SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993457378> ?p ?o ?g. }

Showing items 1 to 100 of ±118 with 100 items per page.

W1993457378 endingPage "15756" @default.
W1993457378 startingPage "15751" @default.
W1993457378 abstract "Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 m NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation. Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 m NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; GST, glutathione S-transferase; GPD, glyceraldehyde-3-phosphate; PCR, polymerase chain reaction1The abbreviations used are: HCV, hepatitis C virus; GST, glutathione S-transferase; GPD, glyceraldehyde-3-phosphate; PCR, polymerase chain reactionwas discovered by cDNA cloning in 1989 and shown to cause chronic liver disease (1Choo Q.-L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6209) Google Scholar, 2Kuo 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 M. Lee W.-S. Kou C. Berger K. Shuster J.R. Overby R. Bradley D.W. Houghton M. Science. 1989; 244: 362-364Crossref PubMed Scopus (3030) Google Scholar). Approximately 4 million Americans and 150 million individuals worldwide are infected with HCV and at risk for cirrhosis and hepatocellular carcinoma (3Alter M.J. Semin. Liver Dis. 1995; 15: 5-14Crossref PubMed Scopus (484) Google Scholar, 4Mansell C.J. Locarnini S.A. Semin. Liver Dis. 1995; 15: 15-32Crossref PubMed Scopus (109) Google Scholar, 5Mamiya N. Worman H.J. Curr. Opin. Infect. Dis. 1997; 10: 3990-3997Crossref Scopus (1) Google Scholar, 6National Institutes of Health Consensus Development Panel Hepatology. 1997; 26: 1S-156SPubMed Google Scholar). Because development of a robust cell culture system for HCV infection has remained elusive (6National Institutes of Health Consensus Development Panel Hepatology. 1997; 26: 1S-156SPubMed Google Scholar), extremely little is known about HCV-host cell interactions and how they influence cell physiology or viral replication.HCV is a positive single-stranded RNA virus and a member of theFlaviviridae family (1Choo Q.-L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6209) Google Scholar, 7Kato N. Hijikata M. Ootsuyama Y. Nakagawa M. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar, 8Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-5455Crossref PubMed Scopus (1524) Google Scholar, 9Okamoto H. Okada S. Sugiyama Y. Kurai K. Iizuka H. Machida A. Miyakawa Y. Mayumi M. J. Gen. Virol. 1991; 72: 2697-6704Crossref PubMed Scopus (393) Google Scholar, 10Takamizawa A. Mori C. Fuke I. Manabe S. Murakami S. Fujita J. Onishi E. Andoh T. Yoshida I. Okayama H. J. Virol. 1991; 65: 1105-1113Crossref PubMed Scopus (0) Google Scholar). Once HCV infects cells, the positive, single-stranded RNA genome is translated into a polyprotein of 3010 to 3033 amino acids, depending upon the strain (7Kato N. Hijikata M. Ootsuyama Y. Nakagawa M. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar, 8Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-5455Crossref PubMed Scopus (1524) Google Scholar, 9Okamoto H. Okada S. Sugiyama Y. Kurai K. Iizuka H. Machida A. Miyakawa Y. Mayumi M. J. Gen. Virol. 1991; 72: 2697-6704Crossref PubMed Scopus (393) Google Scholar, 10Takamizawa A. Mori C. Fuke I. Manabe S. Murakami S. Fujita J. Onishi E. Andoh T. Yoshida I. Okayama H. J. Virol. 1991; 65: 1105-1113Crossref PubMed Scopus (0) Google Scholar). The viral RNA is not capped, and translation occurs via an internal ribosome entry site at the 5′ end of the viral RNA (11Reynolds J.E. Kaminski A. Kettinen H.J. Grace K. Clarke B.E. Carroll A.R. Rowlands D.J. Jackson R.J. EMBO J. 1995; 14: 6010-6020Crossref PubMed Scopus (307) Google Scholar, 12Fukushi S. Kurihara C. Ishiyama N. Hoshino F.B. Oya A. Katayama K. J. Virol. 1997; 71: 1662-1666Crossref PubMed Google Scholar). The mechanism of translation of uncapped viral RNA therefore differs from that used by virtually all cellular mRNAs that are capped at their 5′ ends. The HCV polyprotein is cleaved by both host cell and viral proteases into several smaller polypeptides (7Kato N. Hijikata M. Ootsuyama Y. Nakagawa M. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar, 8Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-5455Crossref PubMed Scopus (1524) Google Scholar, 9Okamoto H. Okada S. Sugiyama Y. Kurai K. Iizuka H. Machida A. Miyakawa Y. Mayumi M. J. Gen. Virol. 1991; 72: 2697-6704Crossref PubMed Scopus (393) Google Scholar, 10Takamizawa A. Mori C. Fuke I. Manabe S. Murakami S. Fujita J. Onishi E. Andoh T. Yoshida I. Okayama H. J. Virol. 1991; 65: 1105-1113Crossref PubMed Scopus (0) Google Scholar, 13Selby M.J. Choo Q.-L. Berger K. Kuo G. Glazer E. Eckart M. Lee C. Chien D. Kuo C. Houghton M. J. Gen. Virol. 