studied. This was also the case, when the virus was translocated to late endosomes at 30 to 60 min p.i. Percoll fractionation of homogenized cellular samples, confirmed the location of the virus in early endosomes 5 min p.i. When immunoblotting of the early endosomal fraction using

Abs against αv and β3 was performed, the fraction was shown to contain neither of these integrin subunits.

1.2. Entry The entry route of HPEV1 was studied using IF labeling with Abs against the virus capsid and different markers of the entry pathways. Double-labeling with anti HPEV1 and anti-EEA1 antibodies at 5 min p.i. confirmed the finding that the virus enters early endosomes. At 15 min p.i., no colocalization could be seen, indicating that the virus had

Results

already been translocated from early endosomes. At 30 min p.i. HPEV1 colocalized with CI-MPR, a marker for late endosomes, and remained in these structures until 60 min p.i. These results indicate that the virus uses the clathrin-dependent route for cell entry, suggested also by the fact that no colocalization could be seen with caveolin-1, a marker for the caveolin-dependent entry route. Cycloheximide treatment during the infection did not prevent localization of HPEV1 in the ER, suggesting that the observed proteins were not newly synthesized viral polypeptides. Double-labeling studies with known markers for Golgi (TGN-46 for the trans-Golgi network and p23 for the cis-Golgi) showed viral proteins to be translocated to the cis-Golgi network after 30 to 60 min p.i., whereas no colocalization with the trans-Golgi was observed. The findings made by confocal-microscopy were further confirmed using inhibitors of the clathrin-dependent entry route. Nocodazole, which blocks endosomal traffic between peripheral early and late endosomes by depolymerising the microtubules (Gruenberg et al., 1989), was found to have no effect on HPEV1 replication. Chlorpromazine, which has been shown to inhibit the initiation of the clathrin-dependent endocytic pathway through the disappearance of clathrin-coated pits from the plasma membrane (Wang et al., 1993), prevented approximately 55% of HPEV1 infection.

2. Replication complex of HPEV1 (II)

To replicate their genome, viruses must modify the intracellular environment according to their needs. Positive-stranded RNA viruses replicate on intracellular membranes, which they modify into membranous replication complexes. The formation of these complexes is thought to in part contribute to the viral cytopathic effect (CPE). HPEVs differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown (Stanway & Hyypia, 1999). Furthermore, early studies suggest that the CPE induced by HPEV1 differs from that seen with many other picornaviruses (Shaver et al., 1961). In this study, we wanted to examine the intracellular site of viral replication in HPEV1-infected cells and to visualize the intracellular changes accompanying the replication.

Results

2.1. Synthesis and localization of viral macromolecules in the infected cell To determine the peak of viral RNA replication, a time-course of HPEV1 viral RNA replication was established. Viral RNA synthesis increased exponentially starting 4 h p.i. and peaked at 6 h p.i. In parallel, the appearance of the proteins of the P2 genomic region was studied by Western blotting with Ab against the 2C protein. Unexpectedly, after 4 h p.i., a single band of 35 kDa, which is the size of 2C, was detected, but no band corresponding to the 2BC protein was found. Immunoprecipitation of in vitro-translated 2BC using the 2C antibody, confirmed that the results were not due to the specificity of the antibody. In PV infected cells the viral 2C protein is exclusively associated with the viral RC (Bienz et al., 1994, Egger et al., 1996). The intracellular localization of viral macromolecules in HPEV1 infected cells was assayed with Abs against the 2C protein and with an FITC-labeled riboprobe to detect plus-strand viral RNA. Early in the infectious cycle, viral RNA as well as 2C protein were found in numerous small granules in the cytoplasm. At 6 h p.i., corresponding to the peak of RNA synthesis, the intracellular distribution of 2C changed drastically into dot- and stick-like formations, encircling the nucleus or accumulating on one side of the cell. At the same time, viral RNA was mainly found in a few large granules and, in some cells, also diffusely in the cytoplasm. Protein 2C presented in long stick-like structures at 8 h p.i., and finally as larger irregular bodies at 10 h p.i, whereas the localization of viral RNA was essentially unchanged at 8 h p.i. but became additionally diffuse at 10 h p.i. Thus, the localization pattern of protein 2C differed from that of viral RNA, and the structures labeled by the 2C Ab were found in excess over the dot-like structures harbouring viral RNA. Because of the unexpected features of the HPEV1 2C protein, we compared its sequence with those of the 2C proteins of two other picornaviruses, PV1 and HAV. The overall identity of the 2C protein of HPEV1 with that of PV1 was 32%, and its identity with that of HAV was 28%. The NTP binding motifs A, B and C were found to be conserved in all three proteins. Interestingly, the cysteine-rich region (aa 269 to 286 in PV1), which is highly conserved in entero- and rhinoviruses (Pfister et al., 2000), could not be found in the HPEV1 or HAV 2C sequences. Furthermore, the asparagine at aa 179 in the PV1 2C sequence, the mutation of which renders PV insensitive to guanidine hydrochloride

Results

(Pincus & Wimmer, 1986), was found to correspond to a glycine in both the HPEV1 and HAV sequences. Our findings may explain the guanidine insensitivity of HPEV1, and suggest that although all three proteins share NTP-binding properties, there could be sequence induced differences in other functions of the proteins.

2.2. Ultrastructural aspects of the infected cell and the site of viral RNA synthesis The ultrastructural changes caused by HPEV1 infection were studied by EM. The most prominent feature of the infected cell was a dilated ER and perinuclear space. The dilated ER membranes had lost most of their ribosomes and appeared as shorter pieces. Cytoskeletal material as well as clusters of small vesicles were often seen in close proximity to the modified ER membranes. The vesicles were 30 to 60 nm in diameter and could be seen both in larger clusters, mainly in the perinuclear area, and in small groups. Classical Golgi stacks were found to be absent in the infected cells and IF studies using Golgi markers further confirmed the disintegration of the Golgi apparatus. The stick-like structures that were seen containing 2C protein in the fluorescence microscope were further analyzed by confocal microscopy. The labeled structures were found to consist of long branching sticks that formed a coherent network rather than individual elongated structure. To identify the labeled structures, we performed an immunogold EM analysis. The immunocytochemical 2C signal was found in longer and shorter elongated structures, compatible with the stick-like structures in the IF samples. Higher magnification showed that the label was accumulated on stretches of membranes as well as on clusters of vesicles, compatible with the vesicles found by conventional EM. A flotation assay confirmed the membrane-binding property of the 2C protein. To visualize the viral genomic RNA at an ultrastructural level, a DIG-labeled strand- specific RNA probe was hybridized to sections of cells, harvested at the peak of RNA synthesis. The hybridized probe, visualized with anti-DIG Abs conjugated to 10-nm- diameter gold particles, localized to membranes of small vesicles. These RNA-carrying vesicles were found to also contain 2C protein and to be compatible with the granules seen in infected cells subjected to FISH and to the aggregates of small vesicles seen by conventional EM.

