Hepatitis C virus (HCV), a member of the Flaviviri-dae family, is known to cause persistent infection andchronic hepatitis, cirrhosis and hepatocellular carcino-mas (21, 46). At present, HCV infections can be treatedwith interferon (IFN) alone or in combination with rib-avirin. However, these standard therapies do not alwaysexert satisfactory effects and nearly a half of the treatedpatients do not eliminate the virus (7, 14, 15, 25, 30,41). There is a great need for the development of a newtreatment(s) for HCV infection.

RNA interference (RNAi) has been known to beinvolved in host defense mechanisms in plants andinsects (16, 53). RNAi can be induced by double-stranded RNA (dsRNA), which is cleaved into 21- to23-bp RNA fragments, known as small interfering RNAmolecules (siRNAs), by an RNase III-like enzyme,

Dicer (2). siRNAs are then incorporated into a proteincomplex called RNA-induced silencing complex(RISC), that recognizes and cleaves mRNA in asequence-specific manner (11). It is now widelyaccepted that siRNAs function in mammals as well (3,10, 32, 37, 38, 45, 52) and prove to be a very powerfultool for silencing specific genes to evaluate their partic-ular physiological functions. Short hairpin RNA mole-cules (shRNAs), which resemble siRNA in their struc-ture, are also known to mediate RNAi. Both siRNAsand shRNAs have been shown to suppress replication ofsome viruses, including human immunodeficiency virus(HIV), poliovirus, and HCV (13, 17, 29, 40, 44, 51). It

Suppression of Hepatitis C Virus Replicon by RNAInterference Directed against the NS3 and NS5BRegions of the Viral Genome

Yuki Takigawa, Motoko Nagano-Fujii, Lin Deng, Rachmat Hidajat, Motofumi Tanaka, Hiroyuki Mizuta, and Hak Hotta*

Department of Microbiology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650–0017, Japan

Received February 10, 2004; in revised form, May 19, 2004. Accepted May 24, 2004

Abstract: RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNAduplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degra-dation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful ther-apeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of anumber of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslatedregion (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), andNS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expressionplasmid (pAVU6�27) and the other utilizing a recombinant lentivirus harboring the pAVU6�27-derivedexpression cassette. Although 5'UTR has been considered to be the most suitable region for therapeuticsiRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only afaint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid- andlentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently thereplication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNAagainst NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together,the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1would be a useful tool to inhibit HCV-1b infection.

Key words: Hepatitis C virus, Nonstructural regions, RNA interference, Lentivirus vector

591

Microbiol. Immunol., 48(8), 591–598, 2004

Abbreviations: FITC, fluorescein isothiocyanate; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; HCV, hepatitis Cvirus; IFN, interferon; NS, nonstructural region; PAGE, poly-acrylamide gel electrophoresis; PBS, phosphate-buffered saline;RISC, RNA-induced silencing complex; RNAi, RNA interfer-ence; SDS, sodium dodecyl sulfate; shRNA, short hairpin RNA;siRNA, small interfering RNA; 5'UTR, 5'-untranslated region.

*Address correspondence to Dr. Hak Hotta, Department ofMicrobiology, Kobe University Graduate School of Medicine,7–5–1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650–0017, Japan.Fax: �81–78–382–5519. E-mail: hotta@kobe-u.ac.jp

was reported that siRNAs and/or shRNAs against 5'-untranslated region (5'UTR) (44), and nonstructuralregions 3 (NS3) (19, 49) and NS5B (29) of the HCVgenome could effectively suppress the replication ofHCV replicons without induction of IFN. In theirapproaches, however, siRNAs or plasmid vectorsexpressing shRNAs should be introduced to the targetcell by using a transfection reagent or electroporation,that cannot be applied to human use.

In the present study, we aimed to express shRNAsusing a lentivirus vector harboring the pAVU6�27expression plasmid (38). We chose a number of differ-ent target regions of the HCV genome, such as 5'UTR,Core, two regions of NS3 (NS3-1 and NS3-2) andNS5B. We report here that shRNAs against NS3-1 andNS5B expressed by recombinant lentiviruses effectivelyinhibited HCV replication.

