濠电姷顣藉Σ鍛村磻閹捐泛绶ゅΔ锝呭暞閸嬪鏌eΟ娆惧殭鏉╂繈姊虹捄銊ユ珢闁瑰嚖鎷�
闂傚倸鍊风粈渚€骞栭锔藉亱闊洦绋戠粣妤呭箹濞n剙濡奸柛銊ュ€块弻銊╂偆閸屾稑顏�: 闂備浇顕уù鐑藉极婵犳艾纾诲┑鐘叉搐缁愭鏌¢崶鈺佹灁闁崇懓绉撮埞鎴︽偐閸欏鎮欏┑鈽嗗亝閿曘垽寮诲☉銏犖ㄩ柕蹇婂墲閻濇牠鎮峰⿰鍐ㄧ盎闁瑰嚖鎷� 闂傚倸鍊风欢姘缚閼姐倖瀚婚柣鏃傚帶缁€澶愬箹濞n剙濡奸柛姘秺楠炴牕菐椤掆偓閻忣噣鏌嶇紒妯荤闁哄被鍔戝顒勫垂椤旇瀵栨繝鐢靛仧閵嗗骞忛敓锟� 闂傚倷娴囧畷鍨叏瀹ュ绀冩い顓熷灣閻ヮ亪姊绘担鍛婃儓妞ゆ垵鍊垮畷婊冣攽閸垻鐓撴繝銏f硾婢跺洭宕戦幘缁樻櫜閹肩补鈧磭顔戠紓鍌欐缁躲倝骞忛敓锟� 闂傚倸鍊烽懗鑸电仚濡炪倖鍨甸崯鏉戠暦閺囥垺鐒肩€广儱鎳愰悾娲倵楠炲灝鍔氭い锔诲灣閻ヮ亣顦归柡灞剧〒娴狅箓鎮欓鍌涱吇缂傚倷鑳舵刊鎾箯閿燂拷 闂傚倸鍊烽懗鍫曘€佹繝鍕濞村吋娼欑壕鍧楁⒑椤掆偓缁夋挳宕归崒鐐寸厸闁告劑鍔庢晶娑㈡煟閹烘洦鍤欐い顓℃硶閹瑰嫰鎼归崷顓濈礃婵犵绱曢崕鎴﹀箯閿燂拷 闂傚倸鍊烽懗鍓佹兜閸洖鐤鹃柣鎰ゴ閺嬪秹鏌ㄥ┑鍡╂Ф闁逞屽厸缁舵艾鐣烽妸鈺佺骇闁瑰濯Σ娲⒑閼姐倕孝婵炲眰鍊曡灒濠电姴娲ょ粈澶愭煥閻曞倹瀚� 濠电姷鏁搁崑鐐哄垂閸洖绠伴悹鍥у斀缂傛碍绻涢崱妯虹伇濠殿喗濞婇弻鏇熷緞閸℃ɑ鐝旂紓浣插亾鐎光偓閸曨剙浠梺鎼炲劀閸曘劍鐏嗛梻浣告啞濡垿骞忛敓锟� 闂傚倸鍊峰ù鍥磻閹扮増鍋ら柡鍐ㄧ墕閸ㄥ倸霉閸忓吋鍎楅柡浣告閺屻劑鎮ら崒娑橆伓 闂傚倷娴囬褏鈧稈鏅犲畷妯荤節濮橆厸鎸冮梺鍛婃处閸撴岸宕h箛娑欑叆闁绘洖鍊圭€氾拷 濠电姷鏁搁崑鐐哄垂閸洖绠归柍鍝勬噹閸屻劑鏌i幘宕囩槏闁荤喐瀚堥弮鍫濆窛妞ゆ挾濯Σ娲⒑閼姐倕孝婵炲眰鍊曡灒濠电姴娲ょ粈澶愭煥閻曞倹瀚� 闂傚倸鍊风粈渚€骞夐敓鐘偓鍐疀濞戞ḿ锛涢梺绯曞墲缁嬫垿寮告笟鈧弻鐔煎箲閹伴潧娈紓浣哄У閸ㄥ潡寮婚敓鐘茬妞ゆ劧绲块々浼存⒑閸濄儱娅愰柟鍑ゆ嫹
濠电姷鏁搁崕鎴犲緤閽樺娲晜閻愵剙搴婇梺绋挎湰缁嬫捇銆呴悜鑺ョ叆闁绘洖鍊圭€氾拷: 闂傚倸鍊风粈渚€骞栭锕€纾归柣鎴f绾偓闂佸憡鍔曞Ο濠傘€掓繝姘叆闁绘洖鍊圭€氾拷 闂傚倷娴囧畷鍨叏閺夋嚚娲Χ閸ワ絽浜炬慨妯煎帶閻忥附銇勯姀锛勬噰闁轰焦鎹囬弫鎾绘晸閿燂拷 闂傚倷娴囧畷鐢稿窗閹扮増鍋¢柕澶堝剻濞戞ǚ妲堥柕蹇曞Х閿涙盯姊虹捄銊ユ珢闁瑰嚖鎷� 闂傚倷鐒﹂惇褰掑春閸曨垰鍨傞梺顒€绉甸崑銈夋煛閸ャ儱鐏柛搴★躬閺屻劑鎮ら崒娑橆伓 闂傚倸鍊烽悞锕傛儑瑜版帒纾归柡鍥╁枑濞呯娀鏌﹀Ο渚▓婵炲吋鐗犻弻銊╂偆閸屾稑顏� 闂傚倸鍊烽悞锕傛儑瑜版帒纾块柛妤冧紳濞差亜惟闁挎棁妫勫鍧楁⒑鐠恒劌娅愰柟鍑ゆ嫹 闂傚倸鍊烽懗鑸电仚缂備胶绮〃鍛存偩閻戣姤鍋ㄧ紒瀣閻庮剟姊虹捄銊ユ珢闁瑰嚖鎷� 闂傚倸鍊烽懗鍫曞磿閻㈢ǹ纾婚柟鎹愵嚙缁€澶愮叓閸ャ劍灏甸柡鍡愬€濋弻銊╂偆閸屾稑顏� 闂傚倸鍊烽懗鍫曘€佹繝鍕濞村吋娼欑壕瑙勪繆閵堝懏鍣圭紒鐘崇墵閺屻劑鎮ら崒娑橆伓 闂傚倸鍊峰ù鍥敋閺嶎厼绀傛繛鍡樻尭绾惧潡鏌$仦璇插姎闁汇倝绠栭弻銊╂偆閸屾稑顏� 闂傚倸鍊烽懗鑸电仚濡炪倖鍨甸崯鏉戠暦閺囥垹绠绘い鏃傜摂濡懘姊虹捄銊ユ珢闁瑰嚖鎷� 缂傚倸鍊搁崐鎼佸磹缁嬫5娲Χ閸♀晜顔旈梺褰掓?