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Journal of Virology, August 2003, p. 8512-8523, Vol. 77, No. 15
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.15.8512-8523.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Replication Complex of Human Parechovirus 1

Camilla Krogerus,1,2* Denise Egger,2 Olga Samuilova,1 Timo Hyypiä,1,3 and Kurt Bienz2

Haartman Institute, Department of Virology, University of Helsinki, FIN-00014 Helsinki,1 Department of Microbiology, University of Oulu, FIN-90014 Oulu, Finland,3 Institute for Medical Microbiology, University of Basel, CH-4003 Basel, Switzerland2

Received 21 October 2002/ Accepted 6 May 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Parechoviruses belong to a recently established picornavirus genus (35) which contains two human pathogens, human parechovirus type 1 (HPEV-1) and HPEV-2, and Ljungan virus, which has been isolated from rodents. HPEV-1 infections are common and usually occur during the first years of life (46). The virus causes mostly gastrointestinal and respiratory symptoms, but it has also been associated with central nervous system infections. When the parechoviruses were first isolated, they were classified in the enterovirus genus as echoviruses 22 and 23. During their original characterization, exceptional growth properties, which were compared to those of enteroviruses, were observed. These included difficulties in adaptation of the viruses to cultures of monkey kidney cells and restriction of the cytopathic effect to peripheral parts of the cell monolayer (57).

Sequence analysis of HPEV-1 revealed that the virus was genetically distant from other picornaviruses, and assignment to an independent group of picornaviruses was suggested (20). Although the organization of the parechovirus genome is similar to that of other picornaviruses, HPEV-1 exhibits some distinct differences in molecular and biological properties (45, 47), such as lack of the maturation cleavage of the capsid protein precursor VP0 to VP2 and VP4 polypeptides (47), inability to cause host cell shut-off (12), and resistance to guanidine hydrochloride (49). As shown for poliovirus (PV), the target of guanidine is the nonstructural protein 2C (34), which is a key protein in the formation and function of the PV replication complex (2, 8, 14, 33, 34, 50).

PV protein 2C, an ATPase (33), and particularly its precursor 2BC have been suggested to be involved in the process of virus-specific membrane alterations (1, 11, 15). They were found to be indispensable for PV replication (33, 34, 50, 51, 53); however, their precise function in viral replication remains elusive.

All positive-stranded RNA viruses studied so far replicate their RNA in membrane-bound replication complexes. They modify the intracellular membranes of their host cells to create a compartment where the replication machinery can assemble. However, clear differences in the utilization of different membrane types and in the morphology of the replication complexes can be found between viral families and even within the families. Reorganized cellular structures include the endoplasmic reticulum (ER) (10, 36, 37, 40, 41, 48, 54), the early and late endosomal lysosomal system (23), the mitochondrial membrane (30), and the Golgi apparatus (28). Although it has been shown that the generation of membrane alterations depends on viral proteins, little is known about the mechanisms by which intracellular membranes are converted and about factors which determine the membrane(s) targeted and the specific modifications induced.

The membranous replication complex of PV, the prototype member of the picornavirus family, has been extensively studied. Membranous vesicles form on the ER in the presence of the COPII complex (40) and assemble into higher-order structures in cis (15). Eventually these vesicles have consumed most intracellular membranes (42) and fill large parts of the cytoplasm (5, 6). Viral RNA and most viral proteins are associated with the surface of the poliovirus vesicles, which provide a structural scaffold for rapid and efficient RNA replication.

The differences between the sequences of the noncapsid proteins of parecho- and enteroviruses (17, 20) and previously reported findings concerning the peculiar cytopathic effect of HPEV-1 (21, 52, 57) prompted us to investigate the ultrastructural changes which accompany the formation of the viral replication complex in HPEV-1-infected cells and to determine the site of viral RNA synthesis. We show that virtually all HPEV-1 replication complexes consisted of membranes carrying a trans-Golgi marker and protein 2C. Protein 2C, which we show to have membrane binding properties, was found to associate additionally with elongated, branching membranous structures which, despite their morphology, were devoid of components of the cytoskeleton. Immunoblots of HPEV-1-infected cells showed that the 2BC precursor protein found after enterovirus infection may not be present in HPEV-1-infected cells. The HPEV-1-induced membrane alterations were more subtle than those seen in enterovirus-infected cells and they did not involve the entire endomembrane system of the host cell. The findings suggest a mechanism for the replication complex formation that is different from that described for PV. The unexpected observation that protein 2C also located to membranes seemingly not involved in viral RNA replication may indicate that HPEV-1 2C has additional functions during the replication cycle compared to 2C of enteroviruses.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and virus. A549 cells (human lung carcinoma; American Type Tissue Collection) and HeLa cells were infected with HPEV-1 (Harris strain) by adsorption at 37°C for 1 h at a multiplicity of infection of 5 to 15. The virus stock was obtained by transfection of the cells with the infectious genomic RNA obtained by transcription from clone pHPEV1 (kindly provided by G. Stanway, University of Essex, Colchester, United Kingdom).