Materials and Methods

Stable HCV subgenomic replicon cell line. A plas-mid pFK2884Gly (26) expressing an HCV subgenomicreplicon and Huh-7 human hepatoma cell line werekindly provided by Dr. R. Bartenschlager (University ofHeidelberg, Heidelberg, Germany). The HCV repliconRNA was synthesized by in vitro transcription usingRiboMAX Large Scale RNA Production Systems(Promega), as reported by Lohmann et al. (26, 27) withslight modifications. Huh-7 cells were maintained inDulbecco’s modification of Eagle’s minimum essentialmedium (DMEM) supplemented with 10% fetal calfserum. Huh-7 cells were electrophoretically transfectedwith the HCV replicon and cultured in the presence ofG418 (500 µg/ml) to obtain a stable HCV replicon cellline (Huh7-FK2884Gly-1).

shRNA and siRNA. We adopted two different meth-ods, plasmid-mediated expression and lentivirus-medi-ated expression, to express shRNAs against selectedregions of the HCV genome. The positions and thesequences of the shRNAs and synthetic siRNA areshown in Fig. 1. It should be noted that the HCVsubgenomic replicon contains the first 48 nucleotides ofthe Core-coding sequence after the 5'UTR sequence(26) and that the shRNA sequence for Core resides inthis region. It was confirmed by sequence analysis thatall the shRNA-targeted sequences were maintained inthe replicon throughout the experiments (data notshown).

a) Plasmid vector-mediated shRNA expression: Plas-mid vectors expressing shRNAs were constructed asfollows. The plasmid pAVU6�27 containing humanU6 promoter (38) was kindly provided by Dr. D.R.Engelke (University of Michigan, Ann Arbor, Mich.,

U.S.A.). Chemically synthesized and annealed DNAfragments corresponding to the shRNA-sequences werecloned into the unique BamHI-XhoI site of pAVU6�27downstream of the U6 promoter. The shRNA expres-sion plasmids or an empty control vector were trans-fected into Huh7-FK2884Gly-1 cells by using FuGene6reagent (Roche) according to the manufacturer’sinstructions.

b) Lentivirus vector-mediated shRNA expression:Lentivirus vectors harboring each of the pAVU6�27-derived shRNA expression cassettes were generatedusing a commercial lentivirus vector system (ViraPowerLentiviral Expression System; Invitrogen). We firstconverted the unique BanIII site of pLenti6/V5 to aunique BamHI site using a BamHI linker. The resultantvector is called pLenti6/V5-Bam. BamHI-XhoI frag-ments of pAVU6�27-derived shRNA expression plas-mids were subcloned into the unique BamHI-XhoI sitesof pLenti6/V5-Bam. Recombinant lentiviruses express-ing shRNAs were then generated according to the man-

592 Y. TAKIGAWA ET AL

Fig. 1. shRNA-targeted regions of the HCV subgenomic repliconand sequences of the shRNAs. (A) Schematic representation ofthe HCV subgenomic replicon and shRNA-targeted regions. Ahatched box indicates part of the HCV Core-coding region (nt342 to 389), and a meshed box the internal ribosome entry site ofencephalomyocarditis virus. The shRNA-targeted regions aredepicted by arrows, with the nucleotide numbers being shown inparentheses. (B) Sequences of shRNAs expressed bypAVU6�27. (C) Sequence of synthetic siRNA.

ufacturer’s protocols. The recombinant viruses thusobtained were inoculated into Huh7-FK2884Gly-1 cellsin culture medium containing 6% polybrene for 24 hr.Titers of the stock viruses were about 1.0�105 focus-forming units (ffu)/ml. The virus-transduced cells wereselected in the presence of blasticidin (6 µg/ml).

c) Synthetic siRNA: Chemically synthesized RNAoligonucleotides were purchased (Japan Bio Service,Saitama, Japan). In order to increase the stability of thesiRNA, 3' overhang of each strand was made of thymi-dine residues instead of uridine (10, 12). Eighty µM

each of sense and antisense RNA oligonucleotides wereheated at 90 C for 1 min and incubated at 37 C for 60min in an annealing buffer (100 mM potassium acetate,30 mM HEPES-KOH (pH 7.4), 2 mM magnesiumacetate). The siRNA thus prepared was transfected toHuh7-FK2884Gly-1 cells using Oligofectamine reagent(Invitrogen) according to manufacturer’s instructions.