缁讹繝寮繝鍥ㄧ叆闁绘洖鍊圭€氾拷 濠电姷鏁搁崑鐐哄垂閸洖绠伴柛顐f礀绾惧綊鏌″搴″箹闁绘帒鐏氶妵鍕箳閹搭垱鏁鹃柣搴㈢啲閹凤拷 闂傚倸鍊烽懗鍫曞储瑜嶉锝夊箚閼割兛姹楅梺鍛婂姦閸犳牜绮堟径鎰叆闁绘洖鍊圭€氾拷 闂傚倸鍊烽悞锕€顪冮崹顕呯唵濞撴埃鍋撴鐐茬箻閺佹捇鏁撻敓锟� 濠电姷鏁告繛鈧繛浣冲洤纾诲┑鐘叉搐缁狀垶鏌ㄩ悤鍌涘 闂傚倸鍊烽懗鍫曞储瑜庨幆鏂库堪閸繄顔嗛梺璺ㄥ櫐閹凤拷 闂傚倷娴囬褏鎹㈤幇顓ф闊洦绋戠粻顖炴煥閻曞倹瀚� 闂傚倸鍊烽悞锕傚箖閸洖纾块柤纰卞墰閻瑩鏌熸潏鎯х槣闁轰礁妫濋弻銊╂偆閸屾稑顏� 闂傚倷娴囧畷鍨叏閺夋嚚娲煛娴g儤娈鹃梺鍓茬厛閸嬪懎鈻嶉悩缁樼叆闁绘洖鍊圭€氾拷 闂傚倸鍊风粈渚€骞栭鈷氭椽鏁冮埀顒€鐜婚崹顔规瀻闁规儳绉村ú顓㈠极閹剧粯鏅搁柨鐕傛嫹
濠电姷鏁搁崑鐐哄垂閸洖绠归柍鍝勬噹閸屻劑鏌ゅù瀣珒闁绘帒锕弻銊╂偆閸屾稑顏�: 闂傚倷鐒﹂惇褰掑春閸曨垰鍨傞梺顒€绉甸崑銈夋煛閸ャ儱鐏柛搴★躬閺屻劑鎮ら崒娑橆伓 闂傚倸鍊峰ù鍥ь浖閵娾晜鍊块柨鏇炲€归崑锟犳煥閺囨浜剧€光偓閿濆懏鍋ラ柡浣规崌閺佹捇鏁撻敓锟� 闂傚倸鍊峰ù鍥敋閺嶎厼绀堟慨妯块哺瀹曟煡鏌涢埄鍐槈闁绘帒鐏氶妵鍕箳閹搭垱鏁鹃柣搴㈢啲閹凤拷 濠电姷鏁搁崑鐐哄垂閸洖绠归柍鍝勬噹閻鏌嶈閸撴盯鍩€椤掑喚娼愰柟纰卞亰楠炲繘鏁撻敓锟� 闂傚倸鍊风粈渚€骞夐敓鐘冲亱闁绘劘灏欓弳锕傛煟閵忊懚鍦不閺嶎厽鐓ラ柣鏇炲€圭€氾拷 濠电姷鏁搁崑鐐哄垂閸洖绠伴悹鍥у斀缂傛碍绻涢崱妯虹伇濠殿喗濞婇弻銊╂偆閸屾稑顏� 闂傚倸鍊搁崐宄懊归崶顬盯宕熼娑樹罕闂佸湱鍋撳鍧楀极娓氣偓閺屻劑鎮ら崒娑橆伓 婵犵數濮甸鏍窗濡ゅ嫭鎳岄梻浣规偠閸斿繐鈻斿☉婊呬罕闂備浇娉曢崳锕傚箯閿燂拷 闂傚倸鍊风粈渚€骞栭锕€纾圭紒瀣紩濞差亜围闁搞儻绲芥禍鍓х棯閺夋妲归悗姘炬嫹 闂傚倸鍊烽悞锕傚垂濠靛鍊块柨鏇炲€告闂佺粯鍔楅弫鍝ョ矆婵犲洦鐓ラ柣鏇炲€圭€氾拷 濠电姴鐥夐弶搴撳亾濡や焦鍙忛柟缁㈠枛鐎氬銇勯幒鎴濐仼闁藉啰鍠栭弻銊╂偆閸屾稑顏� 闂傚倸鍊烽悞锕傚箖閸洖纾块柟缁樺笧閺嗭附淇婇娆掝劅婵炲皷鏅犻弻銊╂偆閸屾稑顏� 濠电姷顣槐鏇㈠磻濞戙埄鏁勯柛銉墮缁愭鏌熼幑鎰靛殭闁告垹濞€閺屻劑鎮ら崒娑橆伓 闂傚倸鍊峰ù鍥磻閹扮増鍋ら柡鍐ㄧ墕閸ㄥ倿鏌ц箛锝呬簼婵炲懐濞€閺屻劑鎮ら崒娑橆伓 闂傚倸鍊峰ù鍥磻閹扮増鍋ら柡鍐ㄧ墕閸ㄥ倹銇勯弽銊х煂缂佺姵妫冮弻銊╂偆閸屾稑顏� 闂傚倸鍊峰ù鍥磻閹扮増鍋ら柡鍐ㄧ墕閸ㄥ倿鏌熷▓鍨灓闁告宀搁弻銊╂偆閸屾稑顏� 闂傚倸鍊风粈渚€骞栭锕€纾归悷娆忓閸ㄦ棃鏌﹀Ο渚Ш闁哄棎鍊濋弻銊╂偆閸屾稑顏� 闂傚倸鍊烽懗鍫曞箠閹捐瑙﹂悗锝庡墮閸ㄦ繈骞栧ǎ顒€濡肩痪鎯с偢閺屻劑鎮ら崒娑橆伓 闂傚倸鍊风粈渚€宕ョ€n€綁骞掗幘鍙樼矒闂佸綊妫跨粈渚€宕欓悩缁樼叆闁绘洖鍊圭€氾拷
当前位置: 首页 > 医学版 > 期刊论文 > 基础医学 > 病菌学杂志 > 2006年 > 第9期 > 正文
编号:11200593
Diverse Effects of Cyclosporine on Hepatitis C Vir
http://www.100md.com 病菌学杂志 2006年第9期