1993; 74: 1103-1113Crossref PubMed Scopus (200) Google Scholar). The major structural proteins are a core protein and two envelope proteins called E1 and E2. The core protein forms the nucleocapsid of the mature virion, and E1 and E2 are present in the viral envelope. A small polypeptide called P7 is also generated as a result of cleavage at the E2-NS2 junction, but its function is not clear. Four major nonstructural proteins called NS2, NS3, NS4, and NS5 are also generated, two of which, NS4 and NS5, are further processed into smaller polypeptides called NS4A, NS4B, NS5A, and NS5B. Most of the nonstructural proteins have enzymatic activities that are critical for viral replication.After cells are infected with a virus, viral proteins can interact with host cell proteins and influence cell physiology. In previous studies, HCV core protein has been shown to bind to lymphotoxin-β receptor and other tumor necrosis factor receptor family members (14Matsumoto M. Hsieh T.Y. Zhu N. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar, 15Zhu N. Khoshanan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3691-3697Crossref PubMed Google Scholar). A truncated form of HCV core protein also interacts with ribonucleoprotein K in the nucleus (16Hsieh T.-Y. Matsumoto M. Chou H.-C. Schneider R. Hwang S.B. Lee A.S. Lai M.M.C. J. Biol. Chem. 1998; 273: 17651-17659Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We now show that HCV core protein binds to a cellular RNA helicase and, in experimental systems, inhibits capped RNA translation. This provides a novel mechanism by which HCV may inhibit mRNA translation in infected cells or recruit a cellular protein to enhance its own replication.DISCUSSIONHCV core protein binds to the human DEAD box protein DBX. DBX rescues the lethal phenotype of ded1-deletion, demonstrating that it can function as a RNA helicase for capped mRNA, replacing the essential yeast DEAD box RNA helicase Ded1p. Our findings that HCV core protein prevents DBX from rescuing ded1-deletion yeast and that it inhibits the translation of capped RNA in vitrostrongly suggest that it may inhibit cellular mRNA translationin vivo. These results, however, cannot establish if translation inhibition occurs as a result of HCV core protein inhibiting DBX RNA helicase activity per se or by an interaction that results in “trapping” DBX at a location near the membrane of the endoplasmic reticulum where in cannot function properly. Inhibition of host cell mRNA translation could theoretically provide viral RNA molecules with enhanced access to ribosomes and the rest of the protein synthesis machinery of the cell, a phenomenon shared by several different viruses (29Knipe D.M. Fields B.N. Knipe D.M. Howley P.M. Field's Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 273-299Google Scholar). A recent report has shown that high levels of expression of HCV structural and nonstructural proteins is toxic to mammalian cells (30Moradpour D. Kary P. Rice C.M. Blum H.E. Hepatology. 1998; 28: 192-201Crossref PubMed Scopus (144) Google Scholar); however, it is not clear if this toxicity results from inhibition of host cell translation. Because the development of a robust cell culture system to study HCV has remained elusive, it would be extremely difficult to directly investigate the effects of HCV infection on host cell mRNA translation. Despite these methodological constraints limiting the ability to directly test the hypothesis, our discovery that HCV core binds to DBX and inhibits capped RNA translation in experimental assays suggests that it can similarly inhibit mRNA translation in infected human cells.DEAD box RNA helicases unwind capped mRNA (20Chuang R.-Y. Weaver P.L. Liu Z. Chang T.-H. Science. 1997; 275: 1468-1471Crossref PubMed Scopus (266) Google Scholar), and inhibition of their function should decrease translation of cellular mRNA. Inhibition of DBX function by HCV core protein may only partially inhibit host mRNA translation in mammalian cells because they contain other putative RNA helicases (31Gee S.L. Conboy J.G. Gene. 1994; 140: 171-177Crossref PubMed Scopus (39) Google Scholar). In contrast, the translation of HCV RNA, which is not capped, utilizes internal ribosome entry sites (11Reynolds J.E. Kaminski A. Kettinen H.J. Grace K. Clarke B.E. Carroll A.R. Rowlands D.J. Jackson R.J. EMBO J. 1995; 14: 6010-6020Crossref PubMed Scopus (307) Google Scholar, 12Fukushi S. Kurihara C. Ishiyama N. Hoshino F.B. Oya A. Katayama K. J. Virol. 1997; 71: 1662-1666Crossref PubMed Google Scholar), and can be unwound by its own RNA helicase, which is part of the HCV NS3 protein (32Yao N. Hesson T. Cable M. Hong Z. Kwong A.D. Le H.V. Weber P.C. Nat. Struct. Biol. 1997; 4: 463-467Crossref PubMed Scopus (420) Google Scholar, 33Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.). 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), and may proceed without DBX. This hypothetical mechanism is reminiscent of that used by poliovirus, which inhibits translation factor eIF-4F (34Rose J.K. Trachsel H. Leong D. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2732-2736Crossref PubMed Scopus (92) Google Scholar, 35Etchison D. Milburn S.C. Edery I. Sonenberg N. Hershey J.W.B. J. Biol. Chem. 1982; 257: 14806-14810Abstract Full Text PDF PubMed Google Scholar) and also has RNA with internal ribosome entry sites (36Pelletier J. Sonenberg N. Nature. 