1 Department of Molecular Immunology, School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
2 Department of Clinical and Biological Sciences, University of Insubria, Varese, Italy
3 Laboratory for Behavioural Genetics, Brain Science Institute, RIKEN, Saitama, Japan
Correspondence
Takashi Onodera
aonoder{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In some reports, PrP has been shown to interact with sulfated glycans (Caughey et al., 1994), RNA aptamers (Weiss et al., 1997
) and large nucleic acids (Akowitz et al., 1994
; Nandi & Leclerc, 1999
), causing the formation of nucleoprotein complexes similar to HIV-1 nucleocapsidRNA complexes formed in vitro (Darlix et al., 1995
). A recent report shows that murine leukaemia virus replication accelerates the infectious process of scrapie (Carp et al., 1999
), suggesting possible in vivo interactions between viruses and PrP. These findings prompted us to analyse the effects of PrP expression on virus replication in CNS cells.
Group B coxsackieviruses (CVBs) are important human pathogens in the family Picornaviridae. Most CVB infections are asymptomatic, but different organs may be affected (e.g. myocardium, pancreas, CNS). Some acute and persistent infections are especially severe in infants (Woodruff, 1980). Despite the accumulation of virological and molecular data, the mechanisms of acute and chronic tissue damage induced by CVBs are not well understood.
Two observations prompted us to investigate the replication of coxsackievirus B3 (CVB3) in CNS cells: (i) CVBs are known to have affinity for newborn tissues and may cause encephalomyelitis in infants (Woodruff, 1980); and (ii) PrPC displays RNA-binding and chaperoning properties that may influence virus replication and assembly (Gabus et al., 2001
). Since CVB3 replication occurred more rapidly in primary cell cultures derived from prion protein gene (Prnp)-deficient (Prnp-/-) mice than from Prnp+/+ animals, we investigated whether PrPC expression would influence the susceptibility to CVB3 infection. Here we report that an established Prnp-/- hippocampal cell line is more susceptible to CVB3 infection than two human cell lines (HeLa and HEp-2) commonly used for the detection and titration of these agents. Furthermore, the role of PrPC in CVB3 resistance was studied following the reintroduction of Prnp into a Prnp-/- cell line.
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METHODS |
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Primary cultures.
Primary cultures were prepared from the brain of 3-day-old C57BL/6 Prnp+/+ and Prnp-/- mice (Kuwahara et al., 1999). Cells were isolated by triturating tissue pieces with a Pasteur pipette 1020 times in 1 ml PBS without Ca2+ and Mg2+ (Nissui) supplemented with 1·0 mM sodium pyruvate and 10 mM HEPES (pH 7·4). Cell suspensions were then digested in 0·5 % trypsin (Gibco-BRL); after settling for 3 min, the supernatant was centrifuged for 1 min at 200 g. Culture dishes were coated with cold poly-L-lysine (0·05 mg ml-1; Sigma), incubated for 1 h at room temperature and washed with sterile water. Cells were seeded in coated dishes at 2x105 cells cm-2 with Neurobasal medium (Gibco-BRL) supplemented with L-glutamine (0·5 mM), glutamate (25 µM) and B27 supplement (Gibco-BRL). Cultures were incubated at 37 °C in an atmosphere containing 5 % CO2.
Cell lines.
As reported previously (Kuwahara et al., 1999), the hippocampus cell line HpL3-4 was established from a Prnp-/- mouse on embryonic day 14. HpL3-4, HeLa and HEp-2 cells were grown at 37 °C with 5 % CO2 in DMEM supplemented with 2 mM glutamine and 10 % HI-FCS.
Virus infection of HpL3-4, HeLa and HEp-2 cell lines.
HpL3-4, HeLa and HEp-2 cells were cultured in six-well plates and infected with CVB3 at an m.o.i. of 5. Cultures were observed by phase-contrast microscopy in order to monitor the development of the cytopathic effect (CPE).
Sensitivity of HpL3-4, HeLa and HEp-2 cells to CVB3-induced CPE.