Results

To further examine whether these vesicles containing both 2C protein and viral RNA, were the actual site of viral genome replication, nascent viral RNA was labeled with Br- UTP and detected by indirect IF using an anti-BrdU Ab. The Br-RNA could be found as punctuate fluorescent signals, mainly in the perinuclear area. To investigate whether the structures involved in viral replication carry the 2C protein, a marker for the replication complex in other picornaviruses, Br-UTP-transfected cells were also stained with Ab against the 2C protein. Surprisingly we found that whereas nascent viral RNA colocalized with 2C in discrete small dots, a large excess of the 2C-labeled components, predominantly the stick-like structures, did not carry viral RNA. Since viral RNA located to small vesicles resembling in size and aspect vesicles of a dispersed Golgi complex, a possible involvement of the Golgi apparatus in the viral replication complex was investigated. Confocal microscopy showed that all of the Br- RNA colocalized with ß1,4 Galactosyltransferase (GalT), a trans-Golgi protein, suggesting that the HPEV1 replication complex could be a Golgi-derived structure.

3. Biochemical characterization of the HPEV1 2A protein (III)

Although in general the non-structural proteins are quite well conserved among different picornaviruses, there is a great deal of variability in the functional properties of the non- structural 2A protein. In vitro translation experiments have shown that the HPEV1 2A lacks the proteolytic activity found in many other picornaviruses (Schultheiss et al., 1995a) and sequence alignment has revealed that the protein differs considerably from the corresponding proteins in other picornaviruses (Hughes & Stanway, 2000, Hyypia et al., 1992). However, no particular function has been identified for HPEV1 2A. The aim of this study was to perform an initial biochemical characterization of the 2A protein in order to gain an insight in its possible function in the viral lifecycle.

3.1. Intracellular location To determine the intracellular localization of the 2A protein during HPEV1 infection, cells infected with HPEV1 were examined by indirect IF using a 2A antibody generated against a GST-2A fusion protein. In infected cells, the 2A protein emerged at 4 h p.i., and it exhibited a rather diffuse cytoplasmic staining, located mainly in the perinuclear area. At 6 h p.i., the 2A signal became more intense and it was evenly distributed throughout

Results

the cytoplasm. In addition, there appeared a weak, diffuse nuclear staining which blurred the border between the cytoplasm and the nucleus. At late stages of infection (8 and 10 h p.i.), two distinct types of staining patterns were observed. Some cells had the same type of diffuse cytoplasmic staining seen early in infection, whereas others displayed a stronger nuclear staining and weaker cytoplasmic staining. Furthermore, the proportion of cells displaying nuclear staining grew over time. The observation that 2A was found mainly in the perinuclear area prompted us to test a possible colocalization of 2A with viral RNA, using FISH. At 6 h p.i., 2A partially colocalized with viral RNA, suggesting that the 2A protein could be present at the perinuclear sites of viral replication.

3.2. RNA-binding activity The localization of the 2A protein further encouraged us to study the possible interactions between 2A and viral RNA. To rule out the possibility that the large GST-tag would interfere with the potential RNA-binding activity of 2A, a 6xHis-tagged protein was used. In a North-western blot assay, the 2A protein was found to bind RNA whereas the control proteins did not. Next, the protein-RNA complexes were cross-linked by UV light, the unbound RNA was removed by RNase A digestion and the cross-linked complexes were analysed by SDS-PAGE and autoradiography. A strong radio-labeled band could be seen in the position expected for the 2A protein. A weaker band migrating at the position of 32 kDa, which probably corresponds to a cross-linked dimer of 2A, was also detected. The results also indicated that binding of 2A resulted in the formation of high molecular weight complexes, which in turn suggests that the protein molecules are bound to RNA in close association with each other. Contiguous arrangement of bound protein molecules is characteristic for a cooperative binding mode. Our next goal was to find out whether the 2A protein has any sequence specificity or preference in binding to RNA. The specificity of the 2A-RNA interaction was assessed by comparing the abilities of different unlabeled RNA substrates to compete with a radio- labeled probe for binding to 2A. The competitors included a poly A, two fragments of 5’- UTR (5’UTR70 and 5’UTR295-365), the 3’-UTR of both positive and negative polarity, and a non-parechovirus sequence derived from pGEM3Z. When the results were analyzed, a difference between the viral 3’UTR and other RNA competitors became

Results

apparent; addition of a 10-fold excess of unlabeled 3’UTR(+) RNA resulted in an approximately 45% reduction in binding of 2A to radio-labeled RNA, unlabeled 3’UTR(−) RNA competitor produced almost the same effect, whereas other competitors produced only an approximately 25% reduction in binding. The notion that 2A has higher affinity to 3’UTR RNA than to other RNAs was further supported by electrophoretic retardation experiments in non-denaturing agarose gels. These experiments allowed us to determine the dissociation constants (Kds) of the 2A-3’UTR and 2A-5’UTR complexes. Saturation of the 3’UTR RNA binding was achieved at about 0.1 μM of the 2A protein, whereas the saturation of the 5’UTR RNA-binding was achieved at significantly higher

protein concentration, 0.4 μM. The Kd values for the 2A-3’UTR and 2A-5’UTR complexes were determined to be 0.03 and 0.1 μM, respectively. Next, we wanted to investigate the influence of the secondary structure of 3’UTR(+) RNA on binding to 2A. It was found that the ability of the denatured RNA to interact with the 2A protein was reduced, suggesting that the secondary structure of the 3’UTR has an influence on its ability to bind the 2A protein. Additional gel-retardation experiments were performed to characterize the 3’UTR RNA- binding capability of the 2A protein in more detail. The binding of 2A to RNA was found not to be random, instead, 2A bound RNA so that RNA molecules were either fully coated with 2A or completely free of protein. This “all or none” RNA-binding pattern is consistent with a cooperative mode of interaction between the protein and the RNA (Li and Palukaitis 1996, Citovsky et al 1990, Rajendran and Nagy 2003, Tsai et al 1999, Marcos et al 1999, Lopez et al 2000, Osman et al 1992). From the linear regression analysis, a Hill coefficient of 1.68 was obtained, which exceeds 1 (a Hill coefficient of 1 indicates no cooperativity) and therefore indicates positive cooperativity. Digestion of the 2A-RNA complex with RNase A and subsequent treatment with buffer containing 1% SDS, revealed an RNA core which migrated faster than the free RNA, indicating that 2A binds to discrete lengths of the RNA. No such RNase-resistant core was observed in the absence of the 2A protein. This finding further confirms the cooperative binding between the 2A protein and RNA. Furthermore, when the 2A protein was denatured prior to incubation with labeled RNA transcript, no retarded RNA was detected on the gels,