1988; 334: 320-325Crossref PubMed Scopus (1381) Google Scholar). In cells, eIF-4F exists as a complex with eIF-4B, which has RNA binding activity, and eIF-4A, which is also a DEAD box RNA helicase (37Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar). HCV and poliovirus infection may both therefore cause a decrease in the unwinding of capped mRNA in host cells.In addition to inhibiting capped mRNA translation in infected host cells, the interaction between HCV core protein and DBX may play other possible roles, including the recruitment of DBX to participate in HCV replication itself. Recruitment of host cells proteins into virions to enhance viral replication has been demonstrated in other systems. For example, the principal structural protein of the human immunodeficiency virus HIV-1 binds to cyclophilins and recruits cyclophilin A into viral particles, which appears to be necessary for efficient viral replication (38Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 18: 1067-1078Abstract Full Text PDF Scopus (700) Google Scholar, 39Franke E.K. Yuan H.E. Luban J. Nature. 1994; 372: 359-362Crossref PubMed Scopus (645) Google Scholar). In a similar fashion, recruitment of DBX into HCV particles by binding to core protein may enhance viral replication. This could theoretically occur by DBX altering viral genomic RNA structure in viral particles in newly infected cells. Testing of this hypothesis is limited at the present time because of the lack of an efficient cell culture system for HCV.HCV core protein has also been shown to bind to lymphotoxin-β receptor and other tumor necrosis factor receptor family members (14Matsumoto M. Hsieh T.Y. Zhu N. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar,15Zhu N. Khoshanan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3691-3697Crossref PubMed Google Scholar) as well as ribonucleoprotein K (16Hsieh T.-Y. Matsumoto M. Chou H.-C. Schneider R. Hwang S.B. Lee A.S. Lai M.M.C. J. Biol. Chem. 1998; 273: 17651-17659Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In our yeast two-hybrid screen, we did not isolate clones for these proteins, possibly because of subtle differences in our bait construct and the different cDNA library we used. The demonstration that other proteins interact with HCV core protein suggests that its expression in cells may have myriad consequences. Other groups (40Ray R.B. Steele R. Meyer K. Ray R. J. Biol. Chem. 1997; 272: 10983-10986Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 41Kim D.W. Suzuki R. Harada T. Saito I. Miyamura T. Jpn. J. Med. Sci. Biol. 1994; 47: 211-220Crossref PubMed Scopus (61) Google Scholar) have also reported that HCV core protein represses transcription from the p53 promoter and other eukaryotic promoters. The overall effect of HCV core protein on cell physiology under natural conditions of infection is, however, difficult to assess at the present time because of lack of a cell culture system for HCV.Finally, it should be noted that the best current treatment regimens for chronic hepatitis C are effective in only a minority of patients (42Liang T.J. N. Eng. J. Med. 1998; 339: 1549-1550Crossref PubMed Scopus (43) Google Scholar). If interactions between HCV and host cell proteins alter cell survival or enhance viral replication, they could be rational targets for antiviral drug design. Regardless of the physiological significance, the tight binding of any polypeptide to a structural or nonstructural protein of HCV may potentially interfere with viral replication. The identification of polypeptides such as DBX that bind to HCV proteins therefore has implications for the design of compounds which may be therapeutically useful in the treatment of patients with chronic hepatitis C. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; GST, glutathione S-transferase; GPD, glyceraldehyde-3-phosphate; PCR, polymerase chain reaction1The abbreviations used are: HCV, hepatitis C virus; GST, glutathione S-transferase; GPD, glyceraldehyde-3-phosphate; PCR, polymerase chain reactionwas discovered by cDNA cloning in 1989 and shown to cause chronic liver disease (1Choo Q.-L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6209) Google Scholar, 2Kuo 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 M. Lee W.-S. Kou C. Berger K. Shuster J.R. Overby R. Bradley D.W. Houghton M. Science. 1989; 244: 362-364Crossref PubMed Scopus (3030) Google Scholar). Approximately 4 million Americans and 150 million individuals worldwide are infected with HCV and at risk for cirrhosis and hepatocellular carcinoma (3Alter M.J. Semin. Liver Dis. 1995; 15: 5-14Crossref PubMed Scopus (484) Google Scholar, 4Mansell C.J. Locarnini S.A. Semin. Liver Dis. 1995; 15: 15-32Crossref PubMed Scopus (109) Google Scholar, 5Mamiya N. Worman H.J. Curr. Opin. Infect. Dis. 1997; 10: 3990-3997Crossref Scopus (1) Google Scholar, 6National Institutes of Health Consensus Development Panel Hepatology. 1997; 26: 1S-156SPubMed Google Scholar). Because development of a robust cell culture system for HCV infection has remained elusive (6National Institutes of Health Consensus Development Panel Hepatology. 