Metabolic labeling of viral RNA. To determine the kinetics of viral RNA replication, A549 cells infected with HPEV-1 were treated with actinomycin D (AMD) (final concentration, 5 µg/ml) to block cellular RNA synthesis 30 min before the cells were pulse labeled for 10 min at the indicated times postinfection with [5,6-3H]uridine (Amersham) (final concentration, 30 µCi/ml). The RNA was acid precipitated, and radioactivity was quantified by liquid scintillation counting.

To label nascent viral RNA for in situ detection, HPEV-1-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) (final concentration, 10.5 mM) in the presence of AMD. The cells were fixed as described below, and bromo-RNA (Br-RNA) was detected by indirect immunofluorescence (IF).

Antibodies (Abs). The polyclonal rabbit antiserum against 2C was produced by immunization of rabbits with glutathione-S-transferase-2C. Plasmid pHPEV1, containing the full-length genomic cDNA clone of HPEV-1, was used as a template for cloning 2C into the pGEX4T-1 vector (Pharmacia). Restriction sites (underlined) were used in the oligonucleotide primers 5'-CACTAGAATTCGGACCTTTTAAAGGATTCAAT-3' and 5'-GCACGTCGACTTACTGATTTTCCAATTGTTGTTT-3' for the cloning of the PCR products into the vector. Protein expression in the Escherichia coli strain BL21(DE3), transformed with 2CpGEX4T-1, was induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside, and the glutathione-S-transferase-2C fusion protein was purified under native conditions on glutathione Sepharose 4B according to the manufacturer's protocol (Pharmacia).

To visualize the Golgi apparatus, anti-giantin mouse monoclonal Abs (MAbs), kindly provided by H.-P. Hauri (26), anti-GM130 MAbs (Transduction Laboratories), and anti-1,4-galactosyltransferase (anti-GalT) rabbit Abs, obtained from E. Berger (56), were used. Cytoskeletal material was detected with mouse anti-tubulin MAbs (Chemicon), mouse anti-vimentin MAbs (Amersham) and phalloidin coupled to Alexa 488 (Molecular Probes). A MAb against the transmembrane protein BAP31, kindly provided by E. Kuismanen (27), was used to identify the membrane-containing fractions in the flotation assay. Bromodeoxyuridine (BrdU)-labeled nascent viral RNA was visualized by an anti-BrdU mouse MAb (Biocell Consult).

The following secondary Abs were used: goat anti-rabbit and anti-mouse Texas-Red-coupled Abs (Molecular Probes), goat anti-rabbit fluorescein isothiocyanate (FITC)-coupled Abs (Jackson Laboratories) and goat anti-mouse Alexa 488-coupled Abs (Molecular Probes), and on Western blots, swine anti-rabbit immunoglobulin G (IgG) and rabbit anti-mouse IgG, both conjugated to horseradish peroxidase (DAKO). To detect viral RNA on electron microscopic sections, goat anti-digoxigenin (DIG) Abs coupled to 10-nm-diameter gold particles (Aurion) were applied. To detect viral protein, 1.4-nm-diameter gold particle-conjugated Fab' fragments, termed Nanogold (Nanoprobes), against rabbit IgG were used.

