Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone

Zijiang Zhao1, Tomoko Date1, Yuhua Li2, Takanobu Kato1, Michiko Miyamoto1, Kotaro Yasui1 and Takaji Wakita1

1 Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan
2 Chengdu Institute of Biological Products, Chengdu 610063, Sichuan Province, PR China

Correspondence
Takaji Wakita
wakita{at}tmin.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus–cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus–cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.

The GenBank/EMBL/DDBJ accession numbers for the sequences described in this study are given in Table 1.

Supplementary figures and tables are available in JGV Online.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Japanese encephalitis virus (JEV) belongs to the genus Flavivirus, which predominantly comprises arthropod-borne viruses. Members of this genus are distributed throughout the world. JEV has a single-strand, positive-sense RNA genome of approximately 11 000 nt, which is translated from a single open reading frame into a polyprotein that is processed by viral and host proteases to yield three structural proteins [capsid, membrane or precursor membrane (prM) and envelope (E)] encoded at the 5' end of the genome, followed by at least seven non-structural proteins (Monath & Heinz, 1996). The flavivirus E protein is the viral haemagglutinin, which induces protective immunity and mediates receptor-specific virus attachment to cell surfaces (Kimura-Kuroda & Yasui, 1983). The E protein plays major roles in determining viral pathogenicity by defining cellular tropism and affecting penetration into susceptible cells (Heinz, 1986; Mandl et al., 1989). X-ray crystallographic resolution of the structure of the E ectodomain in Tick-borne encephalitis virus (TBE virus) reveals that the E protein forms head-to-tail homodimers that lie parallel to the viral envelope (Rey et al., 1995). These homodimers dissociate, leading to irreversible formation of homotrimers on the surface of viral particles under conditions of low pH (Allison et al., 1995; Kuhn et al., 2002; Stiasny et al., 2002).

Studies attempting to elucidate the molecular basis of JEV attenuation have analysed the genomes of both the virulent parental strain SA14 and the attenuated vaccine virus SA14-14-2 (Nitayaphan et al., 1990; Aihara et al., 1991). Several JEV mutants have been obtained through {gamma}-ray irradiation (Chen et al., 1996) or passage in cultured cells (Hasegawa et al., 1992). These studies have indicated that some mutations in the E protein correlate with viral attenuation, including the amino acid mutation from glutamic acid (Glu) to lysine (Lys) at residue 138 of the E protein (E-138). Due to the multiple mutations present, the exact mutated residue changes in the E protein that are primarily responsible for JEV attenuation are not clearly understood. Since infectious Yellow fever virus RNA was transcribed successfully from a full-length cDNA template by using in vitro ligation of two cDNA fragments (Rice et al., 1989), infectious flavivirus clones have been constructed in similar fashion for JEV (Sumiyoshi et al., 1992), dengue virus type 2 (Polo et al., 1997; Gualano et al., 1998), dengue virus type 4 (Lai et al., 1991) and other viruses (Khromykh & Westaway, 1994; Gritsun & Gould, 1998; Shi et al., 2002; Hayasaka et al., 2004). This method seems valuable in studying biological function, polyprotein processing and virulence of mutant viruses with specific mutations in various regions of the viral genome. However, irregular mutations occur frequently in full-length JEV cDNA clones during cloning into plasmid vectors, particularly in the 5' fragment, and this has seriously affected such studies (Sumiyoshi et al., 1995; Yamshchikov et al., 2001). Specific strategies are thus necessary to establish stable, full-length JEV infectious clones, such as insertion of short introns or cloning into bacterial artificial chromosomes (Yamshchikov et al., 2001; Yun et al., 2003).

The present report describes the construction of a stable, full-length cDNA clone of the wild-type JEV AT31 strain into the very low-copy-number plasmid pMW118 by using conventional molecular-cloning technology. Another plasmid, containing a Glu-to-Lys mutation at the E-138 residue, was also constructed. Recombinant JEVs comprising wild-type and mutant viruses were recovered after synthetic RNA transfection, and were characterized with regard to viral virulence, internalization and release.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells, viruses, antibodies and mice.
Vero cells and C6/36 cells were maintained in Eagle's minimum essential medium (MEM) supplemented with 5 % fetal calf serum (FCS). Primary neural cells from cerebral cortices of embryonic BALB/c mice at gestational day 18 (Japan SLC) were maintained as described previously (Wilcox et al., 1990).

