Transfection of prion protein gene suppresses coxsackievirus B3 replication in prion protein gene-deficient cells

Yuko Nakamura1, Akikazu Sakudo1, Keiichi Saeki1, Tomomi Kaneko1, Yoshitsugu Matsumoto1, Antonio Toniolo2, Shigeyoshi Itohara3 and Takashi Onodera1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The susceptibility of prion protein gene (Prnp)-null cells to coxsackievirus B3 (CVB3) was investigated. Primary cultures of murine Prnp-/- brain cells were more sensitive to CVBs than corresponding cells from wild-type mice. The viral susceptibility of a Prnp-null cell line (HpL3-4) derived from the murine hippocampus was compared with that of two established cell lines (HeLa and HEp-2) that are widely employed for CVB3 studies. After infection with CVB3, HpL3-4 cells showed a very rapid and complete cytopathic effect (CPE). CPE developed earlier and viruses replicated at higher titres in HpL3-4 cells compared with HeLa and HEp-2 cells. Under a semi-solid medium, plaques developed rapidly in CVB3-infected HpL3-4 cells. To confirm the effect of Prnp on virus infection, a Prnp-/- cell line and a Prnp-transfected neuronal cell line were analysed. The replication and release of infectious particles of CVB3 in Prnp-/- cells were significantly more effective than those of the Prnp-transfected cell line. Levels of type I interferon (IFN) after CVB3 infection were higher in the Prnp-transfected cell line than in Prnp-/- cells, whereas apoptotic cells were more obvious in the Prnp-/- cells than in those of the Prnp-transfected cell line. These findings suggest that the absence of Prnp retards the induction of CVB3-induced IFNs, resulting in an enhanced CVB3 production and apoptotic cell death. Furthermore, our data indicate that the HpL3-4 cell line may provide a novel and sensitive system for isolation of CVB3 from clinical specimens.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The cellular isoform of the prion protein, PrPC, is encoded by a single gene corresponding to about 250 amino acids and is highly conserved among vertebrates (Prusiner et al., 1998; Zanusso et al., 1998). PrPC is expressed in neurons and glia of the brain and spinal cord, as well as in several peripheral tissues and leukocytes. PrPC mRNA can first be detected in the brains of mice and chickens in early embryogenesis and its level increases with development (Harris et al., 1993; Manson et al., 1992). In the adult central nervous systems (CNS), PrPC and its mRNA are widely distributed, with particularly high concentrations in neocortical and hippocampal neurons, cerebellar Purkinje cells and spinal motor neurons (DeArmond et al., 1987; Kretzschmar et al., 1986). Although the PrPC localization on the cell surface may be consistent with roles in adhesion and recognition, ligand uptake or transmembrane signalling, its function remains to be defined.

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 nucleocapsid–RNA 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses.
CVB3 (Nancy strain) was obtained from ATCC. Virus stocks were prepared in cultures of HeLa cells (ATCC) grown in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 2 mM L-glutamine and 2 % heat-inactivated foetal calf serum (HI-FCS; Gibco-BRL). Supernatants were collected 3 days post-infection (p.i.) and centrifuged at 1000 g for 10 min. Virus titres were determined by plaque formation assay in HeLa cells. Virus aliquots were stored at -80 °C until use.

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 10–20 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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Fig. 2. High susceptibility of HpL3-4 cells to CVB3 infection. Using the same m.o.i. of 5 for CVB3, the highest production of infectious virus was observed in cell lysates of HpL3-4 cells (dashed line and circles). Release of virus into the supernatant of HpL3-4 cell cultures (solid line and circles) was also observed. The virus titres of cell lysates from HEp-2 cells (dashed line and diamonds), supernatants from HEp-2 cells (solid line and diamonds), cell lysates from HeLa cells (dashed line and triangles) and supernatants from HeLa cells (solid line and triangles) were also examined.

 
Plasmid construction and gene transfer.
In the gene transfer of Prnp, the coding region of Prnp cDNA was amplified by PCR using the following primers: 5'-GCGAATTCCGCCACCATGGCGAACCTTGGCTACTGGCTG-3' and 5'-CACGAATTCCACCTCAATTGAAAGAGCTACAGGTGG-3'. The Kozak consensus sequence is boxed and the BglII linker is underlined. The PCR products were cloned into the pT7Blue vector and subcloned into the multicloning site of pIREShyg (Clontech). The resulting constructs of pIREShyg-PrP or pIREShyg were transiently transfected into HpL3-4 cells by the lipofection method using Lipofectamine plus (Gibco-BRL). These cells were selected for more than 10 days in a complete medium containing 400 µg hygromycin B ml-1 (Wako, Japan).