In vitro transcription-translation and immunoprecipitation. To confirm that the anti-2C Ab produced recognized both proteins (2C as well as the precursor 2BC), 2C and 2BC were translated in vitro and subsequently immunoprecipitated with the 2C Ab. A hemagglutinin tag and restriction sites were added to the pC1-neo vector (Promega) by digestion of the plasmid with NheI and NotI and ligation of the oligonucleotide 5'-CTAGCATGGCTTACCCATACGATGTTCCAGATTACGCTGAATTCCCGGGTCTAGACGCGTCGACTCGAGC-3' (sense). Proteins 2C and 2BC were cloned in frame with the N-terminal hemagglutinin tag by digestion of a parent clone and the modified pc1-neo vector with EcoRI and XhoI. In vitro transcription-translation was performed 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 proteins 2C and 2BC were added and again incubated overnight. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis on 12% gels. Radioactive proteins were visualized by exposing the gels to X-ray film (Kodak).

IF and fluorescent in situ hybridization (FISH). Cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.2 to 0.3% Triton X-100 (Sigma) as described previously (9). Indirect IF was performed, and the cells were mounted in glycerol containing 1% N-propyl gallate (Sigma). For the detection of viral genomic RNA, a riboprobe of minus polarity, covering the entire viral sequence, was prepared from plasmid pHPEV1 by transcription with T3 RNA polymerase in the presence of FITC-conjugated UTP (Roche). The probe was subjected to alkaline hydrolysis to generate fragments of approximately 100 nt in length and hybridized to the cells at 42°C overnight as described previously (9, 13). The specimens were mounted in glycerol containing 2.5% (wt/vol) DABCO (Sigma) as an antifade reagent.

Conventional light microscopy was performed with a Nikon E800 microscope, and confocal microscopy was done with a confocal laser scanning microscope (TCS4D; Leica Lasertechnik). For colocalization, pictures were recorded sequentially. Pictures were corrected for contrast and intensity with Adobe Photoshop software.

EM, EM-ISH, and immuno-EM (IEM). For conventional electron microscopy (EM), cells were trypsinized, centrifuged, fixed with 2.5% glutaraldehyde and 2% osmium, and embedded in Poly/Bed 812 (Polysciences, Warrington, Pa.) 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., London, United Kingdom) as described previously (5). A DIG (Roche) UTP-labeled riboprobe was prepared, hydrolyzed as described above (4), and visualized by indirect immunogold labeling with a DIG Ab. The sections for conventional EM were poststained with Reynolds lead citrate, and the LRGold sections were poststained with 4% uranyl acetate and Millonig's lead hydroxide stain. The preparations were viewed in a Philips CM100 EM.

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. 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, cells were dehydrated in an alcohol series and processed for Epon embedding as described previously (44). Sections were poststained with uranyl acetate and lead citrate and examined with a Jeol JEM-1200EX II EM.

Flotation assay. HPEV-1-infected and uninfected A549 cells were suspended in 0.1 M HEPES (pH 7.4) containing 10 mM MgCl and protease inhibitors (Sigma). Cells were homogenized with a Dounce homogenizer, and a postnuclear supernatant (PNS) was obtained by centrifugation at 1,000 x g for 5 min. The PNS was divided into two equal samples; one was treated with 1% Triton X-100 for 30 min on ice and the other half 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-milliliter fractions were collected from the mixture described above and trichloroacetic acid precipitated. This assay was modified from the procedure described in reference 31.

Western blotting. PNS of infected and uninfected cells as well as flotation samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% gels and electrotransferred to a nitrocellulose membrane (Schleicher and Schuell). Protein 2C and BAP31 were visualized by indirect immunodetection with secondary Abs conjugated to horseradish peroxidase. The filters were developed with an enhanced chemiluminescence detection system (Amersham).

Sequence alignment and comparison. For sequence comparison, the HPEV-1 2C protein (20) was aligned with PV1 2C (22) and hepatitis A virus (HAV) 2C (24). The Wisconsin Package, version 10.2 (Genetics Computer Group, Madison, Wis.), was used for sequence entry and analysis, and the phylogenetic tree was calculated by using PHYLIP with 500 bootstrap replicates.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Synthesis of viral macromolecules in HPEV-1-infected cells. The time course of HPEV-1 viral RNA replication was established by liquid scintillation counting (Fig. 1A). Starting at 4 h postinfection (p.i.), viral RNA synthesis increased exponentially and peaked at 6 h p.i. The kinetics of appearance of the proteins of the P2 genomic region was studied by Western blotting with Ab against protein 2C. At 4 h p.i., a single band of 35 kDa was detected, which is the size of protein 2C. Its intensity increased up to 6 h p.i. (Fig. 1B). Unexpectedly, no band corresponding to protein 2BC was found. Therefore, we tested whether the anti-2C Ab used would recognize, via its 2C moiety, protein 2BC, if present. This was the case (Fig. 1C), as the 2C Ab immunoprecipitated in vitro-translated protein 2BC. The findings suggest that protein 2BC is not a stable precursor in HPEV-1-infected cells.