     Laboratory of Human Tumor Viruses, Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto

    Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo

    Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    ABSTRACT

    Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.

    INTRODUCTION

    Hepatitis C virus (HCV), a member of the Flaviviridae family, has a positive-strand RNA genome (1, 26). The genome encodes a large precursor polyprotein, which is cleaved by host and viral proteases to generate at least 10 functional viral proteins: core, envelope 1 (E1), E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (6, 8). NS5B is an RNA-dependent RNA polymerase that is crucial for viral genome replication (1, 26). There is genetic heterogeneity within the HCV genome. Currently, these differences are classified into six genotypes that are further segregated into a series of subtypes (4, 23). In Japan, genotype 1b is predominant; roughly 65% of cases of HCV-related chronic hepatitis involve genotype 1b. By comparison, genotype 2a is present in 17% of these patients (13, 23).

    Sustained infection of HCV is the major cause of chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (16). Rarely, HCV causes fulminant hepatitis (13). The predominant treatment for HCV-infected patients is interferon (IFN) or polyethylene glycol-conjugated IFN alone or in combination with ribavirin (19, 20). However, alternative anti-HCV therapies are needed because virus is not eliminated in about half of the treated patients (19, 20). Lohmann et al. have developed the HCV subgenomic replicon system, in which an HCV subgenomic replicon autonomously replicates in Huh-7 cells (HCV replicon cells) (18). This replicon comprises the HCV 5' untranslated region (5'UTR) containing an internal ribosomal entry site (IRES), the neomycin phosphotransferase gene, the encephalomyocarditis virus (EMCV) IRES, the coding region for HCV NS3 through NS5B, and the HCV 3'UTR (subgenomic replicon), but it lacks the coding region for the core and envelope proteins, as well as p7 and NS2 (Fig. 1). Subsequently, a genome-length (full-genome) replicon has been developed. This construct contains a full-genome length of HCV, including the coding regions for the core protein through NS2 (Fig. 1) (5, 10). We can evaluate HCV replication using these subgenomic or genome-length replicon systems. Previously, we established HCV subgenomic replicon cells carrying HCV genotype 1b NN strain (15, 29). We demonstrated that an immunosuppressant, cyclosporine (CsA), has anti-HCV activity in these cells (29). In addition, we determined the molecular mechanism of the anti-HCV effect of CsA on this replicon; cyclophilin B (CyPB), one of the cellular targets of CsA, is a cellular replication cofactor of the HCV genome (31). CyPB interacts with NS5B to promote its RNA binding activity (for a detailed description, see reference 31). CsA is suggested to suppress HCV genome replication by inhibiting the functional association of CyPB with NS5B. Another group also reported anti-HCV function of CsA using a subgenomic replicon of other genotype 1b strain, HCV-N (22). In this study, we demonstrate that CsA also has a strong anti-HCV activity in other available genotype 1b replicons carrying the Con1 and O strains (12, 18).

    Recently, Wakita and colleagues reported that a replicon of HCV genotype 2a JFH-1 strain, which was isolated from a case of type-C fulminant hepatitis, has a much stronger level of replication activity than genotype 1b replicons in Huh-7 cells (13, 27). A production system of infectious viral particles was recently established with this high-replication-competent strain (17, 27, 34). This viral strain may acquire a growth advantage compared with many other strains, although the underlying mechanism is unknown. In this study, we described a characteristic difference in the replication of JFH1 compared to that of genotype 1b replicons.

    Here, we report that JFH1 replication is less sensitive to CsA than genotype 1b strains, although the interaction of CyPB with NS5B is observed with this replicon. However, genome replication and RNA binding activity of NS5B are independent of CyPB. We have exploited a chemical compound to demonstrate how strain diversity can be generated by underlying differences in the mechanisms of the virus-cell interaction. These findings provide important insight into the mechanisms that mediate the efficacy of antiviral agents.

    MATERIALS AND METHODS

    Cell culture. Huh-7 cells were cultured in Dulbecco's modified Eagle medium (Invitrogen) with 10% fetal bovine serum, nonessential amino acids (Invitrogen), and L-glutamine (Invitrogen). MH-14, #50-1, MH14#W31, SN1, sO (formerly named 1B2R1), JFH1#4-1, and JFH1#2-3 cells (12, 13, 15, 18, 29), carrying subgenomic replicons, and NNC#2, SN1A#2, and SNC#7 cells, carrying full-genome replicons, were cultured in the above medium supplemented with 300- to 500-μg/ml G418 (Invitrogen). In the assay measuring the response to CsA, NIM811, or PSC833 (Fig. 2, 3, and 4), we seeded small numbers of each replicon cells (7 x 103 to 15 x 103 cells/12-well plate) and treated with each drug. Culture medium was changed every 3 days (CsA, NIM811, or PSC833 was supplemented in the fresh medium for the treatment groups). We did not perform any passages in the assay period. At day 7, the cells were 70 to 90% confluent. A schematic representation of the constructs of HCV replicon RNAs, the name of HCV strains from which the replicon RNA sequences are derived, and the name of replicon cell clones used in this study are summarized in Fig. 1. Since many replicon clones were used in this study, we list "strain/genotype/length of the replicon construct" in parentheses after the names of each cell clone in Results and in the figure legends to avoid confusion between names: for example, MH-14 (NN/1b/SG), JFH1#4-1 (JFH1/2a/SG), and SN1A#2 (Con1/1b/FL) cells. The designations SG and FL indicate subgenomic and full-genome replicons, respectively.

    Establishment of replicon cells. MH-14, #50-1, sO, JFH1#4-1, and JFH1#2-3 cells were described previously (12, 13, 15, 29). The replicon RNAs were produced using a MEGAscript T7 kit (Ambion) from pMH14, pSN1, pNNC, pSN1A, and pSNC plasmids for the establishment of the MH14#W31, SN1, NNC#2, SN1A#2, and SNC#7 replicon cells, respectively. For the establishment of MH14#W31, we transfected RNA into the Huh-7 cell strain which was identical to the parental cells of JFH1#4-1 and JFH1#2-3. Each replicon RNA was transfected into Huh-7 cells, following the selection with the medium in the presence of 500- to 1,000-μg/ml G418 for around 4 weeks. The resultant cell colonies were isolated and expanded. The HCV RNA titers in cell clones carrying JFH1 replicons were not significantly different from those in established cell clones carrying genotype 1b replicons.

    Plasmid construction. pSN1, the sequence of which is derived from I377NS3-3' (18), was prepared essentially as described previously (15). pSN1A was generated by inserting the region from the core to NS2 of pM1LE (15) into the upstream coding region for NS3 in pSN1. To obtain pSNC, the EMCV IRES of pSN1A was replaced by the HCV IRES. pNNC was produced by inserting the coding region from NS3 to NS5B of pM1LE into pSNC.

    Real-time reverse transcription-PCR (RT-PCR) analysis. The 5'UTR of HCV genome RNA was quantified using the ABI PRISM 7700 sequence detector (Applied Biosystems) as described previously (29).

    Immunoblot analysis. Immunoblot analysis was performed as described previously (30). The primary antibodies used in this study were anti-core, anti-E2 (kindly provided by M. Kohara, Tokyo Metropolitan Institute of Medical Science), anti-NS3, anti-NS5A (a generous gift from A. Takamizawa, Osaka University), anti-NS5B (NS5B-6; kindly provided by I. Fukuya, Osaka University), anti-CyPA (Upstate Cell Signaling), anti-CyPB (Affinity BioReagents), and anti-tubulin (Oncogene).

