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Identification of a Lactoferrin-derived Peptide Possessing Binding Activity to Hepatitis C Virus E2 Envelope Protein*

  • Akito Nozaki
    Affiliations
    Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan,
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  • Masanori Ikeda
    Affiliations
    Gastroenterological Center, Yokohama City University Medical Center, Yokohama City University School of Medicine, Yokohama 236-0004, Japan, and
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  • Atsushi Naganuma
    Footnotes
    Affiliations
    Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan,
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  • Takashi Nakamura
    Affiliations
    Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan,
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  • Michiharu Inudoh
    Affiliations
    Department of Pediatrics, Tsukuba University School of Medicine, Tsukuba 305-8575, Japan
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  • Katsuaki Tanaka
    Affiliations
    Gastroenterological Center, Yokohama City University Medical Center, Yokohama City University School of Medicine, Yokohama 236-0004, Japan, and
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  • Nobuyuki Kato
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-86-235-7385; Fax: 81-86-235-7392; E-mail: .
    Affiliations
    Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan,
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  • Author Footnotes
    * This work was supported by grants-in-aid for the Second-Term Comprehensive 10-Year Strategy for Cancer Control and for research on hepatitis and BSE from the Ministry of Health, Labor and Welfare and by grants-in-aid for scientific research from the Organization for Pharmaceutical Safety and Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ¶ Present address: The First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan.
Open AccessPublished:January 09, 2003DOI:https://doi.org/10.1074/jbc.M207879200
      Bovine and human lactoferrins (LF) prevent hepatitis C virus (HCV) infection in cultured human hepatocytes; the preventive mechanism is thought to be the direct interaction between LF and HCV. To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to determine which region(s) of LF are important for this binding activity. Several regions of human LF have been expressed and purified as thioredoxin-fused proteins in Escherichia coli. Far-Western blot analysis using these LF fragments and the E2 protein, expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein. The 93 carboxyl amino acids of LFs derived from bovine and horse cells also possessed similar binding activity to the E2 protein. In addition, the amino acid sequences of these carboxyl regions appeared to show partial homology to CD81, a candidate receptor for HCV, and the binding activity of these carboxyl regions was also comparable with that of CD81. Further deletion analysis identified 33 amino acid residues as the minimum binding site in the carboxyl region of LF, and the binding specificity of these 33 amino acids was also confirmed by using 33 maltose-binding protein-fused amino acids. Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in cultured human hepatocytes. In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for binding to the E2 protein. These results led us to consider the development of an effective anti-HCV peptide. This is the first identification of a natural protein-derived peptide that specifically binds to HCV E2 protein and prevents HCV infection.
      HCV
      hepatitis C virus
      LF
      lactoferrin
      aa
      amino acid
      TF
      transferrin
      BSA
      bovine serum albumin
      PNGaseF
      peptide-N-glycosidase F
      RT
      reverse transcription
      LEL
      large extracellular loop
      TRX
      thioredoxin
      MBP
      maltose-binding protein
      ELISA
      enzyme-linked immunosorbent assay
      HBS
      HEPES-buffered saline
      Hepatitis C virus (HCV)1infection frequently causes chronic hepatitis (
      • Choo Q.L.
      • Kuo G.
      • Weiner A.J.
      • Overby L.R.
      • Bradley D.W.
      • Houghton M.
      ,
      • Kuo G.
      • Choo Q.L.
      • Alter H.J.
      • Gitnick G.L.
      • Redeker A.G.
      • Purcell R.H.
      • Miyamura T.
      • Dienstag J.L.
      • Alter M.J.
      • Stevens C.E.
      • Tegtmeier G.E.
      • Bonino F.
      • Colombo W.S.
      • Lee W.S.
      • Kuo C.
      • Berger K.
      • Shuster J.R.
      • Overby L.R.
      • Bradley D.W.
      • Houghton M.
      ) and frequently progresses to liver cirrhosis and hepatocellular carcinoma (
      • Ohkoshi S.
      • Kojima H.
      • Tawaraya H.
      • Miyajima T.
      • Kamimura T.
      • Asakura H.
      • Satoh A.
      • Hirose S.
      • Hijikata M.
      • Kato N.
      • Shimotohno K.
      ,
      • Saito I.
      • Miyamura T.
      • Ohbayashi A.
      • Harada H.
      • Katayama T.
      • Kikuchi Y.
      • Watanabe S.
      • Koi S.
      • Onji M.
      • Ohta Y.
      • Choo Q.L.
      • Houghton M.
      • Kuo G.
      ). HCV is an enveloped positive single-stranded RNA (9.6 kb) virus belonging to the Flaviviridae (
      • Kato N.
      • Hijikata M.
      • Ootsuyama Y.
      • Nakagawa M.
      • Ohkoshi S.
      • Sugimura T.
      • Shimotohno K.
      ,
      • Miller R.H.
      • Purcell R.H.
      ,
      • Tanaka T.
      • Kato N.
      • Cho M.J.
      • Shimotohno K.