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  		FIG. 1. Characterization of viral macromolecules in HPEV-1-infected cells. (A) Kinetics of viral RNA synthesis in infected cells determined by [3H]uridine labeling of nascent viral RNA. DPM, disintegrations per minute. (B) Immunoblots of mock-infected (lane 1) and HPEV-1-infected (lanes 2 to 4) cells (harvested at 4, 6, and 8 h p.i., respectively) were probed with Ab against protein 2C. Protein 2C accumulated in the absence of the precursor protein 2BC. (C) In vitro-translated proteins 2C (lane 1) and 2BC (lane 2) were immunoprecipitated with 2C antiserum and electrophoresed. The autoradiograph of the blot indicates that the 2C Ab was capable of recognizing 2C as well as the precursor protein 2BC.

 
The intracellular localization of viral macromolecules was assayed in parallel with Abs against protein 2C and with an FITC-labeled riboprobe to detect plus-strand viral RNA (Fig. 2). Early in the infectious cycle, viral RNA (Fig. 2a) and the protein 2C (Fig. 2e) were found in numerous small granules in the cytoplasm. At peak RNA synthesis, the pattern of protein 2C was changed drastically into dot- and stick-like formations, encircling the nucleus or accumulating on one side of the cell (Fig. 2f). Viral RNA was mainly found in few large granules and, in some cells, also diffusely in the cytoplasm (Fig. 2b). Protein 2C still presented in long stick-like structures at 8 h p.i., and finally as larger irregular bodies at 10 h p.i. (Fig. 2g and h). The localization of viral RNA was essentially unchanged at 8 h p.i. (Fig. 2c) but became additionally diffuse at 10 h p.i. (Fig. 2d). Thus, the 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 harboring viral RNA (see Fig. 9).



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  		FIG. 2. Location of viral RNA and protein 2C in the infected cell. HPEV-1-infected A549 cells, harvested at 4 (a and e), 6 (b and f), 8 (c and g) and 10 (d and h) h p.i., were subjected to FISH with an FITC-coupled HPEV plus-strand specific riboprobe (a to d) or to IF with Ab against protein 2C (e to h). Bar, 25 µm.

 


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  		FIG. 9. Colocalization of nascent viral RNA with protein 2C or the trans-Golgi marker GalT in HPEV-1-infected, Br-UTP-transfected A549 cells at 6 h p.i. (a) Br-RNA (green) detected with an Ab against Br-dUTP and Alexa 488-conjugated secondary Ab. (b) Protein 2C (red) detected with an Ab against 2C and Texas Red-conjugated secondary Ab. (c) Merge of panels a and b. (d) Br-RNA (red) detected with an Ab against Br-dUTP and Texas Red-conjugated secondary Ab. (e) GalT (green) visualized with an anti-GalT Ab and FITC-conjugated secondary Ab. (f) Merge of panels d and e. Picture sizes, 25 by 25 µm.

 
Analysis of HPEV-1 protein 2C sequence. The unexpected staining, cleavage pattern, and intracellular location of HPEV-1 2C protein prompted us to compare its sequence with those of the 2C proteins of other picornaviruses (Fig. 3A). PV1 and HAV where chosen for sequence comparison as two distinct, extensively studied picornaviruses (Fig. 3B). The overall identity of the 2C protein of HPEV-1 with that of PV was 32%, and its identity with that of HAV was 28%. In Fig. 3B, the A and B nucleoside triphosphate (NTP)-binding motifs, originally described by Walker et al. (55), are indicated, as is the C motif characterized by Gorbalenya et al. (18). The cysteine-rich region (amino acids [aa] 269 to 286 in PV1), which is highly conserved in entero- and rhinoviruses (32), could not be found in the HPEV-1 or HAV 2C sequences. The asparagine at aa 179 in the PV1 2C sequence, the mutation of which renders PV insensitive to guanidine hydrochloride (34), was found to correspond to a glycine in both the HPEV-1 and HAV sequences. These findings suggest that all three proteins share NTP-binding properties and that, due to differences in sequence, there could be differences in other functions of the proteins.