    Immunoprecipitation assay and RNA-protein binding precipitation assay. Immunoprecipitation and RNA-protein binding precipitation were performed as described previously (30, 31).

    RNA interference technique. The condition of small interfering RNA (siRNA) used in this study was described previously (31). Transfection was performed using siLentFect (Bio-Rad), according to the manufacturer's protocol.

    Isolation of replication complex. The HCV replication complex was isolated from cells by treatment with 50-μg/ml digitonin at 27°C for 5 min, following treatment with 0.3-μg/ml proteinase K at 37°C for 5 min as described previously (31).

    Purification of recombinant GST-fused CyPB protein. Glutathione S-transferase (GST) and GST-fused CyPB (GST-CyPB) protein expression was induced in transformed BL21 cells (Amersham) with 1 mM isopropyl--thiogalactopyranoside (IPTG). The cell lysate was incubated with glutathione-Sepharose resin (Amersham) and washed extensively. The recombinant protein was eluted by glutathione (pH 8.0) and subsequently dialyzed.

    In vitro RNA binding assay. In vitro-translated 35S-labeled NS5B proteins and poly(U)-Sepharose (Amersham) or protein G-Sepharose (Amersham) resin as a negative control were incubated in the presence of recombinant GST-CyPB protein at 4°C for 1 h. After being washed, precipitates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by imaging analyzer.

    RESULTS

    CsA suppressed the replication of HCV full-genome replicon. We and another group have reported an anti-HCV activity of CsA using subgenomic replicons (22, 29). HCV structural proteins, especially the core protein, have multiple functions. These proteins interact with many cellular factors and modulate a variety of cellular functions (32). Potentially, these viral proteins could diminish or circumvent the suppression of HCV genome replication by CsA. Core protein and E2 reportedly modulate the activity of IFN signaling (9, 25). To test this possibility, we established a full-genome HCV replicon system with cells transfected with the NN strain (NNC#2 cells [NN/1b/FL]) (Fig. 1). HCV RNA and protein productions were confirmed by real-time RT-PCR and immunoblot analysis (Fig. 2A and B). In addition, we confirmed that this replication was not due to the integration of the replicon construct into the cellular genome (data not shown). Similarly, we generated other full-genome replicon cells carrying sequences from the Con1 strain at the nonstructural coding region of the replicon RNA (SN1A#2 [Con1/1b/FL]) and SNC#7 (Con1/1b/FL) cells (Fig. 1). The replicon of SN1A#2 (Con1/1b/FL) cells possessed the EMCV IRES upstream of the open reading frame for HCV proteins, while that of SNC#7 (Con1/1b/FL) cells contained the HCV IRES (Fig. 1). SNC#7 (Con1/1b/FL) cells exhibited almost the same response as that of SN1A#2 (Con1/1b/FL) cells to CsA treatment (Fig. 2D). Consistent with a previous report (22), the EMCV IRES was not responsible for the anti-HCV activity of CsA. We compared the sensitivity to CsA of full-genome replicons with that of subgenomic replicons. CsA strongly decreased the production of HCV proteins in both the full-genome replicon, NNC#2 (NN/1b/FL) cells and the subgenomic replicon, MH-14 (NN/1b/SG) cells (Fig. 2C). Real-time RT-PCR analysis also revealed a dramatic reduction of the RNA level of full-genome replicons in NNC#2 (NN/1b/FL), SN1A#2 (Con1/1b/FL), and SNC#7 (Con1/1b/FL) cells (Fig. 2D). The 50% inhibitory concentrations (IC50) of CsA in NNC#2 (NN/1b/FL), SN1A#2 (Con1/1b/FL), and SNC#7 (Con1/1b/FL) cells awere estimated to be 0.13, 0.19, and 0.24 μg/ml, respectively. The 90% inhibitory concentrations (IC90) of CsA in these cells were 0.68, 0.94, and 0.81 μg/ml, respectively. The CsA dose-response curves of full-genome replicons and subgenomic replicons were similar (i.e., compare SN1A#2 or SNC#7 [Con1/1b/FL] versus SN1 [Con1/1b/SG], NNC#2 [NN/1b/FL] versus MH-14, #50-1, or MH14#W31 [NN/1b/SG]) (Fig. 3C). These results demonstrate that CsA suppresses the replication of full-genome replicons and subgenomic replicons to almost the same extent. Since CsA concentrations of up to 3 μg/ml did not affect the proliferation of any replicon cells (Fig. 2E and data not shown), the effect of CsA on replication is not due to the cytotoxic effect. In addition, we observed the reduction of production of infectious viral particles in the presence of 3-μg/ml CsA (data not shown) using the viral production system with full-genome JFH1 RNA (27).

    The JFH1 replicon was less sensitive to CsA than were genotype 1b replicons. We compared the sensitivity of HCV replication to CsA in several subgenomic replicon cells. We used MH-14 (NN/1b/SG) and #50-1 (NN/1b/SG) cells carrying subgenomic replicons with HCV NN strain (15, 29), SN1 (Con1/1b/SG) cells carrying the Con1 subgenomic replicon (18), and sO (O/1b/SG) cells bearing the subgenomic O strain (12) as genotype 1b replicon-containing cells. We also employed JFH1#4-1 (JFH1/2a/SG) and JFH1#2-3 (JFH1/2a/SG) cell clones carrying the JFH1 subgenomic replicon (13). Treatment of CsA (1 μg/ml; 7 days) drastically decreased HCV RNA in all the subgenomic replicon cells carrying the HCV genotype 1b strain. HCV RNA levels in SN1 (Con1/1b/SG), MH-14 (NN/1b/SG), sO (O/1b/SG), and #50-1 (NN/1b/SG) cells decreased to 1/134, 1/219, 1/128, and 1/295, respectively (Fig. 3A). Genotype 1b replicon cells appeared highly sensitive to CsA. In contrast, the effect of CsA on HCV RNA levels in replicon cells containing sequences from the JFH1 strain was limited to 1/5 to 1/7 (Fig. 3A). These results of the response to CsA were reproduced in further additional cell clones.

