DOI: 10.1261/rna.2197210. The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

10.1261/rna.2197210

Crossref journal-article

Cold Spring Harbor Laboratory

RNA (246)

Abstract

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5′ untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine–Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2′-OMe oligonucleotides.

Bibliography

Berry, K. E., Waghray, S., & Doudna, J. A. (2010). The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit. RNA, 16(8), 1559â1569.

Authors 3

Katherine E. Berry (first)
Shruti Waghray (additional)
Jennifer A. Doudna (additional)

References 39 Referenced 69

10.1016/j.str.2005.08.008 / Structure / Structure of the hepatitis C virus IRES bound to the human 80S ribosome: Remodeling of the HCV IRES (2005)
10.1038/nrmicro1704
10.1093/nar/20.19.5041
10.1111/j.1365-2893.2007.00887.x / J Viral Hepat / Old and emerging therapies in chronic hepatitis C: An update (2008)
10.1093/nar/gkp588
10.1128/JVI.76.10.5024-5033.2002
10.1017/S1355838202023038 / RNA / The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes (2002)
10.1016/j.molcel.2007.05.019
10.1038/nsmb.1572
10.1002/hep.22759
10.1128/JVI.02403-06
{'key': '2021112105502161000_16.8.1559.12', 'first-page': '955', 'article-title': 'Stability of a stemâ\x80\x93loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA', 'volume': '2', 'year': '1996', 'journal-title': 'RNA'} / RNA / Stability of a stemâloop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA (1996)
10.1073/pnas.0407402101
10.1006/jmbi.1999.3095
10.1017/S1355838201001790
10.1128/JVI.74.14.6242-6250.2000
10.1093/nar/18.9.2828
10.1038/sj.emboj.7601549
10.1128/JB.184.20.5733-5745.2002
10.1016/j.jhep.2005.09.009
10.1021/bi00261a003 / Biochemistry / Immunochemical characterization of mammalian protein synthesis initiation factors (1982)
10.1016/j.cell.2004.09.038
10.1101/gad.12.1.67 / Genes Dev / A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs (1998)
10.1038/emboj.2008.49
10.1093/nar/13.5.1717
10.1002/j.1460-2075.1995.tb00289.x / EMBO J / Unique features of internal initiation of hepatitis C virus RNA translation (1995)
{'key': '2021112105502161000_16.8.1559.27', 'first-page': '867', 'article-title': 'Internal initiation of translation of hepatitis C virus RNA: The ribosome entry site is at the authentic initiation codon', 'volume': '2', 'year': '1996', 'journal-title': 'RNA'} / RNA / Internal initiation of translation of hepatitis C virus RNA: The ribosome entry site is at the authentic initiation codon (1996)
10.1128/jvi.71.1.451-457.1997 / J Virol / Internal entry of ribosomes is directed by the 5â² noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon (1997)
10.1111/j.1365-2958.1992.tb01561.x / Mol Microbiol / Translation initiation in Escherichia coli: Sequences within the ribosome-binding site (1992)
10.1128/JVI.72.6.4775-4782.1998 / J Virol / Specific interaction of eukaryotic translation initiation factor 3 with the 5â² nontranslated regions of hepatitis C virus and classical swine fever virus RNAs (1998)
10.1126/science.1058409
10.1093/nar/gkg139
10.1038/nsmb.1445
10.1128/jvi.66.3.1476-1483.1992 / J Virol / Internal ribosome entry site within hepatitis C virus RNA (1992)
10.1128/jvi.67.6.3338-3344.1993 / J Virol / Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism (1993)
10.1128/jvi.68.11.7301-7307.1994 / J Virol / A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA (1994)
{'key': '2021112105502161000_16.8.1559.37', 'first-page': '526', 'article-title': 'An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5â\x80² noncoding region', 'volume': '1', 'year': '1995', 'journal-title': 'RNA'} / RNA / An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5â² noncoding region (1995)
10.1002/bies.950110406
10.1016/S0092-8674(01)00435-4

Dates

Type	When
Created	15 years, 2 months ago (June 29, 2010, 12:10 a.m.)
Deposited	1 year, 5 months ago (March 27, 2024, 5:17 p.m.)
Indexed	3 months, 2 weeks ago (May 16, 2025, 5:22 p.m.)
Issued	15 years, 2 months ago (June 28, 2010)
Published	15 years, 2 months ago (June 28, 2010)
Published Online	15 years, 2 months ago (June 28, 2010)
Published Print	15 years, 1 month ago (Aug. 1, 2010)