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  		FIG. 3. Sequence comparison of human picornavirus 2C protein. (A) Phylogenetic tree of human picornaviruses based on the 2C genes. HRV, human rhinovirus; CAV, coxsackie A virus; CBV, coxsackie B virus. (B) Sequence alignment of the 2C proteins of HPEV-1, PV, and HAV. The three ATPase motifs are boxed, and 179N in the PV sequence and the corresponding glycine residues in the HPEV-1 and HAV sequences as well as 187M in the PV sequence and its corresponding amino acids in the HPEV-1 and HAV sequences, are highlighted. The cysteine-rich region in the PV sequence is underlined.

 
Ultrastructural aspect of HPEV-1-infected cells. To study the structural changes caused by HPEV-1 infection, ultrathin sections of infected A549 cells were prepared at 6 h p.i., i.e., at the peak of viral RNA synthesis, and analyzed by EM. Infected cells, compared to uninfected cells (Fig. 4a), were easily recognized due to the presence of defined regions with alterations in the cellular architecture (Fig. 4b). Such regions either surrounded the nucleus or occupied a circular area on one side of the nucleus. The most prominent feature of these transformed areas was a dilated ER and perinuclear space (Fig. 4d). The dilated ER membranes had also lost most of their ribosomes and appeared as shorter pieces. Identification of the ER membranes was based on their apparent connection to the perinuclear space, the occasional presence of membrane-bound ribosomes, and the lack of a normal, well-organized ER in their vicinity. Additionally, cytoskeletal material was often seen in close proximity to the dilated ER membranes. Another feature distinguishing infected from uninfected cells was the presence of clusters of small vesicles (Fig. 4c). Modified ER membranes could often be found close to these vesicles. The vesicles were 30 to 60 nm in diameter and were found to aggregate both in larger clusters and in small groups. Larger aggregates were found mainly in the perinuclear area, whereas small groups of vesicles could be found more towards the periphery of the cytoplasm.



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  		FIG. 4. Electron micrographs of A549 cells demonstrate structural changes seen 6 h after HPEV-1 infection. (a) Uninfected cell; (b) infected cell, dashed line delineates region with alterations in the cellular architecture; (c and d) higher magnification shows the main features of cell alterations, including the appearance of clusters of small vesicles (V) (c), dilated ER (arrowheads), and rough ER (rER) (d). N, nucleus. Bars, 0.5 µm.

 
Classical Golgi stacks were absent in infected cells. Double IF with Abs against protein 2C as a marker for infection and the cis-Golgi marker proteins GM130 (Fig. 5a) and giantin (not shown) as well as the trans-Golgi marker GalT (see Fig. 9) confirmed the disintegration of the Golgi apparatus in infected cells.



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  		FIG. 5. Location of protein 2C visualized by confocal scanning laser microscopy. (a) Double labeling of infected cell for protein 2C and Golgi protein GM130 shows the disintegration of the Golgi apparatus in the infected cell. Protein 2C was visualized with Texas Red-conjugated secondary Ab (red), and GM130 was visualized with Alexa 488-conjugated secondary Ab (green). 1, uninfected cell with intact Golgi; 2, HPEV-infected cell with dispersed Golgi complex. (b to f) Optical sectioning through an HPEV-1-infected cell 6 h p.i., stained with Ab against protein 2C. The distance between two consecutive sections is 0.5 µm. Protein 2C is located on branching, stick-like structures. (g to i) Double labeling of infected cells with Abs against protein 2C (red) and the cytoskeleton markers (green) tubulin (g) and vimentin (h) and with Alexa 488-coupled phalloidin that labels actin filaments (i). 1, uninfected cell with intact actin filaments; 2, HPEV-infected cell with depolymerized actin filaments. Picture sizes are as follows: a, 44 by 44 µm; b to f, 25 by 25 µm; g, 19 by 19 µm; h, 30 by 30 µm; i, 50 by 50 µm.

 
Association of protein 2C with membranes. The structures containing protein 2C were analyzed by subjecting IF preparations to optical sectioning with a confocal microscope. The labeled structures were found to consist of long branching sticks that formed a coherent network rather than individual elongated structures (Fig. 5b to f). The branching sticks were also obvious in HeLa cells infected with HPEV-1, indicating that they were not peculiar for A549 cells only (data not shown).