1997; 26: 1S-156SPubMed Google Scholar), extremely little is known about HCV-host cell interactions and how they influence cell physiology or viral replication. HCV is a positive single-stranded RNA virus and a member of theFlaviviridae family (1Choo Q.-L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6209) Google Scholar, 7Kato N. Hijikata M. Ootsuyama Y. Nakagawa M. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar, 8Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-5455Crossref PubMed Scopus (1524) Google Scholar, 9Okamoto H. Okada S. Sugiyama Y. Kurai K. Iizuka H. Machida A. Miyakawa Y. Mayumi M. J. Gen. Virol. 1991; 72: 2697-6704Crossref PubMed Scopus (393) Google Scholar, 10Takamizawa A. Mori C. Fuke I. Manabe S. Murakami S. Fujita J. Onishi E. Andoh T. Yoshida I. Okayama H. J. Virol. 1991; 65: 1105-1113Crossref PubMed Scopus (0) Google Scholar). Once HCV infects cells, the positive, single-stranded RNA genome is translated into a polyprotein of 3010 to 3033 amino acids, depending upon the strain (7Kato N. Hijikata M. Ootsuyama Y. Nakagawa M. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar, 8Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-5455Crossref PubMed Scopus (1524) Google Scholar, 9Okamoto H. Okada S. Sugiyama Y. Kurai K. Iizuka H. Machida A. Miyakawa Y. Mayumi M. J. Gen. Virol. 1991; 72: 2697-6704Crossref PubMed Scopus (393) Google Scholar, 10Takamizawa A. Mori C. Fuke I. Manabe S. Murakami S. Fujita J. Onishi E. Andoh T. Yoshida I. Okayama H. J. Virol. 1991; 65: 1105-1113Crossref PubMed Scopus (0) Google Scholar). The viral RNA is not capped, and translation occurs via an internal ribosome entry site at the 5′ end of the viral RNA (11Reynolds J.E. Kaminski A. Kettinen H.J. Grace K. Clarke B.E. Carroll A.R. Rowlands D.J. Jackson R.J. EMBO J. 1995; 14: 6010-6020Crossref PubMed Scopus (307) Google Scholar, 12Fukushi S. Kurihara C. Ishiyama N. Hoshino F.B. Oya A. Katayama K. J. Virol. 1997; 71: 1662-1666Crossref PubMed Google Scholar). The mechanism of translation of uncapped viral RNA therefore differs from that used by virtually all cellular mRNAs that are capped at their 5′ ends. The HCV polyprotein is cleaved by both host cell and viral proteases into several smaller polypeptides (7Kato N. Hijikata M. Ootsuyama Y. Nakagawa M. Ohkoshi S. Sugimura T. Shimotohno K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9524-9528Crossref PubMed Scopus (1087) Google Scholar, 8Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby R. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-5455Crossref PubMed Scopus (1524) Google Scholar, 9Okamoto H. Okada S. Sugiyama Y. Kurai K. Iizuka H. Machida A. Miyakawa Y. Mayumi M. J. Gen. Virol. 1991; 72: 2697-6704Crossref PubMed Scopus (393) Google Scholar, 10Takamizawa A. Mori C. Fuke I. Manabe S. Murakami S. Fujita J. Onishi E. Andoh T. Yoshida I. Okayama H. J. Virol. 1991; 65: 1105-1113Crossref PubMed Scopus (0) Google Scholar, 13Selby M.J. Choo Q.-L. Berger K. Kuo G. Glazer E. Eckart M. Lee C. Chien D. Kuo C. Houghton M. J. Gen. Virol. 1993; 74: 1103-1113Crossref PubMed Scopus (200) Google Scholar). The major structural proteins are a core protein and two envelope proteins called E1 and E2. The core protein forms the nucleocapsid of the mature virion, and E1 and E2 are present in the viral envelope. A small polypeptide called P7 is also generated as a result of cleavage at the E2-NS2 junction, but its function is not clear. Four major nonstructural proteins called NS2, NS3, NS4, and NS5 are also generated, two of which, NS4 and NS5, are further processed into smaller polypeptides called NS4A, NS4B, NS5A, and NS5B. Most of the nonstructural proteins have enzymatic activities that are critical for viral replication. After cells are infected with a virus, viral proteins can interact with host cell proteins and influence cell physiology. In previous studies, HCV core protein has been shown to bind to lymphotoxin-β receptor and other tumor necrosis factor receptor family members (14Matsumoto M. Hsieh T.Y. Zhu N. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar, 15Zhu N. Khoshanan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3691-3697Crossref PubMed Google Scholar). A truncated form of HCV core protein also interacts with ribonucleoprotein K in the nucleus (16Hsieh T.-Y. Matsumoto M. Chou H.