Funders 0

None

BibTeX

@article{Berry_2010, title={The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit}, volume={16}, ISSN={1469-9001}, url={http://dx.doi.org/10.1261/rna.2197210}, DOI={10.1261/rna.2197210}, number={8}, journal={RNA}, publisher={Cold Spring Harbor Laboratory}, author={Berry, Katherine E. and Waghray, Shruti and Doudna, Jennifer A.}, year={2010}, month=jun, pages={1559–1569} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        5,
        16
      ]
    ],
    "date-time": "2025-05-16T21:22:24Z",
    "timestamp": 1747430544235
  },
  "reference-count": 39,
  "publisher": "Cold Spring Harbor Laboratory",
  "issue": "8",
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        2010,
        8
      ]
    ]
  },
  "abstract": "<jats:p>The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5\u2032 untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine\u2013Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2\u2032-OMe oligonucleotides.</jats:p>",
  "DOI": "10.1261/rna.2197210",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2010,
        6,
        29
      ]
    ],
    "date-time": "2010-06-29T04:10:38Z",
    "timestamp": 1277784638000
  },
  "page": "1559-1569",
  "source": "Crossref",
  "is-referenced-by-count": 69,
  "title": "The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit",
  "prefix": "10.1261",
  "volume": "16",
  "author": [
    {
      "given": "Katherine E.",
      "family": "Berry",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Shruti",
      "family": "Waghray",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Jennifer A.",
      "family": "Doudna",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "246",
  "published-online": {
    "date-parts": [
      [
        2010,
        6,
        28
      ]
    ]
  },
  "reference": [
    {
      "key": "2021112105502161000_16.8.1559.1",
      "doi-asserted-by": "crossref",
      "first-page": "1695",
      "DOI": "10.1016/j.str.2005.08.008",
      "article-title": "Structure of the hepatitis C virus IRES bound to the human 80S ribosome: Remodeling of the HCV IRES",
      "volume": "13",
      "year": "2005",
      "journal-title": "Structure"
    },
    {
      "key": "2021112105502161000_16.8.1559.2",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nrmicro1704"
    },
    {
      "key": "2021112105502161000_16.8.1559.3",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/20.19.5041"
    },
    {
      "key": "2021112105502161000_16.8.1559.4",
      "doi-asserted-by": "crossref",
      "first-page": "2",
      "DOI": "10.1111/j.1365-2893.2007.00887.x",
      "article-title": "Old and emerging therapies in chronic hepatitis C: An update",
      "volume": "15",
      "year": "2008",
      "journal-title": "J Viral Hepat"
    },
    {
      "key": "2021112105502161000_16.8.1559.5",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/gkp588"
    },
    {
      "key": "2021112105502161000_16.8.1559.6",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.76.10.5024-5033.2002"
    },
    {
      "key": "2021112105502161000_16.8.1559.7",
      "doi-asserted-by": "crossref",
      "first-page": "1558",
      "DOI": "10.1017/S1355838202023038",
      "article-title": "The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes",
      "volume": "8",
      "year": "2002",
      "journal-title": "RNA"
    },
    {
      "key": "2021112105502161000_16.8.1559.8",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.molcel.2007.05.019"
    },
    {
      "key": "2021112105502161000_16.8.1559.9",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nsmb.1572"
    },
    {
      "key": "2021112105502161000_16.8.1559.10",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/hep.22759"
    },
    {
      "key": "2021112105502161000_16.8.1559.11",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.02403-06"
    },
    {
      "key": "2021112105502161000_16.8.1559.12",
      "first-page": "955",
      "article-title": "Stability of a stem\u00e2\u0080\u0093loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA",
      "volume": "2",
      "year": "1996",
      "journal-title": "RNA"
    },
    {
      "key": "2021112105502161000_16.8.1559.13",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.0407402101"
    },
    {
      "key": "2021112105502161000_16.8.1559.14",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/jmbi.1999.3095"
    },
    {
      "key": "2021112105502161000_16.8.1559.15",
      "doi-asserted-by": "publisher",
      "DOI": "10.1017/S1355838201001790"
    },
    {
      "key": "2021112105502161000_16.8.1559.16",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JVI.74.14.6242-6250.2000"
    },
    {
      "key": "2021112105502161000_16.8.1559.17",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/18.9.2828"
    },
    {
      "key": "2021112105502161000_16.8.1559.18",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/sj.emboj.7601549"
    },
    {
      "key": "2021112105502161000_16.8.1559.19",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/JB.184.20.5733-5745.2002"
    },
    {
      "key": "2021112105502161000_16.8.1559.20",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.jhep.2005.09.009"
    },
    {
      "key": "2021112105502161000_16.8.1559.21",
      "doi-asserted-by": "crossref",
      "first-page": "4206",
      "DOI": "10.1021/bi00261a003",
      "article-title": "Immunochemical characterization of mammalian protein synthesis initiation factors",
      "volume": "21",
      "year": "1982",
      "journal-title": "Biochemistry"
    },
    {