Results indicating that the RNA-binding activity is determined by the native conformation of the 2A protein. As RNA-RNA duplex regions are generated in the course of picornavirus replication we wanted to test whether 2A could also recognize double-stranded RNA. The 2A protein was incubated with radio-labeled pGEM3Z RNA and unlabeled dsRNAs corresponding to 5’UTR(+)-5’UTR(−) and 3’UTR(+)-3’UTR(−) duplexes and the results were analyzed using a UV cross-linking assay. It was found that the ssRNA-binding activity of 2A was gradually reduced in the presence of increasing concentrations of the 3’UTR(+)- 3’UTR(−) duplex, whereas no significant effect was detected when the 5’UTR(+)- 5’UTR(−) duplex was used as a competitor. Interestingly, comparison of the effects of the competitors on the ssRNA-binding activity of the 2A protein revealed that 2A has a preference in binding in the order: 3’UTR>other ssRNA>3’UTR(+)-3’UTR(−) duplex.

3.3. Mutation analysis The analysis of the hydropathy profile and the distribution of acidic and basic residues revealed two regions (aa 10-24 and 43-56) where the 2A protein has a strong positive charge with a potential to interact with RNA. To examine more closely the contribution of these regions to the RNA binding capacity of the protein, two deletion mutants were constructed. Two additional deletion mutants were designed in accordance with the prediction that the C-terminal region of the 2A protein could be a critical functional element (Hughes and Stanway 2000), one stripped of aa 130-150, and the other of aa 108-150. The RNA-binding activities of the 2A mutants were analyzed using a UV cross- linking assay. The mutant lacking aa 43-56 completely lost its ability to bind to RNA, whereas, deletion of residues 10-24 at the N-terminus did not affect RNA-binding. Interestingly, the mutant lacking the most C-terminal region could bind RNA, but less so than intact 2A, and the mutant protein lacking aa 108-150 could not bind RNA, suggesting that the C-terminus of the protein could play an important role in the RNA interaction. In conclusion, these data provide evidence that the basic N-terminal 43-56 region is essential for interaction with RNA, and that the C-terminus also appears to play a pivotal role in the RNA-binding activity of 2A.

Results

4. ATP hydrolysis and AMP kinase activities of the HPEV1 2C protein

The 2C proteins of picornaviruses display an extensive conservation in amino acid sequences and exhibit homology to the NTPase/helicase superfamily III (Gorbalenya & Koonin, 1989, Gorbalenya et al., 1989, Gorbalenya et al., 1990). However, we found that although HPEV1 2C is a membrane-bound protein present in the viral replication complex, its distribution in the infected cells differs considerably from its enterovirus counterparts. In this study, we wanted to biochemically characterize the HPEV1 2C protein and analyze whether its function could correlate with the unique changes seen in HPEV1-infected cells.

4.1. ATP diphosphohydrolase activity To study the NTPase activity of the 2C protein, a 2C- GST fusion polypeptide was expressed and purified. Hydrolysis of [γ-33P] ATP by GST-2C was investigated by thin- layer chromatographic (TLC) separation of the end products. It was found that the GST- 2C protein constructed could indeed hydrolyse ATP, whereas a mutant, lacking the conserved Lys within the first NTPase/helicase motif, showed a loss of ATP hydrolytic activity. To characterize the position of the enzymatic cleavage site in ATP, experiments were performed with [α-32P] ATP. In the sample containing GST-2C, ATP was hydrolysed to ADP and AMP by hydrolysis of the β-γ and α-β phosphodiester bonds, suggesting that both ATP and ADP are substrates for the protein hydrolytic activity. The activity of the HPEV1 2C protein, as that of many other NTPases, was dependent on divalent cations (Kadare & Haenni, 1997). The optimal Mg2+ concentration for the reaction was in the range 2.5-5 mM and Mn2+ could substitute for Mg2+, although less Pi was released. We found that the pH optimum for ATP hydrolysis by 2C was 7 to 8 and that maximum hydrolysis was achieved at 37°C. Our results concerning NaCl dependence of the reaction indicated that monovalent cations at the concentration up to 150 mM stimulate the enzymatic activity, whereas higher concentrations inhibit ATP hydrolysis. A time-course analysis of ATP hydrolysis by GST-2C, using variable amounts of unlabeled ATP and a constant amount of [α-32P] ATP, demonstrated that 2C does not progressively hydrolyze ATP to AMP, but rather uses both ATP and ADP as substrates.

Results

The Km and kcat values for ATP and ADP indicated that the HPEV1 2C protein has about a 200-fold higher affinity to ATP than PV 2C (Pfister & Wimmer, 1999). The turnover number for ATP hydrolysis by HPEV1 2C was similar to that reported for other viral NTPases (Bisaillon et al., 1997, Morgenstern et al., 1997, Suzich et al., 1993). The substrate specificity of the GST-2C protein was studied through competition experiments. The results showed that the hydrolysis of labeled ATP was competed only by the presence of unlabeled ATP, and that the presence of CTP, GTP or UTP did not affect the release of labeled Pi. dATP had also no effect on radio-labeled ATP hydrolysis. We further investigated the effect of excess of four unlabeled NTPs on HPEV1 2C ATP- binding activity, and found that whereas an addition of excess of unlabeled ATP resulted in significant reduction of binding of radio-labeled ATP to 2C, other NTPs did not reduce protein binding when the same proportional ratio was used. Whereas hydrolytic activity of NTPases is generally stimulated by the presence of single- stranded nucleic acids (Kadare & Haenni, 1997), the ATPase activity of PV 2C has been shown to be sensitive to the presence of RNA (Pfister & Wimmer, 1999). We next sought to determine whether RNA could stimulate HPEV1 2C ATP hydrolysis by adding poly(U) or poly(A) or specific 5’UTR or 3’UTR HPEV1 RNA at different concentrations to the reaction mixture. A 1.3 to 1.5 fold inhibitory effect was found when compared to the activity observed in the absence of RNA.