The sensitivity to CVB3-induced CPE in three different cell lines was evaluated by measuring the virus titre on HeLa cell monolayers using a microtitre assay in 96-well plates. Duplicated wells were infected with 50 µl of serial 10-fold dilutions of virus. CPE was read microscopically on day 5 p.i. and the TCID50 was calculated.
Replication of CVB3 in HpL3-4, HeLa and HEp-2 cells.
Samples of supernatant from cell cultures were collected at different times p.i. (0, 3, 6, 12, 24, 36, 48, 72, 96, 120 and 144 h) and stored at -80 °C. Intracellular virus was released from cells cultured in 60 mm plates by freezing and thawing three times in 1 ml 2 % FCS in PBS. After clarification by low-speed centrifugation, virus-containing medium was stored at -80 °C. Infectious virus was measured in HeLa cells by a microtitre assay as reported above. Virus titration was calculated in triplicate and representative results of four experiments are given (see Fig. 2).
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Plaque formation assay.
The plaque assay was performed on confluent cell monolayers using six-well plates. HpL3-4 cell monolayers were infected with CVB3 and incubated for 1 h at 37 °C. After adsorption for 1 h at 7 °C, cultures were overlaid with 3 % (w/v) methyl cellulose (Sigma) in Eagle's minimal essential medium containing 2 % HI-FCS. Cultures were incubated at 37 °C for 4 days, fixed with formalin and stained with methylene blue.
Immunofluorescence.
Cells growing on glass coverslips (Matsunami, Japan) were fixed with 4 % paraformaldehyde in PBS for 30 min at room temperature, then washed three times with PBS. To block non-specific binding, cells were incubated with 5 % BSA (Sigma) in PBS for 1 h. In order to visualize intracellular protein expression, permeabilization of the cells was performed in a blocking solution containing 0·2 % saponin. The samples were then incubated with anti-PrP 6H4 antibody (Prionics) (diluted 1 : 500) or anti-CVB3 antibody (Chemicon) (diluted 1 : 500) for 1 h at 37 °C in 1 % BSA in PBS and washed with PBS. Fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin antibodies (diluted 1 : 500) were incubated with cells for 1 h at 37 °C. After washing with PBS, indirect immunofluorescence was visualized with a fluorescence microscope.
RT-PCR of type I interferon (IFN).
Total RNA from infected cells was collected from each confluent cell culture in a 60 mm dish using the acid guanidinum thiocyanate/phenol/chloroform method using TRIzol (Gibco-BRL). RT-PCR for type I IFN was performed as described above using primer sets designed for amplification of IFN- (5'-CTCAGGAACAAGAGAGCCTT-3' and 5'-GGAAGACAGGGCTCTCCAGA-3') and IFN-
(5'-AACAACAGGTGGATCCTCCAC-3' and 5'-GGAAGTTTCTGGTAAGTCTTC-3'). PCR products were separated by 1·5 % agarose gel electrophoresis and detected by ethidium bromide staining and UV transillumination.
Antiviral effect of supernatants from CVB3-infected cells.
The supernatants from infected cells were collected at 6 h p.i. and centrifuged at 300 g at 4 °C for 10 min. The IFN-containing supernatant medium was acidified at 4 M HCl at 4 °C for 3 days to destroy residual virus, then neutralized to pH 7·0 with 4 M NaOH. The HpL3-4-phyg cells were incubated with these samples overnight and inoculated with CVB3. The replication of CVB3 in each cell group was determined by an indirect immunofluorescence assay (IFA) with anti-CVB3 antibody.
Bioassay for IFN.
The supernatants of cells were collected at 6 h p.i. The acidified and neutralized supernatants described above were assayed for antiviral activity by protection of L929 cells against vesicular stomatitis virus (VSV)-induced CPE. L929 cells in six-well plates were incubated overnight with the IFN-containing supernatants, inoculated with VSV at 37 °C for 1 h and overlaid with DMEM containing 3 % methyl cellulose and 2 % HI-FCS. The cultured cells were fixed and stained with methylene blue 4 days later. Type I IFN concentrations (IU ml-1) were inferred from an IFN- standard.
Nucleic acid staining.