To test whether cytoskeletal elements formed the elongated stick-like structures associated with protein 2C, infected cells were double labeled for 2C and either tubulin, vimentin, or actin. None of the cytoskeletal elements formed a pattern similar to the stick-like network of 2C, and no colocalization between 2C and cytoskeleton was observed (Fig. 5g to i). However, a variable disorganization of the cytoskeletal elements was observed in infected cells, which was most obvious for actin (compare not infected to infected cells in Fig. 5i), intermediate for vimentin (Fig. 5h), and least pronounced for tubulin (Fig. 5g).

To identify the intricate protein 2C-labeled structures, we performed an immunogold IEM analysis. The immunocytochemical signal was found predominantly on areas of the cell corresponding to altered regions such as that shown in Fig. 4b. The 2C label was found in longer and shorter narrow elongated structures (Fig. 6a), compatible with the stick-like structures in Fig. 5b to f. Higher magnification showed that the label was accumulated on stretches of membranes (Fig. 6b). Protein 2C-specific immunogold labeling was also found on clusters of vesicles (Fig. 6c) compatible with the vesicles found by conventional EM (Fig. 4c).



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  		FIG. 6. Localization of protein 2C in infected cells by IEM at 6 h p.i. (a) Overview of an infected cell showing longer and shorter narrow elongated labeled structures (arrowheads). At higher magnification, gold grains are found on stretches of membranes (arrowheads) (b) and on clusters of vesicles (arrowheads) (c). N, nucleus. Bars, 0.5 µm.

 
To confirm that 2C has membrane-binding properties, homogenized infected cells were analyzed by flotation assay. Figure 7A shows that 2C was found in the membrane-containing fractions, together with the integral membrane protein BAP31. Treatment with 1% Triton abolished the flotation of protein 2C (Fig. 7B).



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  		FIG. 7. Membrane association of protein 2C determined by flotation assay and immunoblotting. (A) PNS of uninfected (I) and infected (II) cells 6 h p.i., before flotation. Lanes 1 to 12 (only even-numbered lanes are numbered), fractions of the sucrose gradient used for the flotation assay of untreated, infected cells. The membrane-binding proteins BAP31 and 2C are found on top of the gradient. (B) Flotation of Triton X-100-treated PNS. Proteins BAP31 and 2C remained at the bottom of the gradient. Lanes are labeled as for panel A.

 
Subcellular location of viral RNA and identification of the viral RNA replication complex. For EM localization of viral genomic RNA, a DIG-labeled strand-specific RNA probe was hybridized to sections of cells harvested at peak RNA synthesis. The hybridized probe, visualized with anti-DIG Abs conjugated to 10-nm-diameter gold particles, localized to membranes of small vesicles (Fig. 8). The RNA-carrying vesicles could be found in small groups distributed through the cytoplasm and in larger clusters in the perinuclear region (Fig. 8, inset). The labeled structures containing genomic RNA (Fig. 8) and protein 2C (Fig. 6c) are compatible with the granules seen in infected cells subjected to FISH (compare Fig. 2b and 8) and to the aggregates of small vesicles shown in Fig. 4c.



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  		FIG. 8. Association of HPEV-1 RNA with small vesicles (arrowheads), shown by EM-ISH with a DIG-labeled riboprobe immunodetected with an anti-DIG Ab and 10-nm-diameter gold-conjugated secondary Ab. The inset shows an example of a cluster of vesicles. Bars, 0.2 µm.

 
To test whether the vesicular structures, found to harbor protein 2C and viral RNA, were involved in replication of viral RNA and thus represented the viral replication complex, nascent viral RNA was labeled with Br-UTP. Br-RNA, detected by indirect IF with an anti-BrdU AB, was found as punctate fluorescent signals (Fig. 9a) and was similar to the FISH pattern but lacking the diffuse spread-out component of the FISH signal. To investigate whether the structures involved in viral replication carry the protein 2C, a marker for the replication complex in other picornaviruses, Br-UTP-transfected cells were also stained with Ab against protein 2C (Fig. 9b). Merging the two confocal images of Fig. 9a and b revealed that nascent viral RNA colocalized with protein 2C in discrete small dots (Fig. 9c). However, a large excess of the 2C-labeled components, predominantly the stick-like structures, did not carry BrdU signal. The number of BrdU-positive structures, which were thus replicating viral RNA-containing structures, was much lower than that of the 2C-containing structures. This is compatible with the findings presented in Fig. 1.