The wild-type JEV strain AT31 was a gift from Dr Nakamura of the Nippon Institute for Biological Science, Japan. Attenuated JEV at222 strain was obtained after 222 passages through primary hamster kidney cells to obtain a vaccine strain, as described previously (Yasui, 2002). Recombinant wild-type rAT virus and mutant rAT-E138K were obtained from supernatants of Vero cells transfected with in vitro-transcribed RNAs. All cDNA sequences of viral RNAs were determined and deposited in GenBank/DDBJ/EMBL (Table 1). The at222 strain contains several mutations, including the Glu-to-Lys mutation at residue E-138. All viruses were propagated once in Vero cells or C6/36 cells and supernatants were obtained and stored at –80 °C until used. Three-day-old BALB/c mice were inoculated intracerebrally with 104 p.f.u. JEV rAT-E138K. The inoculated mice were sacrificed at 8 days of age and their brains were harvested and homogenized. Reverted virus RE-138 was recovered from a single large plaque on Vero cells inoculated with the supernatant of homogenized brains. Rabbit anti-JEV serum was prepared as described previously (Kimura-Kuroda et al., 1993).


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Table 1. Origin, sequence, neurovirulence and neuroinvasiveness of JEV in this study

NA, Not applicable; ND, not done.

 
BALB/c mice were purchased from Japan SLC Inc. All mice were maintained in pathogen-free environments. All experiments were conducted in accordance with the Guidelines for the Care and Use of Animals, 2000 (Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan).

Construction of full-length cDNA and a point-mutant cDNA of JEV strain AT31.
RNA was extracted from the supernatant of C6/36 cells infected with AT31 by using Isogen-LS (Nippon Gene). Oligonucleotide primers were designed based on sequence data for JEV strain JaOArS982 (GenBank/EMBL/DDBJ accession no. M18370; Sumiyoshi et al., 1987) and the sequences of these primers are listed in Supplementary Table S1 (available in JGV Online). The RNA solution was subjected to reverse transcription (RT) using an antisense primer, JEV-10976R-30, and Moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen) at 42 °C for 1 h. Both 5' and 3' fragments of JEV cDNA (nt 1–6125 and 5383–10976, respectively) were amplified by using two pairs of primers: SalT7GJE5P20S and JEV-6125R-30 for the 5' fragment or JEV-5383S-30 and ClaKpnJE3P30R for the 3' fragment, with TaKaRa LA Taq polymerase. PCR conditions comprised 30 cycles of denaturing at 95 °C for 10 s and annealing and extension at 68 °C for 6 min. PCR products containing 5' and 3' fragments of JEV cDNA were cloned into pBR322. Three cloned plasmids were selected for further reconstruction of a full-length JEV cDNA: pJEAT-5'-132 contained the 5' end to the SacI site (nt 2215) region; pJEAT-5'-258 contained the SacI site (nt 2215) to BamHI site (nt 5576) region; and pJEAT-3'-75 contained the BamHI site (nt 5576) to 3' end region (see Supplementary Fig. S1, available in JGV Online). For subcloning purposes, the second SacI site in JEV cDNA was abolished by using PCR-based mutagenesis (Kato et al., 2003a), with an A-to-T mutation at nt 6713 of pJEAT-3'-75, producing pJEAT-3'-75dSac. Each insert from pJEAT-5'-132, pJEAT-5'-258 and pJEAT-3'-75dSac was cloned stably into a very low-copy-number plasmid vector, pMW118 (Nippon Gene), to construct the full-length JEV cDNA and the result was designated pMWJEAT (see Supplementary Fig. S1, available in JGV Online). A single G-to-A point mutation at nt 1389 was also introduced into pMWJEAT by PCR-based mutagenesis and designated pMWJEAT-E138K (Fig. 1a).



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Fig. 1. (a) Schematic representation of the single point mutation in the full-length cDNA pMWJEAT-E138K of JEV AT31 strain, designated rAT-E138K strain. (b) Plaque morphology of wild-type virus AT31, recombinant virus rAT, mutant virus rAT-E138K and reverted virus RE-138. Vero cells infected with virus were fixed in 10 % formalin/PBS solution and stained by using 0·1 % crystal violet solution. (c) Detection of recombinant JEV rAT and rAT-E138K in the cerebral cortex of 3-week-old female BALB/c mice by using IFA, as described in Methods. Positive signals were observed in neuron-like cells in the cerebral cortex of rAT- and rAT-E138K-infected mice.

 
Transfection of synthetic JEV RNA into Vero cells by electroporation.
Plasmid DNAs pMWJEAT and pMWJEAT-E138K were digested by using KpnI. JEV RNA was synthesized by using a MEGAscript T7 kit (Ambion) and treated with DNase I (RQ1 RNase-free DNase; Promega) followed by acid–phenol extraction (Kato et al., 2003b). Trypsinized Vero cells were washed by using Opti-MEM I (Invitrogen) and resuspended in Cytomix buffer at 7·5x106 cells ml–1 (Kato et al., 2003b). Synthesized RNA (10 µg) mixed with 400 µl cell suspension was pulsed at 260 V and 950 µF by using a Gene Pulser II apparatus (Bio-Rad).