    The cellular characteristics of Huh-7 cell strains differ among laboratories. To exclude the possibility that differences between Huh-7 cell strains influence the sensitivity to CsA, we established genotype 1b replicon cells based on the identical Huh-7 cell strain, which were used as parental cells of JFH1#4-1 (JFH1/2a/SG) and JFH1#2-3 (JFH1/2a/SG) cells. The response of the corresponding replicon cells, MH14#W31 (NN/1b/SG), to CsA was almost the same as that of SN1 (Con1/1b/SG), MH-14 (NN/1b/SG), sO (O/1b/SG), and #50-1 (NN/1b/SG) cells (Fig. 3C). Thus, the difference in sensitivity of JFH1 and genotype 1b strains to CsA can be attributed to the characteristic differences of the HCV strains, not to the parental Huh-7 cell strain. In addition, the reduction of NS3 protein in JFH1#4-1 (JFH1/2a/SG) cells following treatment with CsA was less prominent than that in MH14#W31 (NN/1b/SG) cells (Fig. 3B).

    We examined the dose-response curve of HCV RNA against the concentration of CsA (Fig. 3C). The effect of CsA in genotype 1b replicons plateaued at around 1 μg/ml, while in the dose-response curve in JFH1 replicon, the inhibition was not yet saturated (Fig. 3C). As concentrations of CsA up to 3 μg/ml did not affect the proliferation rate of any replicon cells (Fig. 3D and data not shown), the effect of CsA on replication was not due to the cytotoxic effect. The IC50 of CsA in MH-14 (NN/1b/SG), #50-1 (NN/1b/SG), MH14#W31 (NN/1b/SG), SN1 (Con1/1b/SG), sO (O/1b/SG), JFH1#4-1 (JFH1/2a/SG), and JFH1#2-3 (JFH1/2a/SG) cells were estimated to be 0.15, 0.18, 0.16, 0.20, 0.25, 0.67, and 0.43 μg/ml, respectively. The IC90 was 0.86, 0.82, 0.76, 0.88, 0.92, 2.77, and 2.39 μg/ml, respectively. A similar dose-response curve in the JFH1 replicon was obtained by a transient replication assay with the luciferase reporter driven from a JFH1 replicon construct (data not shown) (14).

    JFH1 replicon was less sensitive to a CsA derivative, NIM811. Analysis of several CsA derivatives has revealed that the anti-HCV effect of CsA on the genotype 1b replicon is mediated by the inhibition of CyP (31). We examined the sensitivity of JFH1 replicon to CsA derivatives. CsA is known to have three major cellular targets: CyP, calcineurin (CN)/NF-AT, and P glycoprotein (P-gp) (28, 31). A CsA derivative, NIM811, inhibits CyP and P-gp but not CN/NF-AT, while another derivative, PSC833, inhibits P-gp but neither CyP nor CN/NF-AT (31). The decrease of HCV RNA in MH14#W31 (NN/1b/SG) cells with NIM811 treatment (0.5 μg/ml; 7 days) was more than an order of magnitude greater than that in JFH1#4-1 (JFH1/2a/SG) cells (Fig. 4A). The slope of the dose-response curve of NIM811 treatment of the JFH1 replicon was gentler than that of genotype 1b (Fig. 4B). The IC50 of NIM811 in MH14W#31 (NN/1b/SG) and JFH1#4-1 (JFH1/2a/SG) cells were 0.17 and 0.30 μg/ml, respectively. The IC90 were 0.46 and 0.93 μg/ml, respectively. In contrast, PSC833, which does not inhibit CyP, did not alter HCV RNA level in either genotype 1b or the JFH1 replicon (Fig. 4C). Thus, a CyP inhibitor was less effective at suppressing the replication of the JFH1 replicon than genotype 1b replicons.

    Interactions between CyPB and JFH1 NS5B. Previously, we have shown that CyPB interacts with NS5B to promote HCV genome replication and that CsA inhibits this binding in a genotype 1b replicon (31). Here, we examined the association between CyPB and NS5B in a JFH1 replicon. Immunoprecipitation analysis revealed an interaction of CyPB with NS5B in JFH1#4-1 (JFH1/2a/SG) cells (Fig. 5A). This interaction was dissociated following the treatment of CsA, as observed with the genotype 1b replicon (Fig. 5B).

    Role of CyPB in replication of the JFH1 replicon. Although we observed some differences of expression levels of endogenous CyPB among the replicon cells in the immunoblot analysis (Fig. 6A), there was no particular correlation between endogenous CyPB expression levels and replication sensitivity to CsA among cells. CyPB reportedly regulates HCV genome replication of the genotype 1b replicon (31). We then explored the requirement of CyPB for the replication of JFH1 replicon with RNA interference. Transfecting siRNAs designed to recognize several CyP subtypes [si-CyP(broad)] (Fig. 6B) reduced HCV RNA to <1/5 in MH14#W31 (NN/1b/SG) cells (Fig. 6C). Specific knockdown of CyPB but not CyPA (Fig. 6B) decreased HCV RNA in MH14#W31 (NN/1b/SG) cells, consistent with a previous report (Fig. 6C) (31). In contrast, HCV RNA in JFH1#4-1 (JFH1/2a/SG) cells was not altered following the suppression of either endogenous CyPA or CyPB (Fig. 6B and C). We observed a weak decrease of HCV RNA levels (around one-half) with si-CyP(broad) (Fig. 6C). These data suggests the possibility that the replication of the JFH1 replicon is independent of CyPB, in contrast to the genotype 1b replicon. In the previous study, it was reported that the doubling time, saturation density, and response to cell confluence of the replicon cells carrying JFH1 were different from those in cells carrying a genotype 1b replicon, suggesting the possibility that the coupling relationship between the replication and cell growth was different between genotype 1b and the JFH1 replicon (21). The introduction of either si-CyPB or si-CyP(broad), however, had little effect on cell growth in MH14#W31 (NN/1b/SG) or JFH1#4-1 (JFH1/2a/SG) cells (Fig. 6D). And we did not observe cells being confluent in the experiment period. The above results suggest that the different response to si-CyPB in the two lines is independent of the conditions of cell growth.

    The role of CyPB in the RNA binding activity of JFH1 NS5B. CyPB regulates HCV genome replication of a genotype 1b replicon by promoting the RNA binding activity of NS5B (31). We examined the effect of CyPB on the RNA binding activity of NS5B in JFH1. NS5B in the replication complex was isolated from cells by treatment with digitonin-proteinase K, as described previously (31). This fraction was incubated with poly(U) RNA-Sepharose or protein G-Sepharose as a negative control for the detection of RNA binding NS5B in the replication complex. RNA-bound NS5B in this fraction from MH14#W31 (NN/1b/SG) cells was decreased drastically following treatment with CsA (Fig. 7A, lanes 5 and 6). However, the reduction of RNA binding of NS5B in the replication complex of JFH1#4-1 (JFH1/2a/SG) cells was not as prominent (Fig. 7A, lanes 11 and 12). We confirmed this result by an in vitro RNA binding assay, in which in vitro-synthesized NS5B was incubated with poly(U) RNA-Sepharose, together with recombinant GST-CyPB. The addition of recombinant GST-CyPB increased the binding of genotype 1b NS5B to poly(U) RNA (Fig. 7B and C). However, this augmentation of RNA binding was not observed with NS5B from the JFH1 strain (Fig. 7B and C). From the above results, it is suggested that the RNA binding of JFH1 NS5B is free from regulation by CyPB.