      "key": "2021112105502161000_16.8.1559.22",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/j.cell.2004.09.038"
    },
    {
      "key": "2021112105502161000_16.8.1559.23",
      "doi-asserted-by": "crossref",
      "first-page": "67",
      "DOI": "10.1101/gad.12.1.67",
      "article-title": "A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs",
      "volume": "12",
      "year": "1998",
      "journal-title": "Genes Dev"
    },
    {
      "key": "2021112105502161000_16.8.1559.24",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/emboj.2008.49"
    },
    {
      "key": "2021112105502161000_16.8.1559.25",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/13.5.1717"
    },
    {
      "key": "2021112105502161000_16.8.1559.26",
      "doi-asserted-by": "crossref",
      "first-page": "6010",
      "DOI": "10.1002/j.1460-2075.1995.tb00289.x",
      "article-title": "Unique features of internal initiation of hepatitis C virus RNA translation",
      "volume": "14",
      "year": "1995",
      "journal-title": "EMBO J"
    },
    {
      "key": "2021112105502161000_16.8.1559.27",
      "first-page": "867",
      "article-title": "Internal initiation of translation of hepatitis C virus RNA: The ribosome entry site is at the authentic initiation codon",
      "volume": "2",
      "year": "1996",
      "journal-title": "RNA"
    },
    {
      "key": "2021112105502161000_16.8.1559.28",
      "doi-asserted-by": "crossref",
      "first-page": "451",
      "DOI": "10.1128/jvi.71.1.451-457.1997",
      "article-title": "Internal entry of ribosomes is directed by the 5\u00e2\u0080\u00b2 noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon",
      "volume": "71",
      "year": "1997",
      "journal-title": "J Virol"
    },
    {
      "key": "2021112105502161000_16.8.1559.29",
      "doi-asserted-by": "crossref",
      "first-page": "1219",
      "DOI": "10.1111/j.1365-2958.1992.tb01561.x",
      "article-title": "Translation initiation in Escherichia coli: Sequences within the ribosome-binding site",
      "volume": "6",
      "year": "1992",
      "journal-title": "Mol Microbiol"
    },
    {
      "key": "2021112105502161000_16.8.1559.30",
      "doi-asserted-by": "crossref",
      "first-page": "4775",
      "DOI": "10.1128/JVI.72.6.4775-4782.1998",
      "article-title": "Specific interaction of eukaryotic translation initiation factor 3 with the 5\u00e2\u0080\u00b2 nontranslated regions of hepatitis C virus and classical swine fever virus RNAs",
      "volume": "72",
      "year": "1998",
      "journal-title": "J Virol"
    },
    {
      "key": "2021112105502161000_16.8.1559.31",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1058409"
    },
    {
      "key": "2021112105502161000_16.8.1559.32",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/gkg139"
    },
    {
      "key": "2021112105502161000_16.8.1559.33",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/nsmb.1445"
    },
    {
      "key": "2021112105502161000_16.8.1559.34",
      "doi-asserted-by": "crossref",
      "first-page": "1476",
      "DOI": "10.1128/jvi.66.3.1476-1483.1992",
      "article-title": "Internal ribosome entry site within hepatitis C virus RNA",
      "volume": "66",
      "year": "1992",
      "journal-title": "J Virol"
    },
    {
      "key": "2021112105502161000_16.8.1559.35",
      "doi-asserted-by": "crossref",
      "first-page": "3338",
      "DOI": "10.1128/jvi.67.6.3338-3344.1993",
      "article-title": "Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism",
      "volume": "67",
      "year": "1993",
      "journal-title": "J Virol"
    },
    {
      "key": "2021112105502161000_16.8.1559.36",
      "doi-asserted-by": "crossref",
      "first-page": "7301",
      "DOI": "10.1128/jvi.68.11.7301-7307.1994",
      "article-title": "A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA",
      "volume": "68",
      "year": "1994",
      "journal-title": "J Virol"
    },
    {
      "key": "2021112105502161000_16.8.1559.37",
      "first-page": "526",
      "article-title": "An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5\u00e2\u0080\u00b2 noncoding region",
      "volume": "1",
      "year": "1995",
      "journal-title": "RNA"
    },
    {
      "key": "2021112105502161000_16.8.1559.38",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/bies.950110406"
    },
    {
      "key": "2021112105502161000_16.8.1559.39",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0092-8674(01)00435-4"
    }
  ],
  "container-title": "RNA",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://syndication.highwire.org/content/doi/10.1261/rna.2197210",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2024,
        3,
        27
      ]
    ],
    "date-time": "2024-03-27T21:17:27Z",
    "timestamp": 1711574247000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "http://rnajournal.cshlp.org/lookup/doi/10.1261/rna.2197210"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        2010,
        6,
        28
      ]
    ]
  },
  "references-count": 39,
  "journal-issue": {
    "issue": "8",
    "published-online": {
      "date-parts": [
        [
          2010,
          7,
          13
        ]
      ]
    },
    "published-print": {
      "date-parts": [
        [
          2010,
          8
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1261/rna.2197210"
  ],
  "URL": "http://dx.doi.org/10.1261/rna.2197210",
  "relation": {},
  "ISSN": [
    "1355-8382",
    "1469-9001"
  ],
  "subject": [],
  "container-title-short": "RNA",
  "published": {
    "date-parts": [
      [
        2010,
        6,
        28
      ]
    ]
  }
}