Plaque-reduction neutralization (PRNT) test.
The PRNT test was done as described previously (Zhao et al., 2003). PRNT titre was expressed as the maximum dilution of antibody yielding a 90 % reduction in viral infectivity (PRNT90).

Mouse experiments.
Groups of 3-week-old female BALB/c mice (n=5) were inoculated intracerebrally with 25 µl of a tenfold serially diluted virus solution (AT31, at222, rAT, rAT-E138K and RE-138). Similar groups of mice were inoculated intraperitoneally with 0·1 ml of a tenfold serially diluted virus solution. Mice were observed for 3 weeks after inoculation. End points of neurovirulence and neuroinvasiveness were identified as both mortality ratios and mean survival times. The LD50 was determined for each virus (Reed & Muench, 1938).

Groups of 3-week-old female BALB/c mice (n=30) were inoculated with 105 p.f.u. rAT in 25 µl or 107 p.f.u. rAT-E138K in 25 µl into the left footpad. At 2-day intervals after inoculation, two mice from each group were bled periorbitally before cardiac perfusion with HBSS solution (Invitrogen). Brains, livers, spleens and kidneys of mice were harvested. Tissue suspensions of 10 % (w/v) were prepared by using PBS, then homogenized immediately. After three cycles of freezing and thawing, these 10 % (w/v) tissue suspensions were centrifuged at 20 000 g at 4 °C for 1 h, then filtered through a 0·2 µm filter. Virus titres in filtered supernatant were determined by plaque assay on Vero cells.

Acid resistance of early virus–cell interactions.
Vero cells (1x105) cultured in a six-well tissue-culture plate (Corning) were washed and pre-chilled at 4 °C for 2 h. After inoculation with 400 p.f.u. virus (m.o.i., 0·004 p.f.u. per cell) at 4 °C for 2 h, unbound virus was removed by washing three times with ice-cold MEM supplemented with 2 % FCS (MEM-2). Cells were treated with 2 ml glycine/HCl-buffered saline (8 g NaCl, 0·38 g KCl, 0·10 g MgCl2.6H2O, 0·10 g CaCl2.2H2O and 7·5 g glycine l–1, pH adjusted to 3·0 with HCl) at room temperature for 5 min, as described by Hung et al. (1999), then overlaid with 1·25 % methyl cellulose/MEM for incubation at 37 °C for 5 days. Infected cells without acid treatment were used as controls. Penetration rates of acid-resistant intracellular viruses were calculated as 100x [no. plaques (acid-treated)/no. plaques (controls)].

Heparin assay and heparinase-treatment effect on Vero cells.
Inhibition of JEV binding to Vero cells by heparin (Sigma) was performed as described by Hung et al. (1999), with some modifications. The following two conditions were tested: (i) 400 p.f.u. virus plus heparin was pre-incubated at 37 °C for 1 h, then incubated in Vero cells cultured in a six-well plate at 37 °C for 2 h; and (ii) in total, 400 p.f.u. virus was reacted with heparin at 37 °C for 1 h, then inoculated into Vero cells at 4 °C for 2 h. Unbound virus was removed by three washes with MEM-2. Cells were overlaid with 1·25 % methyl cellulose/MEM to incubate at 37 °C for 5 days. Cells inoculated by using the same procedure without heparin were used as controls. Inhibition was described as: 100x [no. plaques (heparin treatment)/no. plaques (controls)].

The effectiveness of heparinase treatment was analysed on Vero cells that had been treated with different concentrations of heparinase I or III (Sigma Aldrich) at 37 °C for 1 h. Cells cultured in a six-well plate were pre-chilled at 4 °C for 1 h after washing and inoculated with 400 p.f.u. per well (m.o.i., 0·004 p.f.u. per cell) of JEV rAT or rAT-E138K at 4 °C for 2 h. After washing in MEM-2, cells were overlaid with methyl cellulose and incubated at 37 °C for 5 days. Cells not treated with heparinase were used as controls. Inhibition was calculated as described above.

Binding inhibition of recombinant JEV with lectins.
The ability of rAT and rAT-E138K to bind to Vero cells was examined by using concanavalin A (ConA; Sigma), wheatgerm agglutinin (WGA; Sigma) and phytohaemagglutinin P (PHA-P; Sigma) as described by Hung et al. (1999). A total of 400 p.f.u. virus was reacted with lectin at 37 °C for 1 h, then inoculated into Vero cells cultured in a six-well plate for 2 h. After washing in MEM-2, cells were overlaid with methyl cellulose and incubated at 37 °C for 5 days. Cells inoculated by using viral solutions without lectin were used as controls. Inhibition was calculated as: 100x [no. plaques (lectin treatment)/no. plaques (controls)].