      ). The HCV genome encodes a large polyprotein precursor of about 3,000 amino acid (aa) residues, which is cleaved by the host and viral proteases to generate at least ten proteins: the core, E1 (envelope 1), E2, p7, NS2 (nonstructural protein 2), NS3, NS4A, NS4B, NS5A, and NS5B (
      • Hijikata M.
      • Kato N.
      • Ootsuyama Y.
      • Nakagawa M.
      • Shimotohno K.
      ,
      • Hijikata M.
      • Mizushima H.
      • Tanji Y.
      • Komoda Y.
      • Hirowatari Y.
      • Akagi T.
      • Kato N.
      • Kimura K.
      • Shimotohno K.
      ,
      • Grakoui A.
      • Wychowski C.
      • Lin C.
      • Feinstone S.M.
      • Rice C.M.
      ,
      • Lin C.
      • Lindenbach B.D.
      • Pragai B.M.
      • McCourt D.W.
      • Rice C.M.
      ,
      • Mizushima H.
      • Hijikata M.
      • Tanji Y.
      • Kimura K.
      • Shimotohno K.
      ). The most characteristic feature of the HCV genome is its remarkable sequence heterogeneities and variations, and to date at least six major HCV genotypes, which have been further grouped into more than 50 subtypes, have been identified (
      • Bukh J.
      • Miller R.H.
      • Purcell R.H.
      ,
      • Simmonds P.
      ,
      • Purcell R.
      ,
      • Kato N.
      ). The genetic complexity of HCV is thus a major hindrance to the development of the vaccines.
      To date, interferon has been the sole effective antiviral reagent used in the clinical therapy of hepatitis C, but its effectiveness is limited to about 30% of the reported cases (
      • Shiratori Y.
      • Kato N.
      • Yokosuka O.
      • Imazeki F.
      • Hashimoto E.
      • Hayashi N.
      • Nakamura A.
      • Asada M.
      • Kuroda H.
      • Tanaka N.
      • Arakawa Y.
      • Omata M.
      ). Combined treatment of interferon and ribavirin has been shown to be more effective than treatment with interferon alone (
      • McHutchison J.G.
      • Gordon S.C.
      • Schiff E.R.
      • Shiffman M.L.
      • Lee W.M.
      • Rustgi V.K.
      • Goodman Z.D.
      • Ling M.H.
      • Cort S.
      • Albrecht J.K.
      ). The side effects of interferon are also in some cases severe enough to lead to treatment cessation.
      Although the entry mechanism of HCV, as well as that of hepatitis B virus, remains unclear, it was reported recently that human CD81 (
      • Pileri P.
      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
      • Petracca R.
      • Weiner A.J.
      • Houghton M.
      • Rosa D.
      • Grandi G.
      • Abrignani S.
      ) and scavenger receptor class B type I (
      • Scarselli E.
      • Ansuini H.
      • Cerino R.
      • Roccasecca R.M.
      • Acali S.
      • Filocamo G.
      • Traboni C.
      • Nicosia A.
      • Cortese R.
      • Vitelli A.
      ) could be bound by a truncated, soluble form of the E2 protein; such findings suggest that these proteins may act as receptors for HCV on the cell surface. Low density lipoprotein receptor (
      • Agnello V.
      • Abel G.
      • Elfahal M.
      • Knight G.B.
      • Zhang Q.X.
      ) was also reported as a putative HCV receptor in endocytosis experiments using isolated HCV-lipoprotein complexes. However, because of the lack of a reproducible and efficient HCV proliferation system, it is not known whether these candidate receptors for HCV serve as the functional receptor on human hepatocytes (
      • Kato N.
      • Shimotohno K.
      ).
      We previously reported that non-neoplastic human hepatocyte-derived PH5CH8 cells supported HCV replication, although HCV proliferation was at a fairly low level; in that study, we also demonstrated the antiviral effects of interferon-α in HCV-infected PH5CH8 cells (
      • Ikeda M.
      • Sugiyama K.
      • Mizutani T.
      • Tanaka T.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Kato N.
      ). Using a PH5CH8 cell culture system, we found that bovine and human lactoferrin (LF), a milk glycoprotein belonging to the iron transporter family, specifically prevented HCV infection in the cells (
      • Ikeda M.
      • Sugiyama K.
      • Tanaka T.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Kato N.
      ). Recently, Matsuura et al. (
      • Matsuura Y.
      • Tani H.
      • Suzuki K.
      • Kimura-Someya T.
      • Suzuki R.
      • Aizaki H.
      • Ishii K.
      • Moriishi K.
      • Robison C.S.
      • Whitt M.A.
      • Miyamura T.
      ) also showed that bovine LF specifically inhibited infection by the pseudotype vesicular stomatitis virus possessing chimeric HCV E1 and E2 glycoproteins.
      LF has a molecular mass of 80 kDa and consists of two homologous globular lobes (an N-lobe and a C-lobe), each with a single iron (Fe3+) binding site. There is a notable degree of internal homology between the two lobes, i.e. ∼35% identical amino acid residues have been identified in the corresponding portions (
      • Levay P.F.