-C. Schneider R. Hwang S.B. Lee A.S. Lai M.M.C. J. Biol. Chem. 1998; 273: 17651-17659Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We now show that HCV core protein binds to a cellular RNA helicase and, in experimental systems, inhibits capped RNA translation. This provides a novel mechanism by which HCV may inhibit mRNA translation in infected cells or recruit a cellular protein to enhance its own replication. DISCUSSIONHCV core protein binds to the human DEAD box protein DBX. DBX rescues the lethal phenotype of ded1-deletion, demonstrating that it can function as a RNA helicase for capped mRNA, replacing the essential yeast DEAD box RNA helicase Ded1p. Our findings that HCV core protein prevents DBX from rescuing ded1-deletion yeast and that it inhibits the translation of capped RNA in vitrostrongly suggest that it may inhibit cellular mRNA translationin vivo. These results, however, cannot establish if translation inhibition occurs as a result of HCV core protein inhibiting DBX RNA helicase activity per se or by an interaction that results in “trapping” DBX at a location near the membrane of the endoplasmic reticulum where in cannot function properly. Inhibition of host cell mRNA translation could theoretically provide viral RNA molecules with enhanced access to ribosomes and the rest of the protein synthesis machinery of the cell, a phenomenon shared by several different viruses (29Knipe D.M. Fields B.N. Knipe D.M. Howley P.M. Field's Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 273-299Google Scholar). A recent report has shown that high levels of expression of HCV structural and nonstructural proteins is toxic to mammalian cells (30Moradpour D. Kary P. Rice C.M. Blum H.E. Hepatology. 1998; 28: 192-201Crossref PubMed Scopus (144) Google Scholar); however, it is not clear if this toxicity results from inhibition of host cell translation. Because the development of a robust cell culture system to study HCV has remained elusive, it would be extremely difficult to directly investigate the effects of HCV infection on host cell mRNA translation. Despite these methodological constraints limiting the ability to directly test the hypothesis, our discovery that HCV core binds to DBX and inhibits capped RNA translation in experimental assays suggests that it can similarly inhibit mRNA translation in infected human cells.DEAD box RNA helicases unwind capped mRNA (20Chuang R.-Y. Weaver P.L. Liu Z. Chang T.-H. Science. 1997; 275: 1468-1471Crossref PubMed Scopus (266) Google Scholar), and inhibition of their function should decrease translation of cellular mRNA. Inhibition of DBX function by HCV core protein may only partially inhibit host mRNA translation in mammalian cells because they contain other putative RNA helicases (31Gee S.L. Conboy J.G. Gene. 1994; 140: 171-177Crossref PubMed Scopus (39) Google Scholar). In contrast, the translation of HCV RNA, which is not capped, utilizes internal ribosome entry sites (11Reynolds J.E. Kaminski A. Kettinen H.J. Grace K. Clarke B.E. Carroll A.R. Rowlands D.J. Jackson R.J. EMBO J. 1995; 14: 6010-6020Crossref PubMed Scopus (307) Google Scholar, 12Fukushi S. Kurihara C. Ishiyama N. Hoshino F.B. Oya A. Katayama K. J. Virol. 1997; 71: 1662-1666Crossref PubMed Google Scholar), and can be unwound by its own RNA helicase, which is part of the HCV NS3 protein (32Yao N. Hesson T. Cable M. Hong Z. Kwong A.D. Le H.V. Weber P.C. Nat. Struct. Biol. 1997; 4: 463-467Crossref PubMed Scopus (420) Google Scholar, 33Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.). 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), and may proceed without DBX. This hypothetical mechanism is reminiscent of that used by poliovirus, which inhibits translation factor eIF-4F (34Rose J.K. Trachsel H. Leong D. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2732-2736Crossref PubMed Scopus (92) Google Scholar, 35Etchison D. Milburn S.C. Edery I. Sonenberg N. Hershey J.W.B. J. Biol. Chem. 1982; 257: 14806-14810Abstract Full Text PDF PubMed Google Scholar) and also has RNA with internal ribosome entry sites (36Pelletier J. Sonenberg N. Nature. 1988; 334: 320-325Crossref PubMed Scopus (1381) Google Scholar). In cells, eIF-4F exists as a complex with eIF-4B, which has RNA binding activity, and eIF-4A, which is also a DEAD box RNA helicase (37Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar). HCV and poliovirus infection may both therefore cause a decrease in the unwinding of capped mRNA in host cells.In addition to inhibiting capped mRNA translation in infected host cells, the interaction between HCV core protein and DBX may play other possible roles, including the recruitment of DBX to participate in HCV replication itself. Recruitment of host cells proteins into virions to enhance viral replication has been demonstrated in other systems. For example, the principal structural protein of the human immunodeficiency virus HIV-1 binds to cyclophilins and recruits cyclophilin A into viral particles, which appears to be necessary for efficient viral replication (38Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 18: 1067-1078Abstract Full Text PDF Scopus (700) Google Scholar, 39Franke E.K. Yuan H.E. Luban J. Nature. 1994; 372: 359-362Crossref PubMed Scopus (645) Google Scholar). In a similar fashion, recruitment of DBX into HCV particles by binding to core protein may enhance viral replication. This could theoretically occur by DBX altering viral genomic RNA structure in viral particles in newly infected cells. Testing of this hypothesis is limited at the present time because of the lack of an efficient cell culture system for HCV.HCV core protein has also been shown to bind to lymphotoxin-β receptor and other tumor necrosis factor receptor family members (14Matsumoto M. Hsieh T.Y. Zhu N. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar,15Zhu N. Khoshanan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3691-3697Crossref PubMed Google Scholar) as well as ribonucleoprotein K (16Hsieh T.-Y. Matsumoto M. Chou H.-C. Schneider R. Hwang S.B. Lee A.S. Lai M.M.C. J. Biol. Chem. 1998; 273: 17651-17659Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In our yeast two-hybrid screen, we did not isolate clones for these proteins, possibly because of subtle differences in our bait construct and the different cDNA library we used. The demonstration that other proteins interact with HCV core protein suggests that its expression in cells may have myriad consequences. Other groups (40Ray R.B. Steele R. Meyer K. Ray R. J. Biol. Chem. 1997; 272: 10983-10986Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 41Kim D.W. Suzuki R. Harada T. Saito I. Miyamura T. Jpn. J. Med. Sci. Biol. 1994; 47: 211-220Crossref PubMed Scopus (61) Google Scholar) have also reported that HCV core protein represses transcription from the p53 promoter and other eukaryotic promoters. The overall effect of HCV core protein on cell physiology under natural conditions of infection is, however, difficult to assess at the present time because of lack of a cell culture system for HCV.Finally, it should be noted that the best current treatment regimens for chronic hepatitis C are effective in only a minority of patients (42Liang T.J. N. Eng. J. Med. 1998; 339: 1549-1550Crossref PubMed Scopus (43) Google Scholar). If interactions between HCV and host cell proteins alter cell survival or enhance viral replication, they could be rational targets for antiviral drug design. Regardless of the physiological significance, the tight binding of any polypeptide to a structural or nonstructural protein of HCV may potentially interfere with viral replication. The identification of polypeptides such as DBX that bind to HCV proteins therefore has implications for the design of compounds which may be therapeutically useful in the treatment of patients with chronic hepatitis C. HCV core protein binds to the human DEAD box protein DBX. DBX rescues the lethal phenotype of ded1-deletion, demonstrating that it can function as a RNA helicase for capped mRNA, replacing the essential yeast DEAD box RNA helicase Ded1p. Our findings that HCV core protein prevents DBX from rescuing ded1-deletion yeast and that it inhibits the translation of capped RNA in vitrostrongly suggest that it may inhibit cellular mRNA translationin vivo. These results, however, cannot establish if translation inhibition occurs as a result of HCV core protein inhibiting DBX RNA helicase activity per se or by an interaction that results in “trapping” DBX at a location near the membrane of the endoplasmic reticulum where in cannot function properly. Inhibition of host cell mRNA translation could theoretically provide viral RNA molecules with enhanced access to ribosomes and the rest of the protein synthesis machinery of the cell, a phenomenon shared by several different viruses (29Knipe D.M. Fields B.N. Knipe D.M. Howley P.M. Field's Virology. 3rd Ed. Lippincott-Raven Publishers, Philadelphia1996: 273-299Google Scholar). A recent report has shown that high levels of expression of HCV structural and nonstructural proteins is toxic to mammalian cells (30Moradpour D. Kary P. Rice C.M. Blum H.E. Hepatology. 1998; 28: 192-201Crossref PubMed Scopus (144) Google Scholar); however, it is not clear if this toxicity results from inhibition of host cell translation. Because the development of a robust cell culture system to study HCV has remained elusive, it would be extremely difficult to directly investigate the effects of HCV infection on host cell mRNA translation. Despite these methodological constraints limiting the ability to directly test the hypothesis, our discovery that HCV core binds to DBX and inhibits capped RNA translation in experimental assays suggests that it can similarly inhibit mRNA translation in infected human cells. DEAD box RNA helicases unwind capped mRNA (20Chuang R.-Y. Weaver P.L. Liu Z. Chang T.-H. Science. 1997; 275: 1468-1471Crossref PubMed Scopus (266) Google Scholar), and inhibition of their function should decrease translation of cellular mRNA. Inhibition of DBX function by HCV core protein may only partially inhibit host mRNA translation in mammalian cells because they contain other putative RNA helicases (31Gee S.L. Conboy J.G. Gene. 1994; 140: 171-177Crossref PubMed Scopus (39) Google Scholar). In contrast, the translation of HCV RNA, which is not capped, utilizes internal ribosome entry sites (11Reynolds J.E. Kaminski A. Kettinen H.J. Grace K. Clarke B.E. Carroll A.R. Rowlands D.J. Jackson R.J. EMBO J. 1995; 14: 6010-6020Crossref PubMed Scopus (307) Google Scholar, 12Fukushi S. Kurihara C. Ishiyama N. Hoshino F.B. Oya A. Katayama K. J. Virol. 1997; 71: 1662-1666Crossref PubMed Google Scholar), and can be unwound by its own RNA helicase, which is part of the HCV NS3 protein (32Yao N. Hesson T. Cable M. Hong Z. Kwong A.D. Le H.V. Weber P.C. Nat. Struct. Biol. 1997; 4: 463-467Crossref PubMed Scopus (420) Google Scholar, 33Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.). 