Immunoblot analysis. Immunoblotting was per-formed as described previously (36). In brief, Huh7-FK2884Gly-1 cells were lysed in a buffer consisting of50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.2% sodiumazide, 0.1% sodium dodecyl sulfate (SDS), 0.1% NP-40,and 0.5% sodium deoxycholate. The proteins in thelysates were resolved by SDS-polyacrylamide gel elec-trophoresis (SDS-PAGE) and electrophoretically blot-ted onto a polyvinylidene difluoride filter (Bio-Rad).The filters were blocked for 1 hr at room temperaturewith phosphate-buffered saline (PBS) containing 5%skim milk and incubated with mouse monoclonal anti-body against either NS3 (a kind gift from Dr. I. Fuke,Research Foundation for Microbial Diseases, OsakaUniversity, Kagawa, Japan) or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon Inter-national, Inc., Calif., U.S.A.). After being washed withPBS containing 0.05% Tween 20, the filters were incu-bated with peroxidase-labeled goat anti-mouse IgG.The protein bands were visualized by an enhancedchemiluminescence method (ECL; Amersham-Pharma-cia Biotech) according to the manufacturer’s instruc-tion. The intensity of each band was quantified using animage processing and analysis program (Scion Image®;Scion Corporation).

Immunofluorescence analysis. Huh7-FK2884Gly-1cells were cultivated on a cover glass in 24-well tissueculture plates. The cells were washed with PBS, fixedin 3% formaldehyde for 10 min at room temperature,and permeabilized with 0.1% Triton X-100 for 10 minat room temperature. The cells were then incubatedwith a patient’s serum that strongly reacted to HCVproteins at room temperature for 1 hr. After beingwashed with PBS, the cells were incubated with fluores-cein isothiocyanate (FITC)-conjugated goat anti-human

IgG. After being mounted in an antifade reagent (Vec-tor Laboratories, Inc., Burlingame, Calif., U.S.A.), thesamples were observed under a fluorescent microscope(Olympus, Tokyo).

Reverse transcription (RT) and PCR. Total cellularRNA was obtained from Huh7-FK2884Gly-1 cells byusing EASY-Prep (TaKaRa Shuzo, Kyoto, Japan) fol-lowed by isopropanol precipitation. The RNA sampleswere reverse transcribed using avian myeloblastosisvirus reverse transcriptase (TaKaRa) and a randomprimer, and the resultant cDNAs were subsequentlyamplified with Pfu DNA polymerase (Promega) byappropriate PCR cycles (15, 20, 25, 30, 35 cycles), eachconsisting of 1 min at 55 C, 1.5 min at 72 C, and 1 minat 94 C. A set of primers, sub196 (sense; 5'-CCATG-GCGTTAGTATGAGTG-3') and sub470R (antisense;5'-GGCACAACAGACAATCGGCT-3'), were used toamplify a portion of 5'UTR of the HCV genome.Another set of primers, β-actin5 (sense; 5'-CCAAC-CGCGAGAAGATGAC-3') and β-actin3 (antisense; 5'-AGAAGCATTTGCGGTGGAC-3') were used foramplification of β-actin mRNA. The PCR productswere electrophoresed in a 2% agarose gel containingethidium bromide and visualized by UV illumination.

Quantitative RT-PCR was also performed using ABIPRISM® 7000 Sequence Detection System and SYBRGreen PCR Core Reagents (PE Biosystems), accordingto the manufacturer’s protocol. The primers used weresub196 and sub197R (antisense; 5'-GTAGT-GTTGGGTCGCGAAAG-3') for 5'UTR of the HCVgenome. In vitro-synthesized HCV subgenomic repli-con RNA served as a standard for the quantification.