      • Viljoen M.
      ). The three-dimensional structures of human and bovine LFs have been clarified by crystallographic studies (
      • Anderson B.F.
      • Baker H.M.
      • Norris G.E.
      • Rice D.W.
      • Baker E.N.
      ,
      • Moore S.A.
      • Anderson B.F.
      • Groom C.R.
      • Haridas M.
      • Baker E.N.
      ). Although the overall structure of LF is similar to that of transferrin (TF) (∼60% amino acid sequence homology to LF), LF has two distinct features that may be functionally important. First, the association constant of LF for iron is 300 times that of TF (
      • Aisen P.
      • Leibman A.
      ). Second, in contrast to TF, LF possesses strong inhibitory activity against bacterial growth. The antimicrobial activity of LF has been ascribed to the basic N-terminal region (“lactoferricin”) (
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      ). Lactoferricin (24 aa residues) shows activity against a wide range of microorganisms including bacteria and fungi (
      • Bellamy W.
      • Takase M.
      • Yamauchi K.
      • Wakabayashi H.
      • Kawase K.
      • Tomita M.
      ). LF is present in the milk of most mammals, and the LF content in milk changes substantially during the lactation period. The concentrations of LF in mature milk are 0.1–0.4 mg/ml in bovines and 1–3 mg/ml in humans, and LF is especially enriched in the colostrum (0.8 mg/ml in bovines and 10 mg/ml in humans) (
      • Sanchez L.
      • Aranda P.
      • Perez M.D.
      • Calvo M.
      ,
      • Nagasawa T.
      • Kiyosawa I.
      • Kuwahara K.
      ). It is well established that LF plays an important role in the newborn as the primary nonspecific defense against pathogenic microorganisms (
      • Levay P.F.
      • Viljoen M.
      ). Also, it has been reported that rats fed a 2% bovine LF diet displayed no significant side effects (
      • Sekine K.
      • Watanabe E.
      • Nakamura J.
      • Takasuka N.
      • Kim D.J.
      • Asamoto M.
      • Krutovskikh V.
      • Baba-Toriyama H.
      • Ota T.
      • Moore M.A.
      • Masuda M.
      • Sugimoto H.
      • Nishino H.
      • Kakizoe T.
      • Tsuda H.
      ). This low risk of severe side effects presents a major clinical advantage of bovine LF; hence, clinical pilot studies have been performed recently. The results have shown that bovine LF was effective in some patients with chronic hepatitis C (
      • Tanaka K.
      • Ikeda M.
      • Nozaki A.
      • Kato N.
      • Tsuda H.
      • Saito S.
      • Sekihara H.
      ,
      • Iwasa M.
      • Kaito M.
      • Ikoma J.
      • Takeo M.
      • Imoto I.
      • Adachi Y.
      • Yamauchi K.
      • Koizumi R.
      • Teraguchi S.
      ).
      A recent study by our group (
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      ) suggested that the prevention of HCV infection in these cells was because of interactions of LF with HCV rather than with the cells themselves; our study demonstrated that LF inhibited viral entry into the cells by interacting directly with HCV (
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      ). On the other hand, Yi et al. (
      • Yi M.
      • Kaneko S.
      • Yu D.Y.
      • Murakami S.
      ) independently demonstrated that HCV envelope proteins (E1 and E2) could bind to human and bovine LFs, although their binding specificities have not been clarified. E2 protein expressed in mammalian cells specifically binds to human target cells (
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • Dong C.
      • Weiner A.J.
      • Lau J.Y.N.
      • Choo Q.L.
      • Chien D.
      • Pileri P.
      • Houghton M.
      • Abrignani S.
      ), and such binding is associated with HCV particles binding to target cells in vitro, as well as with HCV infection in vivo (
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • Dong C.
      • Weiner A.J.
      • Lau J.Y.N.
      • Choo Q.L.
      • Chien D.
      • Pileri P.
      • Houghton M.
      • Abrignani S.
      ). In addition, the level of antibody response to E2 protein has been shown to correlate with protection against HCV in animal models (
      • Farci P.
      • Shimoda A.
      • Wong D.
      • Cabezon T.
      • De Gioannis D.
      • Strazzera A.
      • Shimizu Y.
      • Shapiro M.
      • Alter H.J.
      • Purcell R.H.
      ) and with occasional clearance of HCV in cases of natural infection (
      • Ishii K.
      • Rosa D.
      • Watanabe Y.
      • Katayama T.
      • Harada H.
      • Wyatt C.
      • Kiyosawa K.
      • Aizaki H.
      • Matsuura Y.
      • Houghton M.
      • Abrignani S.
      • Miyamura T.