4.2. Phosphoryltransfer activity and autophosphorylation Surprisingly, in a reaction containing [γ-33P] ATP and AMP as an unlabeled competitor, about 60% of the substrate ATP was converted into radio-labeled ADP, suggesting that GST-2C can utilize Pi, released during the hydrolysis of ATP, and AMP as an acceptor molecule to generate ADP. In the presence of ADP/ATP as unlabeled competitors, formation of a small amount of radio-labeled ADP could also be seen. In subsequent experiments we analysed the AMP kinase activity of GST-2C in the presence of [32P] and unlabeled AMP/ADP/ATP. Formation of radio-labeled ADP was observed in samples containing unlabeled ADP or ATP, whereas, no radio-labeled ADP was formed in the reaction containing AMP. This could be explained by the activity of the protein being energy-dependent, therefore requiring ATP/ADP hydrolysis.

Results

To determine whether 2C has an associated kinase activity that can cause autophosphorylation, the protein was incubated in a buffer containing [γ-33P] ATP in the presence of Mg2+. Subsequently, half of the sample was treated with λ-protein phosphatase which removes the phosphate group, while the other half was left untreated. When the samples were analyzed by SDS-PAGE, autoradiography revealed that 2C became radio-labeled suggesting that the protein is autophosphorylated during ATP hydrolysis. Autoradiography revealed that 2C autophosphorylation was gradually decreased in the presence of increasing concentrations of unlabeled ATP. GST-2CK146A was used as a control protein and autoradiography showed that the mutant was not autophosphorylated during the reaction.

5. Intracellular localization and effects of individually expressed HPEV1 non-structural proteins

It has been shown that the formation of the enterovirus membranous replication complex as well as other cytopathic changes seen in the infected cell, can be mimicked by the expression of individual non-structural proteins alone or in combination (Sandoval & Carrasco, 1997, Teterina et al., 1997a, Teterina et al., 1997b, van Kuppeveld et al., 1997). As we have shown that the replication complex of HPEV1 differs from that induced by enteroviruses (Krogerus et al., 2003), and that the HPEV1 non-structural proteins have several exceptional features (Samuilova et al., 2004, Samuilova et al., 2006) we last wanted to study the intracellular effects of individually expressed HPEV1 non-structural proteins.

5.1. Localization of individually expressed HPEV1 non-structural proteins To study the intracellular localization of individually expressed HPEV1 non-structural proteins, fusion-proteins were constructed and expressed in A-549 cells. 2A-EGFP was seen diffusely in the cytoplasm and nucleus starting 4 h post-transfection (p.t.), resembling the localization of the virus-encoded polypeptide (Samuilova et al., 2004). No change in the localization pattern was observed during 48 h. The 2B-EGFP protein formed a cytoplasmic reticular pattern while 2C-EGFP and 2BC-EGFP were both seen in accumulations of circular cytoplasmic formations and as a faint diffuse dot-like or reticular cytoplasmic staining starting 4 h p.t. The 3A-EGFP and 3AB-EGFP proteins,

Results

which had an identical distribution, accumulated in the perinuclear area and in some cells additionally formed a reticular pattern in the cytoplasm. The 3D-EGFP protein localized diffusely in the cytoplasm of the transfected cells, and the nuclei of the transfected cells, excluding the nucleoli, were also strongly fluorescent. The 2B, 2C and 3AB proteins were further studied through construction of fusion proteins containing the small (9 aa) HA-tag. As expected, double transfection of EGFP- and HA-fusion proteins showed perfect colocalization. To study the localization of the fusion proteins more precisely, confocal microscopy using cellular markers as well as IEM was performed. The 2B protein was found to localize to the ER, but the ultrastructures of the ER, the Golgi and other parts of the 2B transfected cells were not altered compared to control cells. The 3AB proteins were found to colocalize with the trans-Golgi-network marker p230 but not with the cis-Golgi marker GM130. Again, the morphology of both the Golgi and the ER was found to be intact. The 2C protein could be found mainly on lipid droplets, but also on diverse intracellular membranes. Similar lipid droplets were also found in untransfected control cells. Although the protein also partially localized on Golgi and ER membranes, no obvious change in the morphology of the Golgi or the ER could be observed. Because of the central role that has been ascribed to the 2C and 2BC proteins in enterovirus replication complex formation, CBV-3 HA-2C and HA-2BC fusion proteins were constructed for comparison. The CBV3-2C and -2BC proteins were found to form a reticular pattern with a perinuclear accumulation, which is consistent with earlier findings suggesting that the main target of the enterovirus 2C protein is the ER. As it has been reported for several different viruses that it is the combined action of several non-structural proteins rather than individual proteins that mediates the ultrastructural changes seen in the infected cell (Salonen et al., 2003, Suhy et al., 2000), we further pair-wise co-expressed the HPEV1 non-structural proteins. The localization of the individual proteins did not significantly change in the co-transfected cells.

5.2. 2C protein can associate with viral RNA in infected cells. In view of the above findings, we next wanted to investigate whether the expressed non- structural proteins could be involved in the formation of the viral replication complex.

Results

Cells transfected with non-structural proteins were subsequently infected with HPEV1 and 6 h p.i. the infected cells were identified by the presence of viral RNA or the viral 2C protein in the cell. The pattern of transfected 2A-EGFP, 2B-EGFP or 3D-EGFP did not change upon infection nor could any specific colocalization between the transfected proteins and the viral 2C protein be observed. Interestingly, the viral 2C protein which has been shown to reside on modified ER membranes (Krogerus et al., 2003) did not colocalize with the 2B-EGFP construct which could also be found in the ER. This finding suggests that the 2C-positive structures seen in HPEV1-infected cells are indeed specialized membrane compartments lacking resident ER proteins. As HPEV1 infection results in a disintegration of the Golgi apparatus, it was not surprising that the staining pattern of the expressed 3AB protein changed in the infected cells. The protein formed a diffuse reticular structure and additional vesicular structures appeared. No specific colocalization with either viral 2C protein or with viral RNA could be seen. Interestingly, in 2C transfected, superinfected cells, the structures containing 2C-EGFP partially colocalized with viral RNA. The colocalization did not seem to involve the lipid droplets but rather dot-like structures in the perinuclear area, suggesting that the portion of the protein residing on ER and Golgi membranes could relocate to sites of viral RNA synthesis.

5.3. Effect of the HPEV1 infection and non-structural proteins on the secretory pathway It has been shown that several picornaviruses as well as individually expressed viral non- structural proteins can inhibit ER-to-Golgi transport (Doedens & Kirkegaard, 1995, Moffat et al., 2005). We studied the effect of HPEV1 infection and the individual non- structural proteins on the movement of membrane proteins from the ER into the secretory pathway using a GFP-tagged ts045-VSVG-protein, a well-known membrane-bound secretory marker (Toomre et al., 1999). To investigate whether HPEV1 infection inhibits vesicular trafficking, A-549 cells were first transfected with VSVG-GFP, incubated at 40°C for 24 h, and then infected with HPEV1. The infected cells were further incubated at 40°C for 4 h and then shifted to 32°C for 2 h. The cells were fixed and stained with an antibody against GFP before being subject to FISH, and VSVG-GFP localization was

Results analyzed by confocal microscopy. In HPEV1-infected cells, VSVG-GFP was retained in the ER, suggesting that protein transport was blocked between the ER and the Golgi. However, cells co-transfected with VSVG-GFP and 2B-HA, 2C-HA or 3AB-HA did not exhibit inhibition of protein transport.