Endocytosis inhibition.
Inhibition of rAT and rAT-E138K virus internalization into Vero cells was analysed by using different concentrations of chlorpromazine, cytochalasin D or nystatin (all from Sigma). Vero cells cultured in a six-well plate were treated with these reagents at 37 °C for 1·5 h. These cells were subsequently inoculated with 400 p.f.u. JEV at 37 °C for another 3 h in the presence of the reagents, followed by treatment with glycine/HCl-buffered saline at room temperature for 5 min to inactivate unpenetrated viruses on the cell surface. Inoculated cells without any inhibiting agent were used as controls. Inhibition was calculated as: 100x [no. plaques (agent treatment)/no. plaques (controls)].

Immunohistochemistry.
BALB/c mice (3 weeks old; n=5) received inoculation with 10 p.f.u. JEV rAT or 105 p.f.u. rAT-E138K into the right cerebral hemisphere. Virus titres were detected in the right hemispheres of sacrificed moribund mice. Left hemispheres were embedded immediately in Tissue-Tek embedding medium (Sakura Finetechnical) to examine the presence of JEV antigen with rabbit anti-JEV serum by using an immunofluorescence assay (IFA) as described previously (Zhao et al., 2003).

Statistical analysis.
Statistical analysis was performed by using Student's t-test. Survival rates of inoculated mice were analysed by using Kaplan–Meier methods. Values of P<0·05 were considered statistically significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of a single plasmid DNA containing full-length JEV cDNA
Cloning a full-length JEV cDNA into a single plasmid vector, such as pUC or even a low-copy-number plasmid vector like pBR322 (Sumiyoshi et al., 1992), is difficult without the insertion of short introns (Yamshchikov et al., 2001). We thought that this difficulty might be attributable to toxicity of the proteins produced by JEV cDNA in transformed bacterial cells. To reduce this toxicity, we cloned full-length JEV cDNA into the very low-copy-number vector pMW118, a derivative of pSC101 (see Supplementary Fig. S1, available in JGV Online). The resultant plasmid, pMWJEAT, contained a full-length cDNA of the JEV AT31 strain and was amplified stably in bacterial cells. A mutant plasmid, pMWJEAT-E138K, containing a single Glu-to-Lys point mutation at aa 138 of the E protein, was also constructed (Fig. 1a).

Generation of recombinant JEV on Vero and C6/36 cells
The cDNA-generated recombinant JEV strains rAT and rAT-E138K were obtained from the supernatant of Vero cells transfected with synthetic RNA using the full-length cDNA clone as described in Methods. Viral RNA was extracted from both rAT and rAT-E138K virus solutions and sequences were determined directly after RT-PCR. No mutations were found in the genomic sequences as compared with template plasmid DNA sequences. Parental wild-type AT31 and recombinant rAT viruses displayed similar plaque morphologies at 5 days after infection (diameter ~3·1 mm), whereas rAT-E138K virus exhibited a smaller-plaque morphology (diameter ~0·45 mm) on Vero cells (Fig. 1b). Plaques similar to those of rAT-E138K were observed in all supernatant samples following seven passages on C6/36 cells. Reverted RE-138 virus was obtained as described in Methods. RE-138 virus also exhibited larger-size plaques (~3·1 mm) than those of AT31 and rAT viruses, as shown in Fig. 1(b).

Neurovirulence and neuroinvasiveness of recombinant JEV
Neurovirulence and neuroinvasiveness of JEV strains were determined by direct intracerebral inoculation or intraperitoneal infection using the tested viruses obtained from the supernatants of infected cells, as described in Methods. The JEV rAT strain displayed strong neurovirulence in 3-week-old BALB/c mice, as did the parent AT31 (2·5 and 2·2 p.f.u./LD50, respectively; Table 1). In contrast, the rAT-E138K strain displayed much lower neurovirulence in mice (15 200 p.f.u./LD50) and the attenuated JEV at222 strain displayed the lowest neurovirulence (148 000 p.f.u./LD50). However, RE-138 virus reverted from the rAT-E138K strain displayed strong neurovirulence (2·2 p.f.u./LD50; Table 1), comparable to that seen with rAT and AT31 virus. After sequence analysis of RE-138 virus from the supernatant of infected Vero cells, only residue E-138 had been changed from Lys to Glu, compared with the rAT-E138K virus (data not shown). Furthermore, the JEV rAT strain demonstrated strong neuroinvasiveness, comparable to that of the parental AT31 strain (18 800 and 24 200 p.f.u./LD50, respectively). Both rAT-E138K and at222 strains exhibited weak neuroinvasiveness (>107 p.f.u./LD50 for both strains), as all mice inoculated with 107 p.f.u. of either rAT-E138K or at222 survived (Table 1). These results demonstrate that the Glu-to-Lys mutation at residue E-138 of the JEV AT31 strain strongly attenuates viral neurovirulence and neuroinvasiveness in BALB/c mice.