      ), suggesting that the E2 protein is the major receptor-binding protein. For these reasons, we focused on the interactions between LF and E2 proteins to understand the mechanism by which LF prevents HCV infection of target cells. In this study, we have characterized the binding activity of LF to the E2 protein and have endeavored to determine which region(s) of LF are important for this binding activity. Here, we report the finding of 33 human LF-derived amino acids possessing binding activity to the E2 protein of HCV, which leads to inhibition of HCV infection in target cells.

      DISCUSSION

      Based on the our studies (
      • Ikeda M.
      • Sugiyama K.
      • Tanaka T.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Kato N.
      ,
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      ) and those of other groups (
      • Yi M.
      • Kaneko S.
      • Yu D.Y.
      • Murakami S.
      ), we undertook observations regarding the direct interactions between LF and HCV envelope proteins. In this study, we demonstrated the binding specificity between LF and the E2 protein and identified 33 aa residues from human LF that are primarily responsible for E2 protein binding activity and inhibiting HCV infection of target cells.
      We observed that the deglycosylation of human and bovine LFs enhanced E2 protein binding activity. This observation suggests that a certain N-linked oligosaccharide chain interferes with the interaction between LF and the E2 protein. Comparison with the putative N-linked glycosylation sites between human LF (3 sites; aa 138, 479, and 624) and bovine LF (5 sites; aa 233, 281, 368, 476, and 545) revealed that only one glycosylation site (aa 479 for human LF, aa 476 for bovine LF) is conserved in both LFs. Therefore, it is likely that this conserved glycosylation site, which is located in C-s1, weakens the E2 protein binding activity of human or bovine LF. This may also explain why we were able to detect the strong binding affinity of C-s3 for the E2 protein. However, the reason why the C-lobe did not bind to the E2 protein remains unclear. One possibility would be that the refolding of the TRX-fused C-lobe was not successful. Our results indicate that at least two regions of human LF (the N-lobe and C-s3) are involved in the interaction with the E2 protein, although we could not identify the binding region in the N-lobe. Although N-s3 shows ∼35% aa homology to C-s3, N-s3 is unable to bind to the E2 protein. In addition, 12 aa are shared in common between the N-s3 fragment (aa 256–287) and the C-s3 fragment (aa 600–632) identified as a critical domain for binding to E2 protein. However, a Cys residue at aa 628 appeared to be essential for binding to the E2 protein; it is of note that this Cys is not present in the corresponding position of N-s3. Therefore, some boundary region between N-s1 and N-s2, or between N-s2 and N-s3, may possess the binding ability to the E2 protein. Interestingly, the C-s3 fragment (aa 600–632) was located close to the boundary region between N-s2 and N-s3 in the three-dimensional structure of human LF (
      • Anderson B.F.
      • Baker H.M.
      • Norris G.E.
      • Rice D.W.
      • Baker E.N.
      ), suggesting that the E2 protein is able to bind to both sites of human LF. To clarify this assumption, further analysis will be needed.
      We demonstrated that C-s3-relevant fragments (93 aa) of bovine and horse LFs bound as well to the E2 protein as did C-s3. The C-s3-relevant fragments of bovine and horse LFs show 74 and 71% aa sequence homology to C-s3 of human LF, respectively. These values are significantly higher than those (58 and 53%, respectively) of C-s3-relevant fragments of human and bovine TFs, which possess little binding activity to the E2 protein. The identified critical domain (aa 600–632) of human LF also shows 70% aa sequence homology to bovine LF (aa 597–629), whereas it shows only 42% aa sequence homology to the relevant regions of human or bovine TF (Fig. 10). These data suggest that the binding activity to E2 protein is restricted to the LF family.
      During the process of characterization of a C-s3 LF fragment possessing E2 protein binding activity, we noticed that C-s3 showed partial aa homology with LEL of CD81, which can also bind to E2 protein and is considered as a candidate HCV receptor (
      • Pileri P.
      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
      • Petracca R.
      • Weiner A.J.
      • Houghton M.
      • Rosa D.
      • Grandi G.
      • Abrignani S.
      ). The E2 protein binding activity of CD81 LEL has been well characterizedin vitro (
      • Petracca R.
      • Falugi F.
      • Galli G.
      • Norais N.
      • Rosa D.
      • Campagnoli S.
      • Burgio V.
      • Di Stasio E.
      • Giardina B.
      • Houghton M.
      • Abrignali S.
      • Grandi G.
      ) and in vivo (
      • Flint M.
      • Maidens C.
      • Loomis-Price L.D.
      • Shotton C.
      • Dubuisson J.
      • Monk P.
      • Higginbottom A.
      • Levy S.
      • McKeating J.A.
      ), and the binding specificity of CD81 to E2 protein has been clarified (
      • Higginbottom A.
      • Quinn E.R.
      • Kuo C.C.
      • Flint M.
      • Wilson L.H.
      • Bianchi E.
      • Nicosia A.
      • Monk P.N.
      • McKeating J.A.
      • Levy S.
      ). However, it has been reported that CD81 is not directly involved in the cell fusion caused by HCV (
      • Takikawa S.
      • Ishii K.
      • Aizaki H.
      • Suzuki T.
      • Asakura H.