Since viral RNA, detected by EM-ISH, located to small vesicles resembling in size and aspect vesicles of a dispersed Golgi complex (Fig. 8 and 4c), a possible involvement of the Golgi apparatus in the viral replication complex was investigated. HPEV-1-infected, Br-UTP-transfected cells were double labeled with Ab against the trans-Golgi marker GalT and BrdU. Confocal microscopy showed that all of the Br-RNA colocalized with protein GalT (Fig. 9d to f), suggesting that the HPEV-1 replication complex is a Golgi-derived structure.

In conclusion, the observed labeling patterns suggest that the largest part of the 2C-carrying structures, mostly the stick-like structures, are not involved in viral RNA replication and do not carry viral RNA. Conversely, the viral replication complex, defined by BrdU labeling, consists of vesicle clusters positive for 2C and a trans-Golgi marker.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The replication of positive-stranded RNA viruses, including the picornaviruses, in eukaryotic cells proceeds in connection with cytoplasmic membranes. The viruses utilize different membranes which serve as matrices for the association of their replication complexes. In the present work, we found that the HPEV-1-infected cells presented with defined regions with distinct alterations in the cellular architecture. The major changes consisted of a dilated ER, stripped of its ribosomes, and a disintegrated Golgi apparatus. We were able to identify the HPEV-1 replication complex by locating the nascent BrdU-labeled viral RNA. The replicating viral RNA localized to vesicular structures which carried the nonstructural viral protein 2C and a trans-Golgi marker.

For PV, the replication complex is formed by vesicles proposed to arise on the ER by the use of the COPII machinery (40). The nonstructural protein 2BC has been implicated in the formation of these vesicles (7, 11), possibly by mimicking one of the proteins in the COPII-complex and/or attracting components of the COPII coat (40). The observed lack of the precursor protein 2BC in HPEV-infected cells might thus well contribute to the observed difference in the type, and consequently the morphology, of the membranes used for building up the HPEV replication complex.

We show here that HPEV-1 replicates its RNA on structures that contain the Golgi protein GalT as well as the viral protein 2C, suggesting that protein 2C is involved in RNA replication. In line with a recent suggestion (16), the Golgi-related vesicles of the HPEV replication complex might be derived from vesicles of the retrograde membrane traffic moving from the Golgi towards the ER, since vesicles carrying viral RNA were found intermingled with modified ER membranes and an intact Golgi apparatus was missing. The observed disintegration of the Golgi and the resulting limited amount of vesicles could, however, also be explained by interference of the virus with the anterograde membrane traffic. Whether this interference could be mediated by 2C, present in large amounts on the ER (Fig. 6), remains speculative. Alternatively, it was recently found that Golgi proteins, particularly Golgi enzymes, recycle through the ER and that the ER thus may contain GalT (29, 43). This could mean that the GalT-positive vesicles of the HPEV replication complex derive directly from the ER. However, the observations that recycling takes place predominantly during regeneration of Golgi fields and proteins reemerging from the ER are found in the form of Golgi stacks rather than vesicles (43) argue against this pathway.

The RNA replication of other positive-strand RNA viruses, i.e., members of the flaviviruses, has also been reported to take place on trans-Golgi-derived vesicles which have been proposed to be part of the retrograde transport pathway (28). These vesicles also contain GalT and can be found in close proximity to the rough ER. However, the flavivirus-infected cells present with dispersed trans- but intact cis- and medial-Golgi compartments, whereas with HPEV-1, the entire Golgi is dispersed early in infection.

For PV, the viral nonstructural proteins have been found associated with the replication complex and to be directly or indirectly involved in viral RNA synthesis. Protein 2C is exclusively found on the PV replication complex (6, 14); however, its precise biochemical role in RNA replication still remains uncertain. It is an ATPase (33) with RNA binding properties (3, 38, 39), and it has been suggested to be involved in membrane alterations (1, 11, 15) as well as in decapsidation and encapsidation (25, 53).

In contrast to PV protein 2C, large amounts of HPEV-1 2C were additionally found associated with structures which did not seem to be directly involved in RNA replication because they did not carry nascent viral RNA. The function of this second type of 2C-labeled structures is unclear. EM immunocytochemical findings suggest that these 2C-containing structures are compatible with ER or ER-derived membranes. Their unusual oblong shape in IF preparations was highly suggestive for cytoskeleton-derived structures. However, a relation to the three main components of the cytoskeleton, actin, vimentin, and tubulin, was disproven by the IF results obtained with the corresponding Abs.