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-{alpha} (5'-CTCAGGAACAAGAGAGCCTT-3' and 5'-GGAAGACAGGGCTCTCCAGA-3') and IFN-{beta} (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-{beta} 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CVB infection of primary cultures of brain cells
Primary cultures from the brains of wild-type and Prnp-/- mice consistently allowed replication of CVB3. An earlier and stronger development of CPE was observed in cultures from Prnp-/- mice (Fig. 1b) than in cultures derived from wild-type mice (Fig. 1a). These observations suggested that primary cultures from Prnp-/- mice may be particularly sensitive to CVB infection. By RT-PCR, it was shown that primary cultures from the brains of both Prnp-/- and Prnp+/+ mice and the HpL3-4 and HpL3-4-TRPrP cells expressed high levels of the murine coxsackie–adenovirus receptor (MCAR) transcripts (data not shown).



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Fig. 1. Cytopathic effects induced by CVB3 infection. Primary cultures of brain cells from wild-type C57BL/6 mice and Prnp-/- mice were grown in DMEM. After infection with CVB3 at an m.o.i. of 5, the development of CPE was observed by light microscopy. Two days after infection with CVB3, cells from Prnp-/- mice (b) showed a more complete development of CPE compared with cells from wild-type mice (a). The development of CPE was detected earlier in HpL3-4 cells than in two other cell lines (HeLa and HEp-2 cells). At 36 h p.i., CPE in HpL3-4 cells was most vigorous (c), compared with that induced in HeLa (d) and HEp-2 cells (e). CVB infection of HpL3-4 cells caused typical plaque formation; a representative picture of CVB3-induced plaques is shown in panel (f).

 
CVB replication in HpL3-4, HeLa and HEp-2 cells
To compare the sensitivity of HpL3-4, HeLa and HEp-2 cells to CVB3, the kinetics of virus replication were evaluated in each cell line. Cultures inoculated with virus were examined by phase-contrast microscopy at different times p.i. Although CPE was induced in all cell lines, by 48 h p.i. CPE in HpL3-4 cells was more than 80 % (Fig. 1c) but was only 20 % in HeLa and HEp-2 cells (Fig. 1d, e). Titration of extracellular and intracellular virus at different times p.i. using the same m.o.i. was used to compare the sensitivity of the different cell lines to CVB3. Extracellular and intracellular virus titres were higher in HpL3-4 cells than in HeLa and HEp-2 cell lines (Fig. 2). Thus, in addition to the rapid development of CPE, the HpL3-4 cell line was characterized by the ability to produce virus titres higher than those produced by HeLa and HEp-2 cells.

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 (2–4 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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Fig. 3. CPE in HpL3-4-TRPrP and HpL3-4-phyg. HpL3-4-TRPrP and HpL3-4-phyg cells grown in 60 mm dishes were inoculated with CVB3 (m.o.i.=1). At 24 h p.i., HpL3-4-phyg cells showed marked CPE (b), whereas no significant CPE appeared in HpL3-4-TRPrP cells (a).

 
To determine the causes for the differences in CPE between HpL3-4-TRPrP and HpL3-4-phyg cells, two analyses were performed. First, IFAs for CVB3 replication were carried out to compare the efficiency of CVB3 replication. An IFA with anti-CVB3 antibody was performed to detect intracellular virus replication, as well as for the titration of supernatants, reflecting the release of infectious particles of CVB3. The reduction of CVB3 replication in HpL3-4-TRPrP cells was apparent from the results of the IFA (Fig. 4). At 36 h p.i., rounded cells were seen equally in CVB3-infected HpL3-4-TRPrP and HpL3-4-phyg cells, as shown in Fig. 4(c, d). However, when the same fields were observed by immunofluorescence, HpL3-4-phyg cells showed more positive cells with specific fluorescence (Fig. 4a) than HpL3-4-TRPrP cells (Fig. 4b). The higher level of infectious virus in the supernatant of HpL3-4-phyg cells is shown in Fig. 5.



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Fig. 4. Immunofluorescence of CVB3-infected HpL3-4-TRPrP and HpL3-4-phyg cells. At 36 h p.i. with CVB3 at an m.o.i. of 1, HpL3-4-TRPrP and HpL3-4-phyg cells grown on coverslips were fixed and permeabilized. CVB3 was detected with anti-CVB3 antibody in HpL3-4-phyg (a) and HpL3-4-TRPrP (b) cells. Strong fluorescence was observed in the cytoplasm of HpL3-4-phyg cells. The corresponding phase-contrast images of each cell line are shown in (c) and (d), respectively.

 


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Fig. 5. CVB3 virus production in HpL3-4-TRPrP and HpL3-4-phyg cells. HpL3-4-TRPrP and HpL3-4-phyg cells were infected with CVB3 at an m.o.i. of 1. The supernatants of infected HpL3-4-TRPrP (black bars) and HpL3-4-phyg (white bars) cells were collected at 12 or 24 h p.i. Virus titres were examined by TCID50 assay in HeLa cells. At each point, the virus titres of HpL3-4-phyg cells were higher than those of HpL3-4-TRPrP cells.

 
Antiviral activity in the supernatant of HpL3-4-TRPrP and HpL3-4-phyg cells infected with CVB3
To determine the mechanism of antiviral effects in HpL3-4-TRPrP, levels of type I IFN were determined. At an early stage of infection with CVB3 at an m.o.i. of 1, strong signals of expression of IFN-{alpha} and -{beta} genes were observed (Fig. 6a). The IFN activity in the supernatants of both cell lines was calculated by microassay (Fig. 6b), as described in the Methods. A higher activity was detected in the supernatants from HpL3-4-TRPrP cells compared with HpL3-4-phyg cells. The constitutive production of IFN in non-infected cells was also studied, although the levels of IFN were very low.