      • Matsuura Y.
      • Miyamura T.
      ). Because it has been shown that TRX-fused C-s3 (93 aa) has a comparable E2 protein binding ability with that of TRX-fused CD81 LEL (89 aa), C-s3 may interfere with the binding of the E2 protein to CD81. This may be one of the reasons why LF prevents HCV infection in target cells. However, one major contradiction remains. Regarding the interaction between human CD81 LEL and the E2 protein, it has been shown that aa 186 (Phe) of the CD81 is the critical residue for binding to the E2 protein (
      • Higginbottom A.
      • Quinn E.R.
      • Kuo C.C.
      • Flint M.
      • Wilson L.H.
      • Bianchi E.
      • Nicosia A.
      • Monk P.N.
      • McKeating J.A.
      • Levy S.
      ). This Phe residue is conserved between human CD81 and LF (aa 635) (Fig. 5 A). However, in this study, it appeared that the Phe at aa 635 of human and bovine LFs was unimportant for binding to the E2 protein, because the C-s3 fragment (aa 600–632) possessing the E2 protein binding activity does not contain this Phe residue. In addition, it has been reported that the four Cys of CD81 LEL form two disulfide bridges, the integrity of which would be necessary for CD81-E2 interaction (
      • Oren R.
      • Takahashi S.
      • Doss C.
      • Levy R.
      • Levy S.
      ). However, such a phenomenon was not observed in C-s3 containing five Cys residues, because the C-s3 fragment (aa 600–632) identified as the E2 protein binding domain contains only one Cys. Therefore, these data suggest that the E2 protein region targeted by human LF and CD81 may differ. Preliminarily, our experiment with Far-Western blot analysis showed that the C-s3 fragment (aa 600–632) preferentially bound to aa 411–500 of the E2 protein, which is one of two regions (aa 384–500 and 600–661) identified previously (
      • Yi M.
      • Kaneko S.
      • Yu D.Y.
      • Murakami S.
      ) as regions binding to human LF; however, the C-s3 fragment (aa 600–632) did not bind to aa 501–599 of E2 protein (data not shown). Because it has been indicated that both aa 480–493 and aa 544–551 of the E2 protein are involved in the binding to CD81 (
      • Flint M.
      • Maidens C.
      • Loomis-Price L.D.
      • Shotton C.
      • Dubuisson J.
      • Monk P.
      • Higginbottom A.
      • Levy S.
      • McKeating J.A.
      ), human LF and CD81 may recognize rather different sites on the E2 protein. Further analysis will be necessary to clarify this point.
      Because aa 600–632 of human LF possesses only one Cys at aa 628, it is unlikely that a disulfide bond is required for the E2 protein binding activity of the C-s3 fragment (aa 600–632). However, we cannot exclude the possibility that Cys at aa 628 paired with other Cys residues in TRX during the refolding process of the Far-Western blot analysis; this could have provided E2 protein binding activity. To exclude this possibility, we constructed the MBP-fused C-s3 fragment (aa 600–632), because Cys was not present in the MBP portion including the linker region. Although the MBP-fused C-s3 fragment (aa 600–632) possesses only one Cys, this fusion protein showed similar E2 protein binding activity with that of the TRX-fused C-s3 fragment (aa 600–632). Therefore, the present results suggest that a disulfide bond is not required for binding to the E2 protein, in contrast to the case involving CD81 LEL (
      • Petracca R.
      • Falugi F.
      • Galli G.
      • Norais N.
      • Rosa D.
      • Campagnoli S.
      • Burgio V.
      • Di Stasio E.
      • Giardina B.
      • Houghton M.
      • Abrignali S.
      • Grandi G.
      ). However, it is of note that site-directed mutagenesis to Ala in both terminal regions of the C-s3 fragment (aa 600–632) revealed that Cys at aa 628 is the most critical residue for binding to the E2 protein. To clarify whether only the Cys residue at this position is necessary for binding to the E2 protein, further experiments (e.g. substitution of amino acids other than Ala) will be needed.
      Because it is well known that E1 and E2 proteins form a non-covalently linked heterodimer, which probably represents the surface of infectious virus particles (
      • Deleersnyder V.
      • Pillez A.
      • Wychowski C.
      • Blight K.
      • Xu J.
      • Hahn Y.S.
      • Rice C.M.
      • Dubuisson J.
      ), it is important to clarify whether the C-s3 fragment (aa 600–632) identified in this study binds to the heterodimer of E1 and E2 proteins. To date, aa 441–500 of E2 protein has been identified as the E1 protein heterodimeric binding region (
      • Yi M.K.
      • Nakamoto Y.
      • Kaneko S.
      • Murakami S.
      ). Although our preliminary results estimated that the C-s3 fragment (aa 600–632) binds to aa 411–500 of the E2 protein, the E2 protein binding activity of the C-s3 fragment (aa 600–632) may not be affected by heteromeric complex formation between E1 and E2 proteins, because LF prevents HCV infection by direct interaction between LF and HCV (
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      ).