Detection of JEV antigen in inoculated mouse brain
Positive signals were detected in both rAT- and rAT-E138K-infected mouse brains by IFA, and most positive cells displayed a neuronal morphology (Fig. 1c). The majority of positive signals were distributed in both the cerebral cortex and putaminal areas in brains of rAT-infected mice, whereas most positive cells were present in the frontal area of the cerebral cortex in rAT-E138K-infected mice. No positive signals were identified in the cerebella of either rAT- or rAT-E138K-infected mice (data not shown). These results indicate that the point mutation does not change viral neurotropism in BALB/c mice, although neurovirulence is attenuated.

JEV titre of infected mouse tissues
All mice inoculated with 107 p.f.u. rAT-E138K into the left footpad remained asymptomatic until 14 days post-inoculation, and no virus replication was detected in any tested tissue (Table 2). In contrast, viraemia (163 p.f.u. ml–1) was detected in sera of mice inoculated with 105 p.f.u. of the rAT strain via the footpad on day 2 post-inoculation. Detectable levels of replicated rAT virus in brains of inoculated mice were identified at about 1·2x104 p.f.u. per tissue at 4 days post-inoculation, increasing to 8·6x107 p.f.u. per tissue by day 8. No virus was detected in any other tissue (Table 2).


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Table 2. Organ distribution of JEV in 3-week-old BALB/c mice inoculated with either 1x105 p.f.u. rAT or 1x107 p.f.u. rAT-E138K via the footpad

 
Alteration in acid-resistant viral attachment by Glu-to-Lys mutation at E-138
Some flaviviruses display abolished infectivity with low-pH treatment after adsorption onto target cells (Hung et al., 1999). We thus attached JEVs to Vero cells at 4 °C and then created conditions of low pH to determine the extent to which the adsorbed virus can maintain infectivity. Substantial reduction in plaque formation was detected in the rAT strain under conditions of low pH, with 7·79±2·31 % plaque formation compared with the control experiment (Fig. 2). However, rAT-E138K and at222 strains proved more resistant to low-pH treatment, with plaque formation at 35·95±9·61 and 34·83±4·19 %, respectively. These results indicate that the Glu-to-Lys mutation at E-138 alters the acid resistance of virus–cell attachment on Vero cells.



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Fig. 2. Virus–cell interactions for recombinant JEV on Vero cells. Plaque-formation rates of rAT, rAT-E138K and attenuated at222 viruses bound to Vero cells at 4 °C were examined following treatment with glycine/HCl-buffered saline (pH 3·0) at room temperature for 5 min. Each value represents the mean of nine wells from three separate experiments. Error bars indicate SD. *, P<0·01.

 
Inhibition of virus–cell interactions with heparin or heparinase treatment
Both heparin and heparinase treatments have been used to inhibit the interactions of flavivirus and target cells (Chen et al., 1997; Hung et al., 1999). Infection by the rAT-E138K strain was clearly inhibited by heparin on Vero cells, whereas the rAT strain remained unaffected at both 4 and 37 °C (Fig. 3a, b). Significant differences in heparin inhibition between rAT and rAT-E138K were detected with heparin concentrations of 0·1–100 U ml–1 at 37 °C (P<0·01 or P<0·001; Fig. 3a) and of 0·1–1000 U ml–1 at 4 °C (P<0·05, P<0·01 or P<0·001; Fig. 3b), respectively. Similar results were noted on addition of heparin after virus attachment to Vero cells (data not shown).



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Fig. 3. Inhibition of recombinant JEV rAT ({blacksquare}) and rAT-E138K ({circ}) plaque formation on Vero cells with heparan sulphate were analysed at 37 °C (a) and 4 °C (b). Plaque-formation rates of rAT ({blacksquare}) and rAT-E138K ({circ}) viruses on Vero cells treated with heparinase I (c) or heparinase III (d) at 37 °C for 1 h were examined. Each value represents the mean of nine wells from three separate experiments. Error bars indicate SD. *, P<0·05; **, P<0·01; ***, P<0·001.