The ATPase activity of PV 2C has been reported to be sensitive to RNA (33). If this is applicable to HPEV-1 2C, it might indicate that the 2C moiety present on the replication complex would have its ATPase activity switched off. As estimated from our IF data, the vast majority of protein 2C, present on ER-derived membranes lacking viral RNA, would represent an (active) ATPase, not involved in RNA replication. Thus, it could be the structural environment which regulates one of the presumably several functions of protein 2C.

When we compared the 2C sequence of HPEV-1 with those of PV and HAV, we found that the three sequences showed a rather low degree of homology (32 and 28%, respectively). Conserved amino acids could be found mainly in the middle third of the proteins. Corresponding residues have also been found in the rhino 14, rhino 2, encephalomyocarditis, and foot-and-mouth disease virus 2C sequences as well as in the protein corresponding to 2C in cowpea mosaic virus (34). The sequences of HPEV-1, HAV, and PV all contained the proposed NTP-binding sites, motifs A, B, and C (18, 55). The cysteine-rich region of PV 2C (aa 269 to 286), shown to bind zinc in vitro and to be involved in RNA replication (32), is highly conserved in entero- and rhinoviruses but could not be found in either the HAV or HPEV-1 sequence. Whether the absence of the zinc finger in protein 2C or the instability of the 2C precursor 2BC in HAV and HPEV is a reason for the differences seen in replication complex formation in HAV- (19) and HPEV-1-infected cells (this study) compared to PV remains to be established.

The highly guanidine-resistant mutants of PV contain, in protein 2C, either mutation from N to A or G at aa 179 or mutation from M to L at aa 187 (51). These two mutations can occur in combination with other mutations to render the virus guanidine dependent but have not been found combined together. The asparagine at position 179 in the PV 2C sequence corresponds to a glycine in both HPEV-1 and HAV 2C, whereas the amino acid corresponding to PV 2C methionine187 is an isoleucine in HPEV-1 and a tryptophan in HAV. Whether these differences, in particular the glycine corresponding to the asparagine 179 in PV, explain the variation in guanidine sensitivity between the three viruses remains to be established, e.g., by experimentally mutating the two major sites of guanidine resistance and perhaps also other critical amino acids. HPEV-1 is naturally resistant to guanidine (49), which precludes the use of this inhibitor in characterizing HPEV-1 2C functions. Since guanidine abolishes the ATPase activity of wild-type PV 2C but not of the guanidine-resistant mutant N179G (33), HPEV-1 might well contain a functional ATPase.

In conclusion, the intracellular changes of the HPEV-1-infected cells are less pronounced and clearly different from the ones seen in PV-infected cells (6). The formation of the building blocks of the replication complex, the vesicles, is different for PV and HPEV, as are their donor membranes. The finding that HPEV-1 2C protein additionally associates with structures without apparent function in replication suggests that this protein may have diverse functions during the viral multiplication cycle. Significant variations in sequence exist for certain polypeptides of picornaviruses, leading to remarkable differences in the formation of the replication complex and in effects on the host cell. Whether such differences are reflected in divergent strategies of RNA replication per se remains to be investigated


   ACKNOWLEDGMENTS
 
The study was supported by the Academy of Finland, the Sigrid Juselius Foundation, INTAS project 011-2012, an EMBO short-term fellowship to C.K., and the Swiss National Science Foundation (grant 31-055397.98 to K.B.).

We thank E. Berger, H.-P. Hauri, and E. Kuismanen for Abs, T. Pöyry for supplying the hemagglutinin-tagged 2C and 2BC clones, and G. Stanway for supplying the pHPEV-1 cDNA clone. E. Jokitalo and A. Strandell at the Institute of Biotechnology, Helsinki, Finland, are acknowledged for help with immunoelectron microscopy.


   FOOTNOTES
 
* Corresponding author. Mailing address: Haartman Institute, Department of Virology, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. Phone: 358-9-19126608. Fax: 358-9-19126491. E-mail: camilla.krogerus{at}helsinki.fi. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, August 2003, p. 8512-8523, Vol. 77, No. 15
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.15.8512-8523.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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