      We demonstrated the anti-HCV activity of the MBP-fused C-s3 fragment (aa 600–632) in our HCV infection system using PH5CH8 cells (
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      ,
      • Nozaki A.
      • Kato N.
      ). Although this result suggests that the E2 protein binding activity contributes to the prevention of HCV infection, our results revealed that the anti-HCV activity of the MBP-fused C-s3 fragment (aa 600–632) was severalfold weaker than that of human LF. However, site-directed mutagenesis to an Ala residue within aa 600–605 and aa 625–632 of human LF revealed that several positions strengthened the E2 protein binding activity. This result suggests that some peptides possess stronger binding activity than that of the C-s3 fragment (aa 600–632) and that these peptides could be obtained by screening peptide libraries, e.g. phage libraries. The antiviral activities of such peptides will be evaluated using our cell culture assay system (
      • Ikeda M.
      • Nozaki A.
      • Sugiyama K.
      • Tanaka T.
      • Naganuma A.
      • Tanaka K.
      • Sekihara H.
      • Shimotohno K.
      • Saito M.
      • Kato N.
      ,
      • Nozaki A.
      • Kato N.
      ). Furthermore, such peptides may be useful for the removal of circulating HCV. In any case, the present study broadens the possibilities for developing anti-HCV peptides in the future.

      Acknowledgments

      We are grateful to Dr. H. Kariwa (Hokkaido University) for providing normal bovine breast tissue and normal horse peripheral blood mononuclear cells. We thank Dr. K. Shimotohno (Kyoto University), Dr. K. Sugiyama (National Cancer Center Research Institute, Tokyo, Japan), and Dr. T. Tanaka (National Institute of Neurology and Psychiatry) for helpful suggestions and for discussions about the study. We thank A. Morishita for helpful experimental assistance.

      REFERENCES

        • Choo Q.L.
        • Kuo G.
        • Weiner A.J.
        • Overby L.R.
        • Bradley D.W.
        • Houghton M.
        Science. 1989; 244: 359-362
        • Kuo G.
        • Choo Q.L.
        • Alter H.J.
        • Gitnick G.L.
        • Redeker A.G.
        • Purcell R.H.
        • Miyamura T.
        • Dienstag J.L.
        • Alter M.J.
        • Stevens C.E.
        • Tegtmeier G.E.
        • Bonino F.
        • Colombo W.S.
        • Lee W.S.
        • Kuo C.
        • Berger K.
        • Shuster J.R.
        • Overby L.R.
        • Bradley D.W.
        • Houghton M.
        Science. 1989; 244: 362-364
        • Ohkoshi S.
        • Kojima H.
        • Tawaraya H.
        • Miyajima T.
        • Kamimura T.
        • Asakura H.
        • Satoh A.
        • Hirose S.
        • Hijikata M.
        • Kato N.
        • Shimotohno K.
        Jpn. J. Cancer Res. 1990; 81: 550-553
        • Saito I.
        • Miyamura T.
        • Ohbayashi A.
        • Harada H.
        • Katayama T.
        • Kikuchi Y.
        • Watanabe S.
        • Koi S.
        • Onji M.
        • Ohta Y.
        • Choo Q.L.
        • Houghton M.
        • Kuo G.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549
        • Kato N.
        • Hijikata M.
        • Ootsuyama Y.
        • Nakagawa M.
        • Ohkoshi S.
        • Sugimura T.
        • Shimotohno K.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528
        • Miller R.H.
        • Purcell R.H.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2057-2061
        • Tanaka T.
        • Kato N.
        • Cho M.J.
        • Shimotohno K.
        Biochem. Biophys. Res. Commun. 1995; 215: 744-749
        • Hijikata M.
        • Kato N.
        • Ootsuyama Y.
        • Nakagawa M.
        • Shimotohno K.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5547-5551
        • Hijikata M.
        • Mizushima H.
        • Tanji Y.
        • Komoda Y.
        • Hirowatari Y.
        • Akagi T.
        • Kato N.
        • Kimura K.
        • Shimotohno K.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10773-10777
        • Grakoui A.
        • Wychowski C.
        • Lin C.
        • Feinstone S.M.
        • Rice C.M.
        J. Virol. 1993; 67: 1385-1395
        • Lin C.
        • Lindenbach B.D.
        • Pragai B.M.
        • McCourt D.W.
        • Rice C.M.
        J. Virol. 1994; 68: 5063-5073
        • Mizushima H.
        • Hijikata M.
        • Tanji Y.
        • Kimura K.
        • Shimotohno K.
        J. Virol. 1994; 68: 2731-2734
        • Bukh J.
        • Miller R.H.
        • Purcell R.H.
        Semin. Liver Dis. 1995; 15: 41-63
        • Simmonds P.
        Hepatology. 1995; 21: 570-583
        • Purcell R.
        Hepatology. 1997; 26 Suppl. 1: 11s-14s
        • Kato N.
        Microb. Comp. Genomics. 2000; 5: 129-151
        • Shiratori Y.