Results

HCV Replicon-Silencing Effects of pAVU6�27 Plas-mid-Mediated shRNAs

We selected a number of different target regions ofshRNAs (see Fig. 1). The sequence of 5'UTR shRNAis identical to the one previously reported (44). TheNS5B shRNA covers the same region as reported previ-ously (29) but differs from the reported sequence bythree residues due to the difference in the HCV repli-cons used in the studies. It should be noted that thisNS5B region does not seem to be well conserved sinceonly 6 (26%) of 23 HCV subtype 1b (HCV-1b) isolatesexamined had the identical sequence (unpublisheddata). In addition to those sequences, we selected othertarget sequences from the Core and NS3 regions (NS3-1and NS3-2), that are considerably well conservedamong HCV-1b isolates in Japan. The Core sequencewas identical in 44 (94%) of 47 HCV-1b isolates exam-ined (35, unpublished data) while the NS3-1 and NS3-2

593INHIBITION OF HCV REPLICON BY shRNA AND siRNA

sequences were identical in 52 (68%) and 50 (65%),respectively, of 77 isolates examined (33, 34, unpub-lished data). The Core, NS3-1 and NS3-2 sequenceswere less well conserved across different genotypes,such as HCV-2a and -3a (unpublished data). All ofthose target regions were predicted to form a loop struc-ture, which ensures easy access by RISC. The GC con-tents of the target sequences were 58% for 5'UTR, 42%for Core, 74% for NS3-1, 79% for NS3-2, and 58% forNS5B.

We used pAVU6�27 vector (39) to express shRNAs.The predicted forms and sequences of these shRNAsare shown in Fig. 1B. The shRNA expression plasmidsand a control empty vector were transfected into Huh7-FK2884Gly-1 cells and HCV protein expression wasmonitored 2 days after transfection. shRNAs againstNS3-1 and NS5B suppressed HCV protein expressionmore strongly than did those against 5'UTR, Core andNS3-2 (Fig. 2, top). It should be noted that shRNAsagainst NS3-1 or NS5B did not influence host cell pro-tein expression, as evidenced by the expression levels ofGAPDH (Fig. 2, bottom), suggesting that the suppres-sive effects of shRNAs against NS3-1 and NS5B weredirected specifically to the HCV replicon sequence.Similar results were reproducibly obtained.

HCV Replicon-Silencing Effects of shRNAs Expressedby Recombinant Lentiviruses Harboring the pAVU6�27Expression Cassette

We then generated recombinant lentiviruses harbor-ing each of the pAVU6�27-derived shRNA expression

cassette. The recombinant lentiviruses were inoculatedinto Huh7-FK2884Gly-1 cells. Consistent with theresult obtained with plasmid-mediated expression sys-tem, lentivirus-mediated shRNAs against NS3-1 andNS5B exerted stronger effects compared with the othershRNAs expressed by the lentivirus system (Fig. 3A).shRNAs against NS3-1 or NS5B expressed by recombi-nant lentiviruses did not affect GAPDH expression(Fig. 3A) or cell morphology (data not shown). Inhibi-tion of HCV replicon by shRNAs against NS3-1 andNS5B was demonstrated also by immunofluorescenceanalysis (Fig. 3B).

We also measured the amount of HCV replicon RNAin the cells by semi-quantitative RT-PCR. In mock-infected control Huh7-FK2884Gly-1 cells, the HCVgenome (5'UTR) was clearly detectable after 25 cyclesof PCR amplification (Fig. 4A). On the other hand, incells infected with lentiviruses expressing shRNAsagainst either NS3-1 or NS5B, no amplicon was detect-ed after 25 cycles of PCR amplification and only a faintband, if any, became detectable after 30 cycles ofamplification. Consistently, real time PCR analysisrevealed decreased amounts of HCV replicon RNA in

594 Y. TAKIGAWA ET AL

Fig. 3. HCV replicon-silencing effects of lentivirus-mediatedshRNAs. (A) Recombinant lentiviruses harboring thepAVU6�27-derived shRNA expression cassette described inFig. 2 were each inoculated into Huh7-FK2884Gly-1 cells andlevels of NS3 expression were determined by immunoblotanalysis 7 days after infection. NS3 expression levels normalizedto GAPDH are shown. (B) Immunofluorescence analysis. Huh7-FK2884Gly-1 cells inoculated with the recombinant lentivirusesdescribed in (A) were stained.