Discussion

DISCUSSION

1. Attachment and entry

Viruses can enter cells through different internalization pathways. This variation may largely correlate with the interactions of viruses with specific cell surface receptor molecules. Following attachment to the receptor, the virus penetrates into the host cell. Once inside the cell, the virus uncoats and releases its genome.

The results we obtained strongly support the idea that HPEV1 preferably attaches to αvβ3 integrin at the cell surface. The subsequent entry process seems, however, to be brought about by interactions with one or several additional molecule(s). The concept of multiple receptors or attachment molecules is not strange in the field of virology. The sequence of events leading to human immunodeficiency virus (HIV) fusion and entry likely involves the recruitment of multiple receptor and coreceptor proteins to a specific complex by the viral envelope (Garzino-Demo & Gallo, 2003). Likewise, adenovirus entry into cells generally involves attachment to a primary receptor, followed by interaction with a secondary receptor responsible for internalization (Zhang & Bergelson, 2005). Multiple receptor molecules have been implied in the entry process of several picornaviruses. CBV has been suggested to use multiple receptors to infect human cardiac cells

(Orthopoulos et al., 2004). EV1 binds to α2β1 integrin (Bergelson et al., 1992), but also

interacts with a complex containing β2 microglobulin, since the initiation of infection can be blocked by Abs against this molecule (Ward et al., 1998). CAV9, which was first

shown to utilize the αvβ3 integrin as a receptor (Roivainen et al., 1991, Roivainen et al.,

1994, Williams et al., 2004) and later was found to bind efficiently also to αvβ6 integrin

(Roivainen et al., 1991, Roivainen et al., 1994, Williams et al., 2004), also binds to β2 microglobulin and GRP78 (Triantafilou et al., 2002, Triantafilou et al., 1999). Interestingly, the RGD-integrin interaction is not an absolute requirement for CAV9 infection (Hughes et al., 1995, Roivainen et al., 1991, Roivainen et al., 1994), which further supports the idea that other molecules may also be involved in the entry process. In contrast to CAV9, the RGD motif has been shown to be critical for HPEV1 viability (Boonyakiat et al., 2001).

Discussion

Our results show that chlorpromazine, which prevents the formation of clathrin-coated pits, inhibits HPEV1 infection, supporting the idea that entry takes place through the clathrin route. Nocodazole, which depolymerises microtubules, leading to an accumulation of endocytosed material in early endosomes and carrier vesicles, prevented the colocalization of the virus with late endosomes but had no significant effect on virus replication. These data suggest that internalization of HPEV1 capsids in early endosomes is sufficient for the initiation of a productive replication cycle and that the viral genome may be released at the very early stages of the entry process. Most non-enveloped viruses enter cells via endocytic mechanisms that are either clathrin, caveola or lipid raft mediated (Pelkmans & Helenius, 2003, Smith & Helenius, 2004). FMDVs bind to integrins and are internalized trough the clathrin route to early endosomes. In the low pH of the endosomes, the capsid falls apart releasing the genome (O'Donnell et al., 2005). However, the breakdown of the virion, while necessary for productive infection, is not sufficient, suggesting that additional steps, yet unknown, are required (Knipe et al., 1997, O'Donnell et al., 2005). The clathrin mediated endocytosis of HRV2 has been extensively studied. The virus is dissociated from its receptor in early endosomes (Brabec et al., 2003) and when it reaches late endosomes, it undergoes conformational alterations dependent on low pH, leading to uncoating (Bayer et al., 2001, Huber et al., 2001). The viral RNA is transferred into the cytoplasm across a pore in the endosomal membrane and viral capsid proteins may be transported to lysosomes for degradation (Prchla et al., 1994, Prchla et al., 1995, Schober et al., 1998). For viruses resistant to low pH, like enteroviruses or parechoviruses, the interaction between virus and receptor, coreceptor or an accessory factor must lead to uncoating and release of the genome (Hogle, 2002). The mechanisms releasing the HPEV1 genome into the cytosol, as well as the possible viral and/or cellular factors aiding in these processes remain to be established. Interestingly, HPEV1 capsid proteins were found in the ER at 30 min p.i. and subsequently in the cis-Golgi network (60 min p.i.). In comparison, FMDV capsid proteins were not observed associated with the endoplasmic reticulum or the Golgi following internalization (O'Donnell et al., 2005). It is possible that the appearance of

Discussion viral proteins and/or epitopes in the ER and the Golgi is a step in the antiviral response of the host cell.

2. Replication complex formation The formation of a membranous replication complex is a key feature of positive-stranded RNA viruses. The choice of membrane and the extent of the modifications can be assumed to determine in part the cytopathogenicity of the virus. In the present study, we found that HPEV1 modifies the ER and Golgi membranes and replicates its genome in a vesicular structure containing both the Golgi protein GalT as well as the viral 2C protein. The origin of these Golgi-related vesicles is not clear. It is possible that the vesicles represent the retrograde membrane traffic moving from the Golgi towards the ER (Gazina et al., 2002), since vesicles carrying viral RNA were found intermingled with modified ER membranes and an intact Golgi apparatus was missing. However, the vesicles could also be explained by interference of the virus with the anterograde membrane traffic, possibly mediated by 2C, which could be found in large amounts on modified ER membranes. It was recently found that Golgi proteins, particularly Golgi enzymes, recycle through the ER and that the ER thus may contain GalT (Lippincott-Schwartz et al., 1989, Miles et al., 2001, Seemann et al., 2000a). This could mean that the GalT- positive vesicles of the HPEV replication complex derive directly from the ER. Recent studies regarding the FMDV RC (Knox et al., 2005, Monaghan et al., 2004) are also of much interest since the intracellular morphology of FMDV-infected cells (Knox et al., 2005, Monaghan et al., 2004) seems similar to that observed in HPEV1-infected cells. The FMDV 2C and 3A proteins have been found to partially colocalize in infected cells, and 3A, but not 2C, has been found to colocalize with the ER marker calreticulin (Garcia- Briones et al., 2006). The membranous origin of the FMDV replication complex is, however, yet unknown. For PV, the viral non-structural proteins have been found associated with the replication complex and to be directly or indirectly involved in viral RNA synthesis. 2C protein is exclusively found on the PV replication complex (Bienz et al., 1994, Egger et al., 1996) although its precise biochemical role in RNA replication still remains uncertain.