    DISCUSSION

    Until now, we and another group have utilized subgenomic replicons carrying genotype 1b NN and HCV-N strains to demonstrate that CsA suppresses HCV genome replication (22, 29). This study reveals that CsA is effective on full-genome replicons to almost the same extent. In addition, other available genotype 1b replicons carrying the Con1 and O strains also have a high sensitivity to CsA, consistent with our proposal that HCV genotype 1b is highly sensitive to CsA. However, a fulminant-type genotype 2a replicon, JFH1, was less responsive to CsA, although a high dose of CsA suppressed the replication of this strain.

    CyPB interacts with genotype 1b NS5B to stimulate its RNA binding activity. In contrast, CyPB binds JFH1 NS5B but does not regulate the function of JFH1 NS5B. This is consistent with a previous speculation that genotype 1b and JFH1 replicons utilize the same cellular factors in distinct manners (21). The NS5B sequence of NN strain has 95.0, 95.9, and 70.4% homology to that of Con1, O, and JFH1, respectively (Fig. 8). The region spanning amino acids (aa) 521 to 591 of NS5B, which is involved in the interaction with CyPB (31), is highly conserved among genotype 1b strains NN, Con1, and O while that of JFH1 has 21 substituted residues in this region. The proline at 540 aa, which is important for CyPB binding (31), is conserved but the adjacent residues such as isoleucine at 539 aa and alanine at 541 aa are replaced by leucine and glutamic acid, respectively, in JFH1. Through molecular interactions, CyPB seems to make the conformation of NS5B of genotype 1b strains but not JFH1 suitable for RNA binding (31). The diverse regulation system of NS5B by CyPB among strains may be due to differences in either the sequence or the entire conformation of NS5B. Further study is important for elucidating the regulation mechanism of RNA binding activity of NS5B by CyPB.

    Thus, replication in JFH1 replicon is independent of CyPB. Interestingly, human immunodeficiency virus type 1 (HIV-1) strains also have a diversity of CyP dependence on viral proliferation (3, 33). CyPA plays an important role in the life cycle of HIV-1. The interaction of the HIV-1 capsid protein with CyPA that resides within the target cells of infection is critical for HIV-1 replication (7, 24). In peripheral blood mononuclear cells or Jurkat T cells, CsA suppresses the proliferation of HIV-1 group main (M) strain (3). However, certain strains of group outlier (O), such as MVP5180 and MVP9435, are resistant to CsA (3, 33), suggesting the different dependency of the replication on CyPA. Authors have suggested that MVP5180 and MVP9435 clones adapt to replicate independently of CyPA and that this adaptation provides a significant replication advantage for the virus in vivo (3). In vesicular stomatitis virus (VSV) strains, a role for CyPA in virus replication also has been reported (2). CyPA is required for the infection of the VSV-NJ strain but not the VSV-IND strain. These authors proposed that during evolutionary divergence from the ancestral lineages that initially were dependent on CyPA for replication, VSV-IND may have adapted to reduce its dependency on CyPA (2). In the case of HCV, a fulminant type genotype 2a replicon (JFH1) replicates independently of CyPB. It has previously been reported that JFH1 has a much higher competency of replication in the cells than other strains (13). The adaptation to independence from CyPB may contribute to the high capacity of replication of JFH1.

    Although the JFH1 replicon is less sensitive to CsA, high concentrations of CsA still suppress replication of the JFH1 replicon. Moreover, the introduction of the siRNA designed to recognize several CyP subtypes [si-CyP(broad)] moderately diminishes HCV RNA in the JFH1 replicon. We suspect that a CyP family member other than CyPB is involved in HCV genome replication. Further analysis is needed on the role of other CyP subtypes.

    As there a replicon system for a fulminant-type genotype 1b replicon or chronic-type genotype 2a replicon does not yet exist, we cannot conclude whether chronic-type genotype 2a replicons or fulminant-type replicons are less sensitive to CsA or not. However, there is a clinical report describing cotreatment of patients with chronic hepatitis C with IFN and CsA that resulted in a higher sustained virological rate than with treatment of IFN alone (11). In this report, increase in the sustained virological rate was prominent with patients carrying genotype 1 HCV (51.7% versus 21.9%), while it was relatively weak in patients carrying genotype 2 HCV (66.7% versus 58.3%) (11). Thus, genotype may affect the sensitivity of HCV replication to CsA. However, we cannot exclude the possibility that the diminished sensitivity to CsA is a characteristic only of the fulminant-type genotype 2a strain.

    Our results suggest that sensitivity to CsA and replication dependency to CyPB is different among HCV strains. This finding is an important insight into the diversity of the mechanism of HCV genome replication and its sensitivity to antiviral agents.

    ACKNOWLEDGMENTS

    We thank H. Takahashi and M. Hosaka for preparing replicon cells and generating plasmids. We are grateful to A. Takamizawa at Osaka University, I. Fukuya at Osaka University, and M. Kohara at Tokyo Metropolitan Institute of Medical Science for antibodies and R. Bartenschlager at Heiderberg University for the I377/NS3-3' sequence. We also appreciate Novartis (Basel, Switzerland) for providing the CsA derivatives NIM811 and PSC833.

    This work was supported by grants-in-aid for cancer research and for the second-term comprehensive 10-year strategy for cancer control from the Ministry of Health, Labor, and Welfare; grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology; and grants-in-aid from the Research for the Future from the Japanese Society for the Promotion of Science, the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan, and Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.

    N.I. and K.W. contributed equally to this work.

    REFERENCES

    Bartenschlager, R., and V. Lohmann. 2001. Novel cell culture systems for the hepatitis C virus. Antiviral Res. 52:1-17.

    Bose, S., M. Mathur, P. Bates, N. Joshi, and A. K. Banerjee. 2003. Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype. J. Gen. Virol. 84:1687-1699.

    Braaten, D., E. K. Franke, and J. Luban. 1996. Cyclophilin A is required for the replication of group M human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIVCPZGAB but not group O HIV-1 or other primate immunodeficiency viruses. J. Virol. 70:4220-4227.

    Bukh, J., R. H. Purcell, and R. H. Miller. 1994. Sequence analysis of the core gene of 14 hepatitis C virus genotypes. Proc. Natl. Acad. Sci. USA 91:8239-8243.

    Frese, M., V. Schwarzle, K. Barth, N. Krieger, V. Lohmann, S. Mihm, O. Haller, and R. Bartenschlager. 2002. Interferon-gamma inhibits replication of subgenomic and genomic hepatitis C virus RNAs. Hepatology 35:694-703.