Fig. 2. HCV replicon-silencing effects of pAVU6�27-mediatedshRNAs. pAVU6�27 plasmids expressing shRNAs against5'UTR, Core, NS3-1, NS3-2 and NS5B were each transfectedinto Huh7-FK2884Gly-1 cells and levels of NS3 expressionwere determined by immunoblot analysis 2 days after transfec-tion (top). Control indicates the empty pAVU6�27 vector.GAPDH expression levels were also determined (bottom) andNS3 expression levels normalized to GAPDH were shown.

cells expressing shRNAs against NS3-1 or NS5B thanin the control cell (Fig. 4B), with the control harboring75 HCV RNA copies/cell whereas the shRNA-express-

ing cells harbored 10–11 copies/cell (Fig. 4C).

Gene Silencing Effects of Synthetic siRNAWe chemically synthesized siRNA corresponding to

NS3-1 (Fig. 1C) and examined its gene silencing effectin Huh7-FK2884Gly-1 cells. The 3' overhang of thesiRNA was TT, instead of UU, to ensure the stability ofthe siRNA (10). The result obtained revealed that thesiRNA synthesized against NS3-1 inhibited HCV repli-con in a dose-dependent manner (Fig. 5).

Discussion

Because of the effectiveness and specificity in genesilencing, siRNAs and shRNAs are expected to beapplicable to gene therapies for hard-to-cure diseases,such as HIV infection (6, 22), cancers (43, 48) and cer-tain genetic disorders (31). Both siRNAs and shRNAsinduce cleavage and degradation of RNA molecules in asequence-specific manner (11, 24, 37). HCV is anRNA virus and, in many cases, is difficult to eradicatefrom infected individuals even with an intensive antivi-ral therapy that utilizes IFN and ribavirin (7, 14, 15, 25,30, 41). Although a number of other antiviral com-pounds, including inhibitors against the NS3 proteaseand the RNA-dependent RNA polymerase of NS5B (1,4, 8, 39), are currently being tested for their therapeuticapplicability, such attempts have not always beenpromising.

Synthetic siRNAs have been used both in vitro and invivo to cause gene silencing (23, 29, 50). There are,however, some problems to be solved, such as the needfor high dose administration due to the lack of an effi-cient method to deliver siRNAs to target organs and/orinto target cells. Recently, plasmid vector-mediatedshRNAs were also reported (3, 32, 38, 45). Those plas-

595INHIBITION OF HCV REPLICON BY shRNA AND siRNA

Fig. 4. Comparative RT-PCR analysis of HCV replicon RNA incells inoculated with recombinant lentiviruses harboring thepAVU6�27-derived shRNA expression cassette. (A) Semi-quantitative RT-PCR analysis. Huh7-FK2884Gly-1 cells wereinoculated with the control recombinant lentivirus or virusesexpressing shRNAs against either NS3-1 or NS5B, and analyzedfor HCV replicon RNA 7 days after virus infection. PCR wasperformed over 25, 30 and 35 cycles. RT-PCR for β-actinmRNA was performed in parallel to show an equal amount oftotal RNA in each sample. (B) Quantitative, real time RT-PCRanalysis. The control Huh7-FK2884Gly-1 cells (�) and cellsexpressing NS3-1 shRNA (�) or NS5B shRNA (�) were ana-lyzed. A serial dilution of in vitro-transcribed HCV repliconRNA (dotted lines; 100, 10, 1, 0.1 and 0.01 pg) served as stan-dards for quantification. (C) Estimated numbers of HCV RNAcopies per cell.

