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BIBTEX RIS

Identification and Characterization of the RNA Chaperone Activity of Hepatitis Delta Antigen Peptides

1998; Elsevier BV; Volume: 273; Issue: 41 Linguagem: Inglês

10.1074/jbc.273.41.26455

ISSN

1083-351X

Autores

Zhi-Shun Huang, Huey‐Nan Wu,

Tópico(s)

Hepatitis Viruses Studies and Epidemiology

Resumo

In this study, we identified an activity of the hepatitis delta antigen that both modulates the cis-cleaving activities of hepatitis delta virus (HDV) genomic RNA fragments and facilitates the trans-cleavage reactions between hammerhead ribozymes and the cognate substrates of various lengths in vitro. Hepatitis delta antigen peptides exert their effect by accelerating the unfolding and refolding of RNA molecules and by promoting strand annealing and strand dissociation. In addition, the stimulatory effect of hepatitis delta antigen peptide on hammerhead catalysis is observed whether the peptide is removed or not by phenol/chloroform extraction prior to the initiation of trans-cleavage reaction. Therefore, hepatitis delta antigen peptide acts as an RNA chaperone. The RNA chaperone domain of hepatitis delta antigen overlaps with the coiled-coil domain that is rich in lysine residues. The RNA binding domains of hepatitis delta antigen previously identified are not required for the RNA chaperone activity identified herein. The RNA chaperone activity of hepatitis delta antigen may be important for the regulation of HDV replicationin vivo. In this study, we identified an activity of the hepatitis delta antigen that both modulates the cis-cleaving activities of hepatitis delta virus (HDV) genomic RNA fragments and facilitates the trans-cleavage reactions between hammerhead ribozymes and the cognate substrates of various lengths in vitro. Hepatitis delta antigen peptides exert their effect by accelerating the unfolding and refolding of RNA molecules and by promoting strand annealing and strand dissociation. In addition, the stimulatory effect of hepatitis delta antigen peptide on hammerhead catalysis is observed whether the peptide is removed or not by phenol/chloroform extraction prior to the initiation of trans