The CVB3-infected cells were collected at 24 h p.i. and washed with PBS before centrifugation at 200 g for 20 min at 4 °C. The pellets were resuspended in 100 µl PBS containing ethidium bromide and acridine orange. These samples were observed by fluorescent microscopy.
DNA fragmentation assay.
A DNA laddering technique (Schatzl et al., 1997) was used. Briefly, cells from a 60 mm dish were washed twice with PBS and lysed in 500 µl hypotonic lysis buffer for 5 min [5 mM Tris/HCl (pH 7·5), 20 mM EDTA (pH 8·0), 0·5 % Triton X-100]. The lysates were centrifuged at 10 000 g for 30 min. The supernatants were incubated with 0·3 mg proteinase K ml-1 and 60 µg RNase ml-1 for 30 min at 37 °C and then precipitated in equal volumes of 2-propanol. After centrifugation at 12 000 g for 30 min at 4 °C, the pellets were washed in 70 % ethanol and resuspended in Tris/EDTA buffer [10 mM Tris/HCl (pH 7·4), 1 mM EDTA (pH 8·0)] and subjected to electrophoresis on a 1·5 % agarose gel and stained with ethidium bromide.
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RESULTS |
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Plaque formation in HpL3-4 cell monolayers induced by CVB3 infection
Four days after CVB3 infection, monolayers of HpL3-4 cells produced clear viral plaques (24 mm diameter) under a semi-solid medium (Fig. 1f). Plaque formation on HpL3-4 cells may thus represent a sensitive assay for CVB3 titration.
Reduced CVB3 replication in the presence of PrP
To confirm the observation that Prnp-/- cells showed vigorous CPE following infection with CVB3, HpL3-4-TRPrP (HpL3-4 cells transfected with pIREShyg-PrP) and HpL3-4-phyg (HpL3-4 cells transfected with pIREShyg) cell lines were established. Successful expression of PrPC in HpL3-4-TRPrP cells was confirmed by IFA with anti-PrP antibody 6H4 (data not shown). Immunofluorescence showed expression of PrPC on the membrane surface of HpL3-4-TRPrP but not HpL3-4-phyg cells. CVB3 induced CPE more rapidly and more markedly in HpL3-4-phyg cells compared with HpL3-4-TRPrP cells (Fig. 3).
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DISCUSSION |
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This study suggests two possible roles for PrPC during CVB3 infection. One possibility is that PrPC increases the antiviral activity in neurons, mediated by higher levels of IFN activity. Another is that loss of PrPC stimulates the apoptotic signalling pathway. The results of DNA fragmentation indicated the induction of apoptotic cell death in the CVB3-infected Prnp-/- cell line. After the Prnp transfection, apoptotic cell death was suppressed.
From previous publications it has been shown that, while PrPC prevents Bcl-2-mediated apoptosis (Kuwahara et al., 1999), PrPC prevents Bax-mediated apoptosis (Bounhar et al., 2001
). CVB3-infection promotes apoptotic cell death in the heart, pancreas and in cell lines (Carthy et al., 1998
; Colston et al., 1998
). Although the mechanisms of CVB-induced apoptotic cell death remain unknown, the present study apparently showed pronounced apoptotic cell death in Prnp-/- cells. An ongoing study has shown that CVB3 replication promotes apoptotic cell death via a mitochondria-dependent pathway (Blom et al., 2003
) in Prnp-/- cells (data not shown). Further studies are required to understand this signal transduction via mitochondria.
IFN- is produced in neuronal cells (Ward & Massa, 1995
). PrPC may promote the production of IFNs in neuronal cells and those cytokines could influence CVB3 replication. The absence of PrPC permits virus replication and so induces necrotic and apoptotic cell death. Recently, Yang et al. (2001)
have reported that IFN-
/
promotes cell survival by activating NF-
B. Further investigation of the antiviral activity of PrPC and the inhibition mechanisms of apoptotic cell death are necessary to elucidate fully the function of PrPC in the context of virus infection.
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ACKNOWLEDGEMENTS |
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Received 12 March 2002;
accepted 15 August 2003.
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