Fig. 5. Dose-dependent silencing effects of synthetic siRNAagainst NS3-1. Huh7-FK2884Gly-1 cells were left untransfected(0 nM) or transfected with a range of 5 to 4,000 nM of syntheticsiRNA against NS3-1. The cells were analyzed for NS3 expres-sion 2 days after transfection (top). GAPDH expression levelswere also determined (bottom) and NS3 expression levels nor-malized to GAPDH are shown.

mid vectors utilize promoter sequences for RNA poly-merase III to ensure synthesis of either shRNAs (stem-loop type) or sense and anti-sense small RNA mole-cules (tandem type) that form siRNA after beingannealed. Those expression plasmids can be incorpo-rated into the genome of recombinant viruses so thatthe plasmids can be easily introduced into the targetcells through virus infection.

In the present study, we aimed to establish an effi-cient method to suppress replication of an HCV subge-nomic replicon by using shRNAs. We selected a num-ber of different regions of the HCV genome as the tar-gets: 5'UTR, Core, NS3-1, NS3-2 and NS5B (see Fig.1). In addition, we used two different ways to expressshRNAs: (i) plasmids expressing shRNA, and (ii)recombinant lentiviruses harboring the shRNA expres-sion cassette. Our result demonstrated that shRNAsagainst NS3-1 and NS5B expressed by recombinantlentivirus were most effective in suppressing the HCVsubgenomic replicon. By using recombinant lentivirus-es, the shRNA expression cassette could be introducedinto �90% of the cells in the culture. It should also beemphasized that lentivirus vectors have certain advan-tages over the other virus vectors. Lentivirus vectorscan ensure integration of an shRNA expression cassetteinto the chromosome of the cell so that shRNA is stablyexpressed for a long period of time. Moreover,lentivirus vectors can introduce the expression cassetteinto the chromosome of even non-dividing cells, such asneurons and mature hepatocytes (9, 18, 42, 47).

Viruses can change their own genomic sequences togain resistance against antiviral drugs (13, 24). HCV, inparticular, is prone to genetic mutation. It is possiblethat a given shRNA becomes ineffective after a longtime of treatment with the same shRNA. However, cer-tain regions of the viral genome are put under constraintof sequence conservation because of the importance oftheir gene products and/or their particular secondarystructures being formed. As for HCV, 5'UTR has beenconsidered to be among the most suitable targets forshRNAs because of its extremely high degree ofsequence conservation (5). NS3-1 is also likely a suit-able target as it is well conserved among clinical isolatesof HCV-1b (33, 34, unpublished data). In fact, thesequence encodes Asp-1107, which is one of the cat-alytic triad (His-Asp-Ser) of the NS3 serine protease ofHCV (20, 28) and is, therefore, indispensable for theenzymatic activity. In this connection, we observed thatthe suppressive effect of shRNA against NS3-1 lastedfor more than one month after infection with shRNA-expressing recombinant lentivirus (unpublished data).On the other hand, the shRNA sequence against NS5Bis not well conserved among different clinical isolates

and, therefore, may not be a suitable target in a clinicalsetting.

In summary, we have generated recombinantlentiviruses expressing shRNAs to specifically suppressreplication of an HCV subgenomic replicon in Huh-7cells. Our results suggest the possibility that the NS3-1sequence is a good target for gene silencing by shRNAand/or siRNA, and that the recombinant lentivirusexpressing shRNA against NS3-1 is a good tool tostudy molecular events in HCV-infected cells. Thissystem may also be applicable to development of a newantiviral therapy.

The authors are grateful to Dr. D.R. Engelke, University ofMichigan, Ann Arbor, Mich., U.S.A., and Dr. R. Bartenschlager,University of Heidelberg, Heidelberg, Germany, for providingpAVU6�27 and pFK5B2884Gly, respectively. Thanks are alsodue to Dr. I. Fuke, Research Foundation for Microbial Diseases,Osaka University, Kagawa, Japan, for providing mouse mono-clonal antibody against NS3. This work was supported in part bya Grant-in-Aid for Special Scientific Research from the Ministryof Education, Culture, Sports, Science and Technology, Japan,and a Grant-in-Aid from the Japan Society for the Promotion ofScience.

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