 
Next, heparan sulphate on the surface of Vero cells was eliminated by treatment with heparinase I or III. Plaque formation for the JEV rAT strain on Vero cells was not inhibited by heparinase I or III treatment (Fig. 3c, d). However, plaque formation by rAT-E138K virus was reduced by both heparinase I and III (Fig. 3c, d). Differences in the plaque formation of infected Vero cells treated with heparinase I were small (P<0·05 at 4 U ml–1; Fig. 3c). Treatment with >1 U heparinase III ml–1 on Vero cells reduced plaque formation of rAT-E138K by >40 %, but had no effect on rAT virus, representing a significant difference (P<0·001 for approx. 1–4 U heparinase III ml–1; Fig. 3d). These data demonstrate that the point mutation increases dependency on heparan sulphate B residues for virus attachment to the cell surface.

Inhibition of virus–cell interactions with lectins
Regulation of viral entry by carbohydrates on dengue virus type 2 glycoproteins has been reported (Hung et al., 1999). To explore the effect of the point mutation in JEV glycoproteins on this regulation, various lectins were used to inhibit virus–cell interactions. No significant differences in plaque formation were seen following ConA treatment between JEV rAT and rAT-E138K strain infection on Vero cells (Fig. 4a). However, significant differences in plaque formation were found between rAT and rAT-E138K strains when virus was treated with >5 µg WGA ml–1 pre-inoculation (P<0·01 or P<0·001; Fig. 4b). Finally, PHA-P did not reduce plaque formation in either JEV rAT or rAT-E138K strains at any tested concentration (Fig. 4c). These results indicate that the point mutation affects inhibition of virus–cell interactions with WGA, but exerts less effect with ConA and PHA-P. The higher efficiency of WGA inhibition against the rAT strain than against the rAT-E138K strain suggests that residues of {beta}-N-acetylmuramic acid or {alpha}-N-acetylneuraminic acid on rAT virus are more involved in virus–cell interactions (Fig. 4b).



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Fig. 4. Inhibition of viral internalization of rAT ({blacksquare}) and rAT-E138K ({circ}) viruses into Vero cells with lectins: (a) concanavalin A; (b) wheatgerm agglutinin; and (c) phytohaemagglutinin P. Plaque-formation rates for each sample were examined. Each value represents the mean of three separate experiments. Error bars indicate SD. *, P<0·01; **, P<0·001.

 
Detection of endocytosis of recombinant JEV on Vero cells
Plaques of both rAT and rAT-E138K were effectively reduced by chlorpromazine on Vero cells (Fig. 5a). Plaque formation was inhibited more efficiently for rAT-E138K than for rAT at a concentration of 5 µg chlorpromazine ml–1 (P<0·05). At a higher concentration of 20 µg ml–1, about 20 % of Vero cells dissociated due to cytotoxicity. This result indicates that clathrin-dependent endocytosis is involved in JEV infection. Plaque formation by rAT virus was reduced by about 20 % by cytochalasin D at concentrations within the range 1–20 µg ml–1, but the rAT-E138K strain was unaffected. Thus, macropinoendocytosis by Vero cells is more important for rAT than for rAT-E138K (P<0·05; Fig. 5b). Furthermore, nystatin, an inhibitor of caveolae-mediated endocytosis, did not inhibit plaque formation of either the rAT or rAT-E138K strains (Fig. 5c). These results demonstrate that both rAT and rAT-E138K strains predominantly utilize penetration via clathrin-dependent endocytosis, rather than caveolae-mediated endocytosis, in Vero cells. The macropinoendocytosis function may also be involved in penetration of rAT virus, but not in that of rAT-E138K.



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Fig. 5. Inhibition of recombinant JEV internalization on Vero cells. Endocytosis inhibition of rAT ({blacksquare}) and rAT-E138K ({circ}) viruses was examined by using different concentrations of chlorpromazine (a), cytochalasin D (b) and nystatin (c). Each value represents the mean of three separate experiments. Error bars indicate SD. *, P<0·05.

 
Recombinant virus release
Following JEV infection of Vero cells at an m.o.i. of 5 p.f.u. per cell, significantly higher titres of rAT virus were detected in supernatant compared with cell-associated virus at 24–48 h post-inoculation (P<0·05; Fig. 6a). In contrast, higher titres of rAT-E138K virus were detected consistently in the intracellular fraction than in the supernatant. These significant differences in rAT-E138K virus titres between intra- and extracellular fractions were seen at all time points examined (P<0·05; Fig. 6b). However, total amounts of replicated rAT virus and rAT-E138K virus were similar (Fig. 6c). These data demonstrate clearly that the point mutation significantly inhibits virus release from infected Vero cells, but does not affect virus replication.