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Fig. 6. Expression and activity of IFN in the presence and absence of PrP. (a) RT-PCR for type I IFN. Total RNA samples were obtained from both HpL3-4-TRPrP and HpL3-4-phyg cells after CVB3 infection (m.o.i.=1). PCR products were amplified with IFN-{alpha}-, IFN-{beta}- or GAPDH-specific primers and were separated by 1·5 % agarose gel electrophoresis. No product was detected when reverse transcriptase was omitted. Lanes 1–4, HpL3-4-TRPrP cells; lanes 5–8, HpL3-4-phyg cells. Lanes 1 and 5, uninfected cells at 0 h; lanes 2 and 6, infected cells at 3 h p.i.; lanes 3 and 7, 24 h p.i.; lanes 4 and 8, 48 h p.i. (b) IFN activity in the supernatant. To determine the activity of IFN in the samples of HpL3-4-TRPrP and HpL3-4-phyg cells, microassays were performed. The activity of type I IFN was measured as described in the Methods.

 
Apoptotic cell death induced by CVB3
From the results of these experiments, the presence of PrPC apparently correlates with susceptibility to virus replication and consequent apoptotic cell death. The higher levels of IFN in HpL3-4-TRPrP cells may inhibit CVB3 replication and CPE production. Although IFN would interfere with virus replication, CPE was influenced by the mechanism of apoptotic cell death. To study this, apoptosis was examined by staining DNA extracted from cells following electrophoresis, while the fragmented (apoptotic) nuclei were evaluated in virus-infected HpL3-4-phyg cells (Fig. 7a). Fragmented nuclei were frequently observed in HpL3-4-phyg cells, whereas none was observed in HpL3-4-TRPrP cells after virus infection (Fig. 7a). The DNA laddering assay also showed DNA fragmentation only in HpL3-4-phyg cells infected with CVB3 (Fig. 7b).



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Fig. 7. Detection of DNA fragmentation in HpL3-4 cells infected with CVB3. (a) Ethidium bromide and acridine orange staining of CVB3-infected HpL3-4-TRPrP and HpL3-4-phyg cells (m.o.i.=5). (b) DNA electrophoresis of CVB3-infected HpL3-4-TRPrP and HpL3-4-phyg cells. The DNA samples in lanes 1–4 were derived from HpL3-4-TRPrP cells, whereas those in lanes 5–8 were derived from HpL3-4-phyg cells. Lanes 1 and 5, uninfected cells at 0 h; lanes 2 and 6, infected cells at 12 h p.i.; lanes 3 and 7, 24 h p.i.; lanes 4 and 8, 48 h p.i.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CVB3s are high-risk human pathogens, especially for infants (Woodruff, 1980). The neonatal mouse brain is one of the most susceptible organs to CVB3 infection. Several studies have shown that CVB can induce severe infections in the CNS of newborns (Rotbart, 1995; Xu & Crowell, 1996). In this study, we observed that CVB3 infection of primary cultures of brain cells from Prnp-/- mice was followed by a rapid and intense development of CPE. The expression of MCAR is known to play a major role in the cellular tropism of CVB3 (Selinka et al., 1998). MCAR transcripts have been detected at high levels in cells cultured from the neonatal murine brain. In primary cultures of murine Prnp-/- brain cells, the expression level of MCAR appeared to decrease with age (data not shown). Considering these in vivo data, it was expected that primary brain cells from newborn mice would be highly susceptible to CVB3. Recently, PrP has been demonstrated to have RNA-binding properties analogous to those of certain retrovirus gene products (Darlix et al., 1995). The difference in CVB3 susceptibility between the Prnp-/- cells and wild-type cells indicates a possible relationship between PrP expression and susceptibility to virus infection in neurons. In the present experiments, PrP-deficient HpL3-4 cells were more susceptible to CVB3-induced CPE and produced higher virus titres than the HeLa and HEp-2 cell lines used for comparison. Similarly, this susceptibility and high level of virus production were also observed in an assay using VSV (data not shown). HpL3-4 cells were also able to form plaques after CVB3 infection. Comparative studies with Prnp+/+ cells indicate that viral susceptibility of the HpL3-4 cell line not only reflects the high-level expression of MCAR (a feature common to all neuronal cell types investigated so far) but also the lack of the PrP gene product. Although further studies are warranted to define the relationship between PrP and susceptibility to CVB3, the described Prnp-/- cell line may represent a useful tool for detecting and titrating these agents in human and animal samples.

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-{beta} 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-{alpha}/{beta} promotes cell survival by activating NF-{kappa}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.


   ACKNOWLEDGEMENTS
 
This work was partly supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, Tokyo, Japan, and from the ‘Fondazione Banca del Monte’, Milano, Italy.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 12 March 2002; accepted 15 August 2003.



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