    Grakoui, A., C. Wychowski, C. Lin, S. M. Feinstone, and C. M. Rice. 1993. Expression and identification of hepatitis C virus polyprotein cleavage products. J. Virol. 67:1385-1395.

    Hatziioannou, T., D. Perez-Caballero, S. Cowan, and P. D. Bieniasz. 2005. Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells. J. Virol. 79:176-183.

    Hijikata, M., H. Mizushima, T. Akagi, S. Mori, N. Kakiuchi, N. Kato, T. Tanaka, K. Kimura, and K. Shimotohno. 1993. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J. Virol. 67:4665-4675.

    Hosui, A., K. Ohkawa, H. Ishida, A. Sato, F. Nakanishi, K. Ueda, T. Takehara, A. Kasahara, Y. Sasaki, M. Hori, and N. Hayashi. 2003. Hepatitis C virus core protein differently regulates the JAK-STAT signaling pathway under interleukin-6 and interferon-gamma stimuli. J. Biol. Chem. 278:28562-28571.

    Ikeda, M., M. Yi, K. Li, and S. M. Lemon. 2002. Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J. Virol. 76:2997-3006.

    Inoue, K., K. Sekiyama, M. Yamada, T. Watanabe, H. Yasuda, and M. Yoshiba. 2003. Combined interferon 2b and cyclosporin A in the treatment of chronic hepatitis C: controlled trial. J. Gastroenterol. 38:567-572.

    Kato, N., K. Sugiyama, K. Namba, H. Dansako, T. Nakamura, M. Takami, K. Naka, A. Nozaki, and K. Shimotohno. 2003. Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro. Biochem. Biophys. Res. Commun. 306:756-766.

    Kato, T., T. Date, M. Miyamoto, A. Furusaka, K. Tokushige, M. Mizokami, and T. Wakita. 2003. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125:1808-1817.

    Kato, T., T. Date, M. Miyamoto, M. Sugiyama, Y. Tanaka, E. Orito, T. Ohno, K. Sugihara, I. Hasegawa, K. Fujiwara, K. Ito, A. Ozasa, M. Mizokami, and T. Wakita. 2005. Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system. J. Clin. Microbiol. 43:5679-5684.

    Kishine, H., K. Sugiyama, M. Hijikata, N. Kato, H. Takahashi, T. Noshi, Y. Nio, M. Hosaka, Y. Miyanari, and K. Shimotohno. 2002. Subgenomic replicon derived from a cell line infected with the hepatitis C virus. Biochem. Biophys. Res. Commun. 293:993-999.

    Liang, T. J., and T. Heller. 2004. Pathogenesis of hepatitis C-associated hepatocellular carcinoma. Gastroenterology 127:S62-S71.

    Lindenbach, B. D., M. J. Evans, A. J. Syder, B. Wolk, T. L. Tellinghuisen, C. C. Liu, T. Maruyama, R. O. Hynes, D. R. Burton, J. A. McKeating, and C. M. Rice. 2005. Complete replication of hepatitis C virus in cell culture. Science 309:623-626.

    Lohmann, V., F. Korner, J. Koch, U. Herian, L. Theilmann, and R. Bartenschlager. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110-113.

    Manns, M. P., J. G. McHutchison, S. C. Gordon, V. K. Rustgi, M. Shiffman, R. Reindollar, Z. D. Goodman, K. Koury, M. Ling, and J. K. Albrecht. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358:958-965.

    McHutchison, J. G., S. C. Gordon, E. R. Schiff, M. L. Shiffman, W. M. Lee, V. K. Rustgi, Z. D. Goodman, M. H. Ling, S. Cort, J. K. Albrecht, et al. 1998. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N. Engl. J. Med. 339:1485-1492.

    Miyamoto, M., T. Kato, T. Date, M. Mizokami, and T. Wakita. 2006. Comparison between subgenomic replicons of hepatitis C virus genotypes 2a (JFH-1) and 1b (Con1 NK5.1). Intervirology 49:37-43.

    Nakagawa, M., N. Sakamoto, N. Enomoto, Y. Tanabe, N. Kanazawa, T. Koyama, M. Kurosaki, S. Maekawa, T. Yamashiro, C. H. Chen, Y. Itsui, S. Kakinuma, and M. Watanabe. 2004. Specific inhibition of hepatitis C virus replication by cyclosporin A. Biochem. Biophys. Res. Commun. 313:42-47.

    Ohno, O., M. Mizokami, R. R. Wu, M. G. Saleh, K. Ohba, E. Orito, M. Mukaide, R. Williams, and J. Y. Lau. 1997. New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a. J. Clin. Microbiol. 35:201-207.

    Sokolskaja, E., D. M. Sayah, and J. Luban. 2004. Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity. J. Virol. 78:12800-12808.

    Taylor, D. R., S. T. Shi, P. R. Romano, G. N. Barber, and M. M. Lai. 1999. Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285:107-110.

    Tellinghuisen, T. L., and C. M. Rice. 2002. Interaction between hepatitis C virus proteins and host cell factors. Curr. Opin. Microbiol. 5:419-427.

    Wakita, T., T. Pietschmann, T. Kato, T. Date, M. Miyamoto, Z. Zhao, K. Murthy, A. Habermann, H. G. Krausslich, M. Mizokami, R. Bartenschlager, and T. J. Liang. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11:791-796.

    Waldmeier, P. C., K. Zimmermann, T. Qian, M. Tintelnot-Blomley, and J. J. Lemasters. 2003. Cyclophilin D as a drug target. Curr. Med. Chem. 10:1485-1506.

    Watashi, K., M. Hijikata, M. Hosaka, M. Yamaji, and K. Shimotohno. 2003. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 38:1282-1288.

    Watashi, K., M. Hijikata, A. Tagawa, T. Doi, H. Marusawa, and K. Shimotohno. 2003. Modulation of retinoid signaling by a cytoplasmic viral protein via sequestration of Sp110b, a potent transcriptional corepressor of retinoic acid receptor, from the nucleus. Mol. Cell. Biol. 23:7498-7509.

    Watashi, K., N. Ishii, M. Hijikata, D. Inoue, T. Murata, Y. Miyanari, and K. Shimotohno. 2005. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol. Cell 19:111-122.

    Watashi, K., and K. Shimotohno. 2003. The roles of hepatitis C virus proteins in modulation of cellular functions: a novel action mechanism of the HCV core protein on gene regulation by nuclear hormone receptors. Cancer Sci. 94:937-943.

    Wiegers, K., and H. G. Krausslich. 2002. Differential dependence of the infectivity of HIV-1 group O isolates on the cellular protein cyclophilin A. Virology 294:289-295.