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Fig. 6. Replication and secretion of rAT ({blacksquare}, {square}) and rAT-E138K ({bullet}, {circ}) viruses inoculated into Vero cells. JEV rAT and rAT-E138K strains were inoculated at an m.o.i. of 5 p.f.u. per cell into Vero cells (a–c) or primary neuron cells of BALB/c mice (d–f). Viruses released into the supernatant or retained in the cytoplasm were detected by plaque assay. IC, Intracellular; EC, extracellular; Total, total of intra- and extracellular virus. *, P<0·05.

 
On primary neural cells inoculated with JEV at an m.o.i. of 5 p.f.u. per cell, higher titres of rAT virus were found in supernatant than in cell-associated fractions from 18 h post-inoculation (Fig. 6d). Again, significant differences in rAT virus titres were identified between intra- and extracellular virus titres at 24–72 h post-inoculation (P<0·05; Fig. 6d). In contrast, higher titres of rAT-E138K virus were also present in the intracellular fraction than in supernatant (Fig. 6e), and significant differences in virus titres were found between intra- and extracellular fractions at 12–72 h post-inoculation (P<0·05; Fig. 6e). As with the results for Vero cells, no significant differences in total amounts of replicated virus were observed between rAT and rAT-E138K (Fig. 6f). These results indicate clearly that the point mutation significantly inhibits secretion of replicated virions from both infected Vero cells and primary neural cells.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Previous studies have indicated that irregular mutations often taken place at the 5'-end region of full-length JEV cDNA clones (Arroyo et al., 2001; Sumiyoshi et al., 1992; Yun et al., 2003). Through insertion of short introns, the JEV infectious clone was constructed and was able to be propagated stably in Escherichia coli (Yamshchikov et al., 2001). In our study, several unforeseen mutations also occurred when the full-length cDNA of AT31 virus was cloned into pBR and pUC vectors (T. Wakita, T. Date, Y. Li & K. Yasui, unpublished data). Focusing on this problem, the 5'-end fragment of JEV AT31 cDNA was cloned into the very low-copy-number pMW118 vector. By inserting another 3' fragment of JEV, a full-length cDNA of JEV AT31, pMWJEAT, was successfully constructed and amplified. Sequence analysis of viral RNA isolated from the recombinant JEV rAT strain revealed no mutation compared with the template pMWJEAT.

By analogy with three-dimensional structure models of TBE virus (Rey et al., 1995; Mandl et al., 2001) and Dengue virus (Zhang et al., 2003b), the E glycoprotein of JEV is predicted to contain an extended structure with seven {beta}-sheets, two {alpha}-helices and three domains (Kolaskar & Kulkarni-Kale, 1999). The E glycoprotein is the viral haemagglutinin and induces protective immune responses and mediates receptor-specific viral attachment to cell surfaces. Previous reports have indicated that mutations in the JEV E protein affect viral neurovirulence or neuroinvasiveness in vivo (Yu et al., 1981; Ni & Barrett, 1998) or viral binding and entry into cultured cells in vitro (Hasegawa et al., 1992; Lee & Lobigs, 2002). The molecular basis of viral attenuation in flaviviruses has been analysed by using site-directed mutagenesis of infectious cDNA clones (Kinney et al., 1997; Hurrelbrink et al., 1999), production of chimeric viruses (Arroyo et al., 2001) and deletions in the 3' untranslated regions of genomes (Whitehead et al., 2003). Residue 138 of the E protein has been considered to be located on the E0 {beta}-strand in domain I and to be exposed on the surface of the E protein (Lee et al., 2004). The data described in the present manuscript demonstrate that mutation of the E-138 residue in JEV from Glu to Lys inhibits viral spread from cell to cell, explaining the small-plaque morphology. Previous studies have indicated that mutations in domain III of the flavivirus E protein modulate virus binding and entry into host cells (Hung et al., 2004; Liu et al., 2004; Modis et al., 2004). The dimer-to-trimer structural transition of the E protein induced by low-pH conditions is considered crucial for virus–cell binding and entry processes (Stiasny et al., 1996; Chen et al., 1997; Arroyo et al., 2001) and this binding may be affected by mutations in the E protein. The E-138 mutation is not located in domain III, but enhances virus–cell attachment greatly under acidic conditions (Fig. 2).