Discussion

In contrast to PV 2C, HPEV1 2C protein was additionally found to associate with structures which did not seem to be directly involved in RNA replication. Our IEM findings suggest that these 2C-containing structures are compatible with ER or ER- derived membranes but their function however remains elusive. Two different structural environments could be explained by the need to regulate one of the presumably several functions of the 2C protein. When we compared the sequences of HPEV1, HAV and PV all contained the proposed NTP-binding sites, motifs A, B and C (Gorbalenya et al., 1990, Walker et al., 1982). However, the cysteine-rich region of PV 2C (aa 269 to 286), shown to bind zinc in vitro and to be involved in RNA replication (Pfister et al., 2000), is highly conserved in entero- and rhinoviruses but could not be found in either the HAV or HPEV1 sequence. Whether the absence of the zinc finger in the 2C protein, or the instability of the 2C precursor 2BC, is a reason for the differences seen in replication complex formation in HPEV1-infected cells compared to PV, remains to be established.

3. The biochemical properties of the 2A protein

Our study on the 2A protein represents the first analysis of the biochemical properties and cellular localization of the HPEV1 2A protein. Using the generated antibody, we showed that at early stages of HPEV1 infection the 2A protein can be found diffusely distributed throughout the cytoplasm, whereas later, 2A could also be detected in the nucleus of some infected cells. Despite the presence of the HPEV1 2A protein in the nucleus, no canonical nuclear localization signal in the 2A sequence was identified. Interestingly, a diffuse cytoplasmic distribution and association of the 2A protein with the nucleus was recently demonstrated for EMCV (Aminev et al., 2003a, Aminev et al., 2003b), although in general the presence of viral proteins in the nucleus appears to be uncharacteristic for proteins of cytoplasmic viruses. The function of the nuclear localization is not clear although one might speculate that it could be related to the modulation of host cell functions. Since the 2A protein is only 17 kDa, passive diffusion could account for its nuclear entry, possible aided by a virus-induced modification of nuclear-cytoplasmic trafficking (Belov et al., 2000, Rowland & Yoo, 2003, Tijms et al., 2002, Weidman et al., 2003).

Discussion

The results of our competition binding experiments showed that neither unrelated nor other related RNAs were as effective as the 3’UTR RNA in forming the RNA-protein complex. This finding indicates that 2A exhibits two types of RNA-binding properties, sequence specific and non-specific. Discrimination between target and non-target RNAs could be achieved on the basis of both nucleotide sequence and RNA structures. The fact that 2A bound the denatured 3’UTR(+) RNA with lower affinity than native RNA supports the involvement of a secondary structure in specific binding. The secondary structure of the 3’UTR(+) could, therefore, be a regulatory factor in the interaction with 2A. Several different experiments indicated that the 2A protein binds to RNA in an “all or none” behaviour which is consistent with cooperative binding (Citovsky et al., 1990, Li & Palukaitis, 1996, Rajendran & Nagy, 2003). Furthermore, the binding of 2A to 3’UTR(+) was clearly dependent on the native conformation of the protein. In addition to the ssRNA-binding activity of 2A, we found that the protein also binds dsRNA. Most dsRNA-binding proteins are known to recognize the target without obvious RNA sequence specificity (Fierro-Monti & Mathews, 2000). However, the 2A protein was found to bind duplex RNA containing 3’UTR(+)-3’UTR(-), but not other dsRNA molecules studied. This finding could be explained by sequence and structural factors recognized by 2A. No obvious nucleic acid-binding motif could be found in the 2A protein. Mutagenesis revealed that the N-terminal basic-rich-region (aa 43-56) as well as the C-terminus are important for RNA-binding. It seems unlikely that the protein lost its affinity to RNA simply due to an alteration in the conformation of the deletion mutants, but this possibility cannot be completely ruled out. It is generally thought that conserved sequences and structures at the 3’-terminus of viral genomic RNA function as cis-acting signals that interact with viral proteins to initiate minus-strand RNA synthesis during viral replication. As our results indicate that 2A preferably binds to positive sense 3’UTR RNA, one could suggest that 2A is involved in recognition of specific sequences during the RNA synthesis, possibly keeping the 3’-end of RNA immobilized so that other viral and/or host proteins could form a complex for initiation of RNA synthesis.

Discussion

4. The ATP diphosphohydrolase and AMP kinase activities of the 2C protein

The picornavirus 2C proteins are well conserved ATPases with multiple functions in the viral life cycle. They have been implied in replication (Barton et al., 1995, Barton & Flanegan, 1997, Hadaschik et al., 1999, Klein et al., 2000, Molla et al., 1991, Pincus & Wimmer, 1986, Tolskaya et al., 1994, Wimmer et al., 1993), membrane modification (Aldabe & Carrasco, 1995, Barco & Carrasco, 1995, Cho et al., 1994, Teterina et al., 1997b), encapsidation (Vance et al., 1997) and regulation of virus-encoded proteases (Banerjee et al., 2004). We found that the HPEV1 2C protein has ATP- diphosphohydrolase and AMP kinase activity and that the protein is autophosphorylated as a result of these activities. The finding that GST-2C has ATP diphosphohydrolase activity that can hydrolyse both β-γ and α-β bonds of ATP is interesting as PV and EV 9 2C proteins have been found to only cleave γ-phosphate of ATP (Klein et al., 1999, Pfister & Wimmer, 1999, Rodriguez & Carrasco, 1993). The picornavirus 2C proteins belong to the helicase superfamily 3 (SF3), the type member of which is Simian virus 40 large tumor antigen (SV40 Tag), a large protein essential for the expression and replication of the Simian virus genome as well as for the establishment and maintenance of cell transformation (Fanning, 1992, Fanning & Knippers, 1992). As direct evidence of helicase activity of the picornavirus 2C proteins is still lacking, one could speculate that ATP hydrolysis by 2C may also be important for generating energy that is used for conformational changes in proteins, in transport, sorting or packaging of viral RNA as well as in viral replication and the formation of the viral replication complex. Although the specific role of the HPEV1 2C AMP kinase activity in virus replication is yet unclear, one may speculate that it represents an adenosine-diphosphate-generating system and allows utilization of ADP for different processes, e.g. replication during the host cell stationary phase when NTP levels may be low. Interestingly, EMCV has been shown to exhibit a marked preference for nucleoside diphosphates over NTPs as substrates for viral RNA synthesis (Koonin & Agol, 1983, Koonin & Agol, 1984). EMCV could utilize NDP and NMP for the viral RNA synthesis, and NDP and NMP kinases appear to be specifically associated with viral replication complexes (Koonin &

Discussion

Agol, 1983, Koonin & Agol, 1984). The AMP-kinase activity of the HPEV1 2C protein may also be involved in modulation of cellular reactions and 2C may indirectly affect processes by changing the delicately balanced intracellular nucleotide levels. The significance of the autophosphorylation of the 2C protein is not yet clear. One might suggest that it could lead to a conformational change in the protein which could further regulate its function.