    Zhong, J., P. Gastaminza, G. Cheng, S. Kapadia, T. Kato, D. R. Burton, S. F. Wieland, S. L. Uprichard, T. Wakita, and F. V. Chisari. 2005. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. USA 102:9294-9299.(Naoto Ishii, Koichi Watas)
    婵犵數鍎戠徊钘壝洪悩璇茬婵犻潧娲ら閬嶆煕濞戝崬鏋ゆい鈺冨厴閺屾稑鈽夐崡鐐差潾闁哄鏅滃Λ鍐蓟濞戞ǚ鏋庨煫鍥ㄦ尨閸嬫挻绂掔€n亞鍔﹀銈嗗坊閸嬫捇鏌涢悩宕囥€掓俊鍙夊姇閳规垿宕堕埞鐐亙闁诲骸绠嶉崕鍗炍涘☉銏犵劦妞ゆ帒顦悘锔筋殽閻愬樊鍎旀鐐叉喘椤㈡棃宕ㄩ鐐靛搸婵犵數鍋犻幓顏嗗緤閹灐娲箣閻樺吀绗夐梺鎸庣箓閹峰宕甸崼婢棃鏁傜粵瀣妼闂佸摜鍋為幐鎶藉蓟閺囥垹骞㈤柡鍥╁Т婵′粙鏌i姀鈺佺仩缂傚秴锕獮濠囨晸閻樿尙鐤€濡炪倖鎸鹃崑鐔哥閹扮増鈷戦柛锔诲帎閻熸噴娲Χ閸ヮ煈娼熼梺鍐叉惈閹冲氦绻氶梻浣呵归張顒傜矙閹烘鍊垫い鏂垮⒔绾惧ジ鏌¢崘銊モ偓绋挎毄濠电姭鎷冮崟鍨杹閻庢鍠栭悥鐓庣暦濮椻偓婵℃瓕顦抽柛鎾村灦缁绘稓鈧稒岣块惌濠偽旈悩鍙夋喐闁轰緡鍣i、鏇㈡晜閽樺鈧稑鈹戦敍鍕粶濠⒀呮櫕缁瑦绻濋崶銊у幐婵犮垼娉涢敃銈夊汲閺囩喐鍙忛柣鐔煎亰濡偓闂佽桨绀佺粔鎾偩濠靛绀冩い顓熷灣閹寸兘姊绘担绛嬪殐闁哥姵鎹囧畷婵婄疀濞戣鲸鏅g紓鍌欑劍宀e潡鍩㈤弮鍫熺厽闁瑰鍎戞笟娑㈡煕閺傚灝鏆i柡宀嬬節瀹曟帒顫濋鐘靛幀缂傚倷鐒﹂〃鍛此囬柆宥呯劦妞ゆ帒鍠氬ḿ鎰磼椤旇偐绠婚柨婵堝仱閺佸啴宕掑鍗炴憢闂佽崵濞€缂傛艾鈻嶉敐鍥╃煋闁割煈鍠撻埀顒佸笒椤繈顢橀悩顐n潔闂備線娼уú銈吤洪妸鈺佺劦妞ゆ帒鍋嗛弨鐗堢箾婢跺娲寸€规洏鍨芥俊鍫曞炊閵娿儺浼曢柣鐔哥矌婢ф鏁Δ鍜冪稏濠㈣埖鍔栭崑锝夋煕閵夘垰顩☉鎾瑰皺缁辨帗娼忛妸褏鐣奸梺褰掝棑婵炩偓闁诡喗绮撻幐濠冨緞婢跺瞼姊炬繝鐢靛仜椤曨厽鎱ㄦィ鍐ㄦ槬闁哄稁鍘奸崹鍌炴煏婵炵偓娅嗛柛瀣ㄥ妼闇夐柨婵嗘噹閺嗙喐淇婇姘卞ⅵ婵﹥妞介、鏇㈡晲閸℃瑦顓婚梻浣虹帛閹碱偆鎹㈠┑瀣祦閻庯綆鍠栫粻锝嗙節婵犲倸顏柟鏋姂濮婃椽骞愭惔锝傛闂佸搫鐗滈崜鐔风暦閻熸壋鍫柛鏇ㄥ弾濞村嫬顪冮妶鍡楃瑐闁绘帪绠撳鎶筋敂閸喓鍘遍梺鐟版惈缁夋潙鐣甸崱娑欑厓鐟滄粓宕滃顒夋僵闁靛ň鏅滈崑鍌炴煥閻斿搫孝閻熸瑱绠撻獮鏍箹椤撶偟浠紓浣插亾濠㈣泛鈯曡ぐ鎺戠闁稿繗鍋愬▓銈夋⒑缂佹ḿ绠栭柣鈺婂灠閻g兘鏁撻悩鑼槰闂佽偐鈷堥崜姘额敊閹达附鈷戦悹鍥b偓铏亖闂佸憡鏌ㄦ鎼佸煝閹捐绠i柣鎰綑椤庢挸鈹戦悩璇у伐闁哥噥鍨堕獮鍡涘磼濮n厼缍婇幃鈺呭箵閹烘繂濡锋繝鐢靛Л閸嬫捇鏌熷▓鍨灓缁鹃箖绠栭弻鐔衡偓鐢登瑰暩閻熸粎澧楅悡锟犲蓟濞戙垹绠抽柡鍌氱氨閺嬪懎鈹戦悙鍙夊櫣闂佸府绲炬穱濠囧箻椤旇姤娅㈤梺璺ㄥ櫐閹凤拷

   闂佽娴烽弫濠氬磻婵犲洤绐楅柡鍥╁枔閳瑰秴鈹戦悩鍙夋悙婵☆偅锕㈤弻娑㈠Ψ閵忊剝鐝栭悷婊冨簻閹凤拷  闂傚倷鑳舵灙缂佺粯顨呴埢宥夊即閵忕姵鐎梺缁樺姉閸庛倝宕曞畝鍕厽闁逛即娼ф晶顔姐亜鎼搭垱瀚�  闂備浇宕垫慨鏉懨洪妶鍥e亾濮樼厧鐏︽い銏$懇楠炲鏁冮埀顒傜矆閸曨垱鐓熸俊顖濐嚙缁茶崵绱撳蹇斿  闂傚倷鑳堕幊鎾诲触鐎n剙鍨濋幖娣妼绾惧ジ鏌曟繛鐐珔闁告濞婇弻鈩冨緞鐎n亞鍔稿┑鈽嗗灲閹凤拷   闂傚倷娴囬~澶嬬娴犲绀夌€光偓閸曨剙浠遍梺闈浤涢崨顖ょ床婵犵數鍋涘Λ娆撳礉閺嶎偆涓嶆繛鍡樻尰閻撴洘绻涢崱妯兼噭閻庢熬鎷�   闂傚倷绀侀幉鈥愁潖缂佹ɑ鍙忛柟缁㈠枛閻鏌涢埄鍐槈缂備讲鏅犻弻鐔碱敍濠婂喚鏆銈冨劵閹凤拷