Flaviviruses are considered to be internalized after binding to glycosaminoglycan (GAG) residues on cells, and other molecules are also involved in virus entry for some viruses (Mandl et al., 2001; Lee et al., 2004; Liu et al., 2004). The binding reaction can be inhibited in vitro by using heparin (Chen et al., 1997; Hurrelbrink & McMinn, 2001; Lee & Lobigs, 2002). However, the molecular effects of heparin on virus-binding activity with JEV attenuation are not well understood. Previous studies have indicated that mutations of residues E-49, E-138, E-306 or E-389 on the JEV E protein reduce the efficiency of viral binding to heparan sulphate residues on target cells (Lee et al., 2004; Liu et al., 2004). Furthermore, cell-adapted TBE virus infection of BHK-21 cells is inhibited by GAGs, whereas this is not the case for the parental virus (Mandl et al., 2001). Mutation at residue E-138 in both JEV and West Nile virus shows clearly that this residue is a strong determinant of viral binding to GAG residues on target cells (see Supplementary Table S2, available in JGV Online). In our study, binding by rAT-E138K was inhibited significantly by heparin, whereas the rAT strain was unaffected. Cells digested with heparinase III displayed specifically reduced binding activity to rAT-E138K (Fig. 3). This indicates that rAT-E138K virus binding to cell surfaces is more dependent on heparan sulphate B residues than rAT virus. The better interaction of heparin with rAT-E138K virus in comparison with rAT virus is explained by the fact that heparin is negatively charged and the substitution of negatively charged Glu for positively charged Lys provides a more highly charge-mediated interaction, based on the consideration that residue 138 of the E protein is exposed on the surface (Lee et al., 2004); alternatively, this mutation may expose other positively charged residues on the surface of the virion. Residue E-138 is negatively charged and conserved among the JEV serocomplex, but not among other flaviviruses (See Supplementary Fig. S2, available in JGV Online). Furthermore, a Glu-to-Lys mutation was found not only in JEV, but also in West Nile virus. Taken together, the E-138 residue is suggested to be an important determinant of GAG binding and virulence of the JEV serocomplex.

Flaviviruses generally bud into the lumen of the endoplasmic reticulum in infected cells and are subsequently secreted through the vesicle-transport pathway. A recent study has indicated that a single Pro-to-Ser point mutation at position 63 in the prM protein of TBE virus may induce a reduction in virus-particle secretion from RNA-transfected BHK-21 cells (Yoshii et al., 2004). Interestingly, the Glu-to-Lys mutation at E-138 also affects the efficiency of viral release from infected cells. Changes in not only infection efficiency, but also viral secretion efficiency, may play important roles in virus attenuation. Some studies have reported that about 180 glycoprotein E monomers are present on the surface of a mature flavivirus particle with the (pre)M–E heterodimer (Lescar et al., 2001; Kuhn et al., 2002; Zhang et al., 2003a, b). The Glu-to-Lys mutation changes the charge of the side chain from negative to positive. About 180 positively charged side chains at E-138 residues would change to 180 negative charges in each rAT-E138K virus particle. Clearly, this would affect both the binding of rAT-E138K virus to target cells and intracellular transport of viral particles. Furthermore, the E-138 residue exists near the hinge region between domains I and II, and the three-dimensional structure of the E protein might therefore change due to mutation of this residue. Taken together, these mechanisms may attenuate JEV virulence cooperatively in vivo, but further study is needed to clarify this point.

Neuroinvasiveness of both at222 and rAT-E138K viruses was attenuated (Table 1). The blood–brain barrier (BBB) can inhibit viral invasion into mouse brains, and both rAT-E138K and at222 viruses proved unable to enter the brain. A recent publication demonstrated that West Nile virus replication in peripheral tissues triggers a Toll-like receptor inflammatory response that alters the permeability of the BBB (Wang et al., 2004). Increased BBB permeability allows West Nile virus to cross the BBB. West Nile virus is a flavivirus that is genetically related very closely to JEV. Similar mechanisms might thus be important for the neuroinvasiveness of JEV. The data in Table 2 demonstrate clearly that rAT-E138K virus does not cause viraemia and, thus, inflammatory responses might be insufficient to replace BBB permeability. This issue warrants further analysis in future.

Previous investigators of the JEV SA14-14-2 strain have demonstrated that change of residue E-279 from methionine to lysine in domain II affects viral neurovirulence significantly (Monath et al., 2002). Our studies indicate that residue E-138, which is located at the link region between domains I and II, is an important determinant of neurovirulence and neuroinvasivenes in JEV. However, the at222 virus contains additional mutations besides E-138 and demonstrated characteristics of further attenuation, compared with rAT-E138K with the single mutation at E-138. The function of point mutations at other residues in the JEV E protein should thus be examined by using full-length cDNA clones in future studies.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr J. Nakamura of the Nippon Institute for Biological Science, Japan, for providing the JEV AT31 strain. We also wish to thank Dr Satoshi Koike for his helpful advice. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, by a Grant from Toray Industries, Inc., by the Program for Promotion of Fundamental Studies in Health Sciences of the Pharmaceuticals and Medical Devices Agency (PMDA) and by the Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.


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METHODS
RESULTS
DISCUSSION
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Received 25 September 2004; accepted 21 April 2005.



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