5. The role of the non-structural proteins in RC formation

Recently, new data has been obtained about the intracellular alterations induced by different picornaviruses as well as by individual picornaviral proteins (Knox et al., 2005, Krogerus et al., 2003, Monaghan et al., 2004). Among the picornaviruses, RNA replication complexes from enterovirus-infected HeLa cells have been the best studied. However the precise mechanism of replication complex formation remains elusive. When expressed in isolation, the enterovirus 2C and 2BC proteins have been shown to induce extensive rearrangements of intracellular membranes and vesicles resembling those seen during viral infection (Aldabe & Carrasco, 1995, Cho et al., 1994). The 2C and 2BC proteins of HAV have also been shown to cause membrane rearrangements (Teterina et al., 1997a), although the relationship of these to the formation of the viral replication complex is not clear. The aim of our study was to examine the possible involvement of the 2C protein, and other non-structural proteins, in replication complex formation and to study their individual localization and possible membrane modifications. None of the proteins studied was able to, alone or in combination, induce changes in the intracellular morphology similar to those seen in HPEV1-infected cells. Individually expressed HPEV1 2C and 2BC proteins were found to associate with lipid droplets but also with ER and Golgi membranes. However, no apparent rearrangements of intracellular membranes could be seen. This suggests that 2C alone is not sufficient to generate the membranous changes seen during HPEV1 infection, although the finding that transfected 2C can associate with viral RNA in superinfected cells suggests that the protein may interact with cellular and/or viral factors present in the replication complex. However, it has been

Discussion shown for other viruses that several or all of the replicative proteins might be needed for correct localization of the replicative machinery (Salonen et al., 2003). Recently, more information has been obtained on the nature of lipid droplets as active cellular organs with close ties to the ER (Martin & Parton, 2005, Tauchi-Sato et al., 2002). Interestingly, the HCV non-structural NS5A protein, has been shown to associate with lipid droplets as well as with Golgi and ER membranes when expressed in isolation (Shi et al., 2002). HCV has several common features when compared to picornaviruses and the NS5A protein is similar to the picornaviral 2C protein (Teterina et al., 2006). However, in cells containing HCV subgenomic replicons the protein is not associated with lipid droplets, but rather with a structure of modified membranes termed the “membranous web”, presumably derived from ER membranes (Egger et al., 2002b, Mottola et al., 2002). Another study has associated the protein with lipid rafts and caveolin-2, a protein which is found also on lipid droplets (Shi et al., 2003). In HPEV1- infected cells, the 2C protein colocalizes both with a trans-Golgi marker, and with modified ER membranes (Krogerus et al., 2003). The relationship of the 2C protein with lipid rafts has not been investigated. It is however tempting to speculate that during HPEV1 replication complex formation the 2C protein might interact with certain ER- proteins or a subregion of the ER, as well as with other viral components, to form a specialized membrane compartment. Our results indicate that similarly to other picornaviruses studied, HPEV1 inhibits cellular secretion. However, the individual protein responsible for this feature seems to be neither 3A, like in enteroviruses (Doedens et al., 1997), nor 2BC, like in FMDV (Moffat et al., 2005). At present we cannot rule out the possibility that other precursor proteins, unique to HPEV1, and/or the concomitant translation of the viral polyprotein, are responsible for inhibiting cellular secretion in HPEV1-infected cells. Despite overall similarities, different picornaviruses employ remarkably different mechanisms in replication complex formation. It seems also that the functions of individual proteins in the viral replication cycle and the induction of intracellular pathology cannot be directly derived from the properties of analogous proteins of other picornaviruses.

Summary and Concluding Remarks

SUMMARY AND CONCLUDING REMARKS The HPEVs belong to the picornavirus family of positive-stranded RNA viruses. Although the genome shares the general properties of other picornaviruses there are considerable differences and the genus has several unique features when compared to other picornaviruses.

We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. The results further indicated that the genome, which is to be translated and replicated, is released during the internalization events. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein, which was found to be a membrane binding protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein, which shows very little homology to other picornaviral 2A proteins, was found to be a RNA-binding protein that preferably bound to positive sense 3’UTR RNA. This binding was dependent on the native conformation of the protein and the protein was found to also bind duplex RNA containing 3’UTR(+)-3’UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region (aa 43-56) as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Other NTP:s and dATP were not suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. In an attempt to study the possible intracellular pathology brought about by the individual HPEV1 non-structural proteins, fusion proteins were constructed and expressed in cells alone or in combinations. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein which could be found mainly on the surface of lipid droplets but also on diverse intracellular membranes was partly relocated to viral replication complexes in transfected,

Summary and Concluding Remarks superinfected cells. The effect of the non-structural proteins on the anterograde membrane traffic was also studied and it was found that although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on membrane trafficking. Our results suggest that the HPEV1 replication strategy is somewhat different from that of many other picornaviruses, however, some similarities might be found with other recently studied members of the family. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Svenskt sammandrag

SVENSKT SAMMANDRAG Picornavirusen är en stor familj av små RNA-virus utan yttre membran. De kanske mest kända picornavirusen är poliovirusen, men till gruppen hör även rhinovirusen, som ger upphov till de flesta febersnuvor, enterovirusen som orsakar neurologiska, respiratoriska och gastrointestinala symptom samt hepatit A-virus. Bland familjemedlemmarna finns även djurpatogener. Human parechovirus 1 och 2 (HPEV1 och HPEV2) upptäcktes 1956. Redan vid den ursprungliga karakteriseringen uppmärksammades dock dessa virus ovanliga tillväxtegenskaper samt cytopatologi. Med hjälp av sekvensanalys har man senare kunnat visa att parechovirusen har flera unika molekylbiologiska egenskaper. HPEV1-infektioner är mycket vanliga, speciellt bland små barn, och ofta asymptomatiska, men de kan dock även orsaka ett flertal olika kliniska symptom, däribland gastroenterit, respiratoriska infektioner, infektioner av centrala nervsystemet och myokardit. HPEV1 har ett positiv-strängat RNA-genom som är 7100 nukleotider långt. RNAt kodar för ett ca 230 aminosyror långt polyprotein som bearbetas av virusets egna proteaser till mindre enheter. Slutprodukterna indelas i kapsidprotein (VP0, VP1 och VP3) och icke- kapsidprotein (2A, 2B, 2C, 3A, 3B, 3C, 3D). Sextio kopior av vart och ett av de tre kapsidproteinen bygger upp den ikosahedrala kapsid som innehåller virusets genom. Till icke-kapsidproteinen hör t.ex. enzym som är nödvändiga för virusets replikation. I mitt första delprojekt undersökte vi HPEV1:s interaktion med cellens yta. Vi fann att