        • Kato N.
        • Yokosuka O.
        • Imazeki F.
        • Hashimoto E.
        • Hayashi N.
        • Nakamura A.
        • Asada M.
        • Kuroda H.
        • Tanaka N.
        • Arakawa Y.
        • Omata M.
        Gastroenterology. 1997; 113: 558-566
        • McHutchison J.G.
        • Gordon S.C.
        • Schiff E.R.
        • Shiffman M.L.
        • Lee W.M.
        • Rustgi V.K.
        • Goodman Z.D.
        • Ling M.H.
        • Cort S.
        • Albrecht J.K.
        N. Engl. J. Med. 1998; 339: 1485-1492
        • Pileri P.
        • Uematsu Y.
        • Campagnoli S.
        • Galli G.
        • Falugi F.
        • Petracca R.
        • Weiner A.J.
        • Houghton M.
        • Rosa D.
        • Grandi G.
        • Abrignani S.
        Sience. 1998; 282: 938-941
        • Scarselli E.
        • Ansuini H.
        • Cerino R.
        • Roccasecca R.M.
        • Acali S.
        • Filocamo G.
        • Traboni C.
        • Nicosia A.
        • Cortese R.
        • Vitelli A.
        EMBO J. 2002; 21: 5017-5025
        • Agnello V.
        • Abel G.
        • Elfahal M.
        • Knight G.B.
        • Zhang Q.X.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12766-12771
        • Kato N.
        • Shimotohno K.
        Curr. Top. Microbiol. Immunol. 2000; 242: 261-278
        • Ikeda M.
        • Sugiyama K.
        • Mizutani T.
        • Tanaka T.
        • Tanaka K.
        • Sekihara H.
        • Shimotohno K.
        • Kato N.
        Virus Res. 1998; 56: 157-167
        • Ikeda M.
        • Sugiyama K.
        • Tanaka T.
        • Tanaka K.
        • Sekihara H.
        • Shimotohno K.
        • Kato N.
        Biochem. Biophys. Res. Commun. 1998; 245: 549-553
        • Matsuura Y.
        • Tani H.
        • Suzuki K.
        • Kimura-Someya T.
        • Suzuki R.
        • Aizaki H.
        • Ishii K.
        • Moriishi K.
        • Robison C.S.
        • Whitt M.A.
        • Miyamura T.
        Virology. 2001; 286: 263-275
        • Levay P.F.
        • Viljoen M.
        Haematologica. 1995; 80: 252-267
        • Anderson B.F.
        • Baker H.M.
        • Norris G.E.
        • Rice D.W.
        • Baker E.N.
        J. Mol. Biol. 1989; 209: 711-734
        • Moore S.A.
        • Anderson B.F.
        • Groom C.R.
        • Haridas M.
        • Baker E.N.
        J. Mol. Biol. 1997; 274: 222-236
        • Aisen P.
        • Leibman A.
        Biochim. Biophys. Acta. 1972; 257: 314-323
        • Bellamy W.
        • Takase M.
        • Yamauchi K.
        • Wakabayashi H.
        • Kawase K.
        • Tomita M.
        Biochim. Biophys. Acta. 1992; 1121: 130-136
        • Sanchez L.
        • Aranda P.
        • Perez M.D.
        • Calvo M.
        Biol. Chem. Hoppe-Seyler. 1988; 369: 1005-1008
        • Nagasawa T.
        • Kiyosawa I.
        • Kuwahara K.
        J. Dairy Sci. 1972; 55: 1651-1659
        • Sekine K.
        • Watanabe E.
        • Nakamura J.
        • Takasuka N.
        • Kim D.J.
        • Asamoto M.
        • Krutovskikh V.
        • Baba-Toriyama H.
        • Ota T.
        • Moore M.A.
        • Masuda M.
        • Sugimoto H.
        • Nishino H.
        • Kakizoe T.
        • Tsuda H.
        Jpn. J. Cancer Res. 1997; 88: 523-526
        • Tanaka K.
        • Ikeda M.
        • Nozaki A.
        • Kato N.
        • Tsuda H.
        • Saito S.
        • Sekihara H.
        Jpn. J. Cancer Res. 1999; 90: 367-371
        • Iwasa M.
        • Kaito M.
        • Ikoma J.
        • Takeo M.
        • Imoto I.
        • Adachi Y.
        • Yamauchi K.
        • Koizumi R.
        • Teraguchi S.
        Am. J. Gastroenterol. 2002; 97: 766-767
        • Ikeda M.
        • Nozaki A.
        • Sugiyama K.
        • Tanaka T.
        • Naganuma A.
        • Tanaka K.
        • Sekihara H.
        • Shimotohno K.
        • Saito M.
        • Kato N.
        Virus Res. 2000; 66: 51-63
        • Yi M.
        • Kaneko S.
        • Yu D.Y.
        • Murakami S.
        J. Virol. 1997; 71: 5997-6002
        • Rosa D.
        • Campagnoli S.