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), and may proceed without DBX. This hypothetical mechanism is reminiscent of that used by poliovirus, which inhibits translation factor eIF-4F (34Rose J.K. Trachsel H. Leong D. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2732-2736Crossref PubMed Scopus (92) Google Scholar, 35Etchison D. Milburn S.C. Edery I. Sonenberg N. Hershey J.W.B. J. Biol. Chem. 1982; 257: 14806-14810Abstract Full Text PDF PubMed Google Scholar) and also has RNA with internal ribosome entry sites (36Pelletier J. Sonenberg N. Nature. 1988; 334: 320-325Crossref PubMed Scopus (1381) Google Scholar). In cells, eIF-4F exists as a complex with eIF-4B, which has RNA binding activity, and eIF-4A, which is also a DEAD box RNA helicase (37Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Crossref PubMed Scopus (497) Google Scholar). HCV and poliovirus infection may both therefore cause a decrease in the unwinding of capped mRNA in host cells. In addition to inhibiting capped mRNA translation in infected host cells, the interaction between HCV core protein and DBX may play other possible roles, including the recruitment of DBX to participate in HCV replication itself. Recruitment of host cells proteins into virions to enhance viral replication has been demonstrated in other systems. For example, the principal structural protein of the human immunodeficiency virus HIV-1 binds to cyclophilins and recruits cyclophilin A into viral particles, which appears to be necessary for efficient viral replication (38Luban J. Bossolt K.L. Franke E.K. Kalpana G.V. Goff S.P. Cell. 1993; 18: 1067-1078Abstract Full Text PDF Scopus (700) Google Scholar, 39Franke E.K. Yuan H.E. Luban J. Nature. 1994; 372: 359-362Crossref PubMed Scopus (645) Google Scholar). In a similar fashion, recruitment of DBX into HCV particles by binding to core protein may enhance viral replication. This could theoretically occur by DBX altering viral genomic RNA structure in viral particles in newly infected cells. Testing of this hypothesis is limited at the present time because of the lack of an efficient cell culture system for HCV. HCV core protein has also been shown to bind to lymphotoxin-β receptor and other tumor necrosis factor receptor family members (14Matsumoto M. Hsieh T.Y. Zhu N. VanArsdale T. Hwang S.B. Jeng K.S. Gorbalenya A.E. Lo S.Y. Ou J.H. Ware C.F. Lai M.M.C. J. Virol. 1997; 71: 1301-1309Crossref PubMed Google Scholar,15Zhu N. Khoshanan A. Schneider R. Matsumoto M. Dennert G. Ware C. Lai M.M.C. J. Virol. 1998; 72: 3691-3697Crossref PubMed Google Scholar) as well as ribonucleoprotein K (16Hsieh T.-Y. Matsumoto M. Chou H.-C. Schneider R. Hwang S.B. Lee A.S. Lai M.M.C. J. Biol. Chem. 1998; 273: 17651-17659Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In our yeast two-hybrid screen, we did not isolate clones for these proteins, possibly because of subtle differences in our bait construct and the different cDNA library we used. The demonstration that other proteins interact with HCV core protein suggests that its expression in cells may have myriad consequences. Other groups (40Ray R.B. Steele R. Meyer K. Ray R. J. Biol. Chem. 1997; 272: 10983-10986Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 41Kim D.W. Suzuki R. Harada T. Saito I. Miyamura T. Jpn. J. Med. Sci. Biol. 1994; 47: 211-220Crossref PubMed Scopus (61) Google Scholar) have also reported that HCV core protein represses transcription from the p53 promoter and other eukaryotic promoters. The overall effect of HCV core protein on cell physiology under natural conditions of infection is, however, difficult to assess at the present time because of lack of a cell culture system for HCV. Finally, it should be noted that the best current treatment regimens for chronic hepatitis C are effective in only a minority of patients (42Liang T.J. N. Eng. J. Med. 1998; 339: 1549-1550Crossref PubMed Scopus (43) Google Scholar). If interactions between HCV and host cell proteins alter cell survival or enhance viral replication, they could be rational targets for antiviral drug design. Regardless of the physiological significance, the tight binding of any polypeptide to a structural or nonstructural protein of HCV may potentially interfere with viral replication. The identification of polypeptides such as DBX that bind to HCV proteins therefore has implications for the design of compounds which may be therapeutically useful in the treatment of patients with chronic hepatitis C. We thank T.-H. Chang (Ohio State University) for providing reagents and invaluable advice, S. P. Goff (Columbia University) for helpful discussions, M. Houghton (Chiron Corp.) for providing pHCV-1, and P. J. Mustacchia (Columbia University) and T. W. Chun (State University of New York) for reviewing the manuscript." @default.
W1993457378 created "2016-06-24" @default.
W1993457378 creator A5062381493 @default.