HPEV1 binder vid αvβ3-integrin på cellens yta och internaliseras med hjälp av clathrin- medierad endocytos. I mitt andra delprojekt undersökte vi, med hjälp av främst elektronmikroskopiska tekniker, de förändringar som HPEV1 orsakar i den infekterade cellen. Med hjälp av RNA-sonder kunde vi lokalisera den plats i cellen där virusets genom replikeras. Våra studier visade att HPEV1:s replikationskomplex skiljer sig avsevärt från de replikationskomplex som kan iakttas vid infektion med andra picornavirus. Vid elektronmikroskopi av HPEV1-infekterade celler kunde vi se att dessa hade ett avvikande, dilaterat endoplasmatiskt retikulum samt en disintegrerad Golgiapparat. Vi fann att 2C-proteinet, som vid andra pikornavirusinfektioner är lokaliserat uteslutande till virusets replikationskomplex, i HPEV1-infekterade celler därutöver kan återfinnas i en

Svenskt sammandrag

struktur som inte deltar i virusets RNA syntes. Vi kunde även konstatera att HPEV1:s RNA syntetiseras på ytan av oregelbundna vesikler som verkar vara av Golgiursprung. I det tredje delprojektet undersökte vi 2A-proteinet. Parechovirusens 2A-kodande gensekvens skiljer sig markant från motsvarande gensekvens hos andra picornavirus. För att utreda 2A:s möjliga funktioner producerade vi antikroppar mot proteinet och lokaliserade det i den infekterade cellen med hjälp av immunofluorescens- och konfokalmikroskopi. Merparten av proteinet återfanns i cellens cytoplasma, men i senare skeden av infektionen även i cellkärnan. Med hjälp av olika biokemiska analyser utredde vi 2A-proteinets förmåga att binda till RNA. Vi fann att proteinet har största affinitet för den icke-kodande sekvensen i slutet på HPEV1:s RNA. Med hjälp av mutationsanalys kunde vi bestämma att proteinets amino-ända är nödvändig för protein-RNA-bindingen. I det fjärde delprojeketet undersökte vi 2C-proteinets förmåga att hydrolysera tri-fosfat- nukleotider. Vi fann att proteinet kunde hydrolysera ATP och ADP, men inte CTP eller GTP. Till vår förvåning fann vi att 2C proteinet även fungerade som ett monofosfat- kinas; i en energikrävande reaktion kunde proteinet tillverka ADP av AMP och fosfat. Därtill kunde vi konstatera att proteinet som en följd härav blev fosforylerat, något som eventuellt kan reglera proteinets funktion. I det femte delprojektet uttryckte vi de enskilda 2B-, 2BC-, 2C-, 3A- och 3AB-proteinen i cellodlingar och undersökte de förändringar proteinen orsakar i de intracellulära strukturerna. Vi fann att 2B-proteinet lokaliserades till det endoplasmatiska retiklet medan 2BC och 2C återfanns på ytan av lipidvesikler och 3A och 3AB i Golgiapparaten. Inget av proteinen var förmöget att i sig orsaka de förändringar som observeras i HPEV1- infekterade celler. Resultaten tyder på att HPEV1:s replikationsstrategi skiljer sig markant från replikationsstrategin hos andra studerade picornavirus samt att relativt små skillnader i virusens genom kan leda till stora skillnader i virusens intracellulära patologi.

Acknowledgements

ACKNOWLEDGEMENTS The work for my thesis was carried out at the Department of Virology at the Haartman Institute. I would like to thank the present Head of the Department, Professor Kalle Saksela as well as his predecessor Professor Emeritus Antti Vaheri, for providing a creative and enthusiastic environment for research. I want to express my gratitude to the supervisor of the thesis, Professor Timo Hyypiä, for taking me in as a young medical student and for always believing in the thesis project even when I had my doubts. The reviewers of my thesis, Professor Kristiina Mäkinen and Docent Maija Vihinen- Ranta, are thanked for constructive comments on the manuscript that greatly helped to improve it. I would like to thank Dr. Päivi Joki-Korpela for introducing me into the field and for being a patient teacher. Dr. Olga Samuilova is acknowledged for teaching me everything I know about biochemistry. Olya, your help has been invaluable! Prof. Kurt Bienz and Dr. Denise Bienz-Egger at the Institute of Medical Microbiology in Basel, Switzerland are acknowledged for showing me what science is really about. Thank you Kurt and Denise for making our stay in Basel so very pleasant and for your continuous support! Many thanks also to Rainer, René, Andrea and Claudia for making me feel so welcome in the lab. Dr. Tuija Pöyry is thanked for her patience with me during those first years in the lab and for always finding the time to help and answer questions. Dr. Eija Jokitalo is thanked for valuable help with EM preparations, extremely helpful discussion and for a positive attitude. Arja Strandell is acknowledged for her skilful technical assistance. I also want to thank other collaborators including Dr. Varpu Marjomäki, Prof. Jyrki Heino, Dr. Igor Fabrichniy and Prof. Glyn Stanway. I also wish to express my deepest gratitude to the “girls in the lab”: Anne, Annu, Hannamari, Maaria, Olya, Pia, Päivi, Vilja and Åse: Thanks to you research was fun! In addition, I would like to thank all the former members of our group, including Heli, Veera, Tommi, Juhana, Tiiu, Seija and Leena P. Maaria and Tiiu, thank you for helping me with the practical aspects of my work.

Acknowledgements

Finally, I want to thank all the people at the Haartman Institute for help and advice whenever needed. Special thanks go to Fang Zhao at the Department of Pathology for help with the electron microscope and the confocal microscope. Family, relatives and friends are acknowledged for all their love and support. Thank you mamma Leena and pappa Björn for everything. Thank you Samuel for always being there for me and for making my life so extremely enjoyable. This thesis was financially supported by the Academy of Finland, the Finnish Cultural Foundation, the Paulo Foundation, Virustautien tutkimussäätiö, the University of Helsinki, the European Molecular Biology Organization, the Sigrid Juselius Foundation, Victoria stiftelsen, Nylands nation and Finska läkaresällskapet.

Helsinki, February 2007

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