        • Moretto C.
        • Guenzi E.
        • Cousens L.
        • Chin M.
        • Dong C.
        • Weiner A.J.
        • Lau J.Y.N.
        • Choo Q.L.
        • Chien D.
        • Pileri P.
        • Houghton M.
        • Abrignani S.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1759-1763
        • Farci P.
        • Shimoda A.
        • Wong D.
        • Cabezon T.
        • De Gioannis D.
        • Strazzera A.
        • Shimizu Y.
        • Shapiro M.
        • Alter H.J.
        • Purcell R.H.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15394-15399
        • Ishii K.
        • Rosa D.
        • Watanabe Y.
        • Katayama T.
        • Harada H.
        • Wyatt C.
        • Kiyosawa K.
        • Aizaki H.
        • Matsuura Y.
        • Houghton M.
        • Abrignani S.
        • Miyamura T.
        Hepatology. 1998; 28: 1117-1120
        • Inudoh M.
        • Nyunoya H.
        • Tanaka T.
        • Hijikata M.
        • Kato N.
        • Shimotohno K.
        Vaccine. 1996; 14: 1590-1596
        • Inudoh M.
        • Kato N.
        • Tanaka Y.
        Microbiol. Immunol. 1998; 42: 875-877
        • Kato N.
        • Pfeifer-Ohlsson S.
        • Kato M.
        • Larsson E.
        • Rydnert J.
        • Ohlsson R.
        • Cohen M.
        J. Virol. 1987; 61: 2182-2191
        • Kato N.
        • Hijikata M.
        • Ootsuyama Y.
        • Nakagawa M.
        • Ohkoshi S.
        • Shimotohno K.
        Mol. Biol. Med. 1990; 7: 495-501
        • Georgescu M.M.
        • Kirsch K.H.
        • Akagi T.
        • Shishido T.
        • Hanafusa H.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10182-10187
        • Naganuma A.
        • Nozaki A.
        • Tanaka T.
        • Sugiyama K.
        • Takagi H.
        • Mori M.
        • Shimotohno K.
        • Kato N.
        J. Virol. 2000; 74: 8744-8750
        • Hijikata M.
        • Mizushima H.
        • Akagi T.
        • Mori S.
        • Kakiuchi N.
        • Kato N.
        • Tanaka T.
        • Kimura K.
        • Shimotohno K.
        J. Virol. 1993; 67: 4665-4675
        • Efthymiadis A.
        • Shao H.
        • Hubner S.
        • Jans D.
        J. Biol. Chem. 1997; 272: 22134-22139
        • Nozaki A.
        • Kato N.
        Acta Med. Okayama. 2002; 56: 107-110
        • Spik G.
        • Coddeville B.
        • Montreuil J.
        Biochimie (Paris). 1988; 70: 1459-1469
        • Powell M.J.
        • Ogden J.E.
        Nucleic Acids Res. 1990; 18: 4013
        • Huang K.X.
        • Huang Q.L.
        • Wilduang M.R.
        • Croteau R.
        • Scott A.I.
        Protein Expr. Purif. 1998; 13: 90-96
        • Kato N.
        • Nozaki A.
        • Naganuma A.
        • Ikeda M.
        • Tanaka K.
        Curr. Top. Biochem. Res. 2000; 3: 164-173
        • Oren R.
        • Takahashi S.
        • Doss C.
        • Levy R.
        • Levy S.
        Mol. Cell. Biol. 1990; 10: 4007-4015
        • Petracca R.
        • Falugi F.
        • Galli G.
        • Norais N.
        • Rosa D.
        • Campagnoli S.
        • Burgio V.
        • Di Stasio E.
        • Giardina B.
        • Houghton M.
        • Abrignali S.
        • Grandi G.
        J. Virol. 2000; 74: 4824-4830
        • Flint M.
        • Maidens C.
        • Loomis-Price L.D.
        • Shotton C.
        • Dubuisson J.
        • Monk P.
        • Higginbottom A.
        • Levy S.
        • McKeating J.A.
        J. Virol. 1999; 73: 6235-6244
        • Higginbottom A.
        • Quinn E.R.
        • Kuo C.C.
        • Flint M.
        • Wilson L.H.
        • Bianchi E.
        • Nicosia A.
        • Monk P.N.
        • McKeating J.A.
        • Levy S.
        J. Virol. 2000; 74: 3642-3649
        • Takikawa S.
        • Ishii K.
        • Aizaki H.
        • Suzuki T.
        • Asakura H.
        • Matsuura Y.
        • Miyamura T.
        J. Virol. 2000; 74: 5066-5074
        • Deleersnyder V.
        • Pillez A.
        • Wychowski C.
        • Blight K.
        • Xu J.
        • Hahn Y.S.
        • Rice C.M.
        • Dubuisson J.
        J. Virol. 1997; 71: 697-704
        • Yi M.K.
        • Nakamoto Y.
        • Kaneko S.
        • Murakami S.
        Virology. 1997; 231: 119-129