W1993457378 creator A5071188846 @default.
W1993457378 date "1999-05-01" @default.
W1993457378 modified "2023-09-29" @default.
W1993457378 title "Hepatitis C Virus Core Protein Binds to a DEAD Box RNA Helicase" @default.
W1993457378 cites W1480601143 @default.
W1993457378 cites W1495304774 @default.
W1993457378 cites W1559808711 @default.
W1993457378 cites W1581048386 @default.
W1993457378 cites W1644202690 @default.
W1993457378 cites W1776345984 @default.
W1993457378 cites W1971227936 @default.
W1993457378 cites W1971792395 @default.
W1993457378 cites W1973782344 @default.
W1993457378 cites W1977186059 @default.
W1993457378 cites W1984083837 @default.
W1993457378 cites W1992180542 @default.
W1993457378 cites W2006309458 @default.
W1993457378 cites W2013982103 @default.
W1993457378 cites W2020597127 @default.
W1993457378 cites W2020746683 @default.
W1993457378 cites W2027779897 @default.
W1993457378 cites W2036654773 @default.
W1993457378 cites W2047220299 @default.
W1993457378 cites W2048186445 @default.
W1993457378 cites W2048731469 @default.
W1993457378 cites W2052014046 @default.
W1993457378 cites W2053885170 @default.
W1993457378 cites W2062639699 @default.
W1993457378 cites W2066124462 @default.
W1993457378 cites W2069136255 @default.
W1993457378 cites W2073903623 @default.
W1993457378 cites W2079377965 @default.
W1993457378 cites W2079395665 @default.
W1993457378 cites W2085795760 @default.
W1993457378 cites W2086369650 @default.
W1993457378 cites W2092403756 @default.
W1993457378 cites W2094215066 @default.
W1993457378 cites W2124528668 @default.
W1993457378 cites W2125353176 @default.
W1993457378 cites W2142946131 @default.
W1993457378 cites W2151603470 @default.
W1993457378 cites W2171991181 @default.
W1993457378 doi "https://doi.org/10.1074/jbc.274.22.15751" @default.
W1993457378 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10336476" @default.
W1993457378 hasPublicationYear "1999" @default.
W1993457378 type Work @default.
W1993457378 sameAs 1993457378 @default.
W1993457378 citedByCount "160" @default.
W1993457378 countsByYear W19934573782012 @default.
W1993457378 countsByYear W19934573782013 @default.
W1993457378 countsByYear W19934573782014 @default.
W1993457378 countsByYear W19934573782015 @default.
W1993457378 countsByYear W19934573782016 @default.
W1993457378 countsByYear W19934573782017 @default.
W1993457378 countsByYear W19934573782018 @default.
W1993457378 countsByYear W19934573782019 @default.
W1993457378 countsByYear W19934573782020 @default.
W1993457378 countsByYear W19934573782021 @default.
W1993457378 countsByYear W19934573782022 @default.
W1993457378 countsByYear W19934573782023 @default.
W1993457378 crossrefType "journal-article" @default.
W1993457378 hasAuthorship W1993457378A5062381493 @default.
W1993457378 hasAuthorship W1993457378A5071188846 @default.
W1993457378 hasBestOaLocation W19934573781 @default.
W1993457378 hasConcept C104317684 @default.
W1993457378 hasConcept C120665830 @default.
W1993457378 hasConcept C121332964 @default.
W1993457378 hasConcept C159047783 @default.
W1993457378 hasConcept C161223559 @default.
W1993457378 hasConcept C194575299 @default.
W1993457378 hasConcept C2164484 @default.
W1993457378 hasConcept C2522874641 @default.
W1993457378 hasConcept C2776408679 @default.
W1993457378 hasConcept C2779568538 @default.
W1993457378 hasConcept C54355233 @default.
W1993457378 hasConcept C67705224 @default.
W1993457378 hasConcept C68592252 @default.
W1993457378 hasConcept C73616013 @default.
W1993457378 hasConcept C86803240 @default.
W1993457378 hasConceptScore W1993457378C104317684 @default.
W1993457378 hasConceptScore W1993457378C120665830 @default.
W1993457378 hasConceptScore W1993457378C121332964 @default.
W1993457378 hasConceptScore W1993457378C159047783 @default.
W1993457378 hasConceptScore W1993457378C161223559 @default.
W1993457378 hasConceptScore W1993457378C194575299 @default.
W1993457378 hasConceptScore W1993457378C2164484 @default.
W1993457378 hasConceptScore W1993457378C2522874641 @default.
W1993457378 hasConceptScore W1993457378C2776408679 @default.
W1993457378 hasConceptScore W1993457378C2779568538 @default.
W1993457378 hasConceptScore W1993457378C54355233 @default.
W1993457378 hasConceptScore W1993457378C67705224 @default.
W1993457378 hasConceptScore W1993457378C68592252 @default.
W1993457378 hasConceptScore W1993457378C73616013 @default.
W1993457378 hasConceptScore W1993457378C86803240 @default.
W1993457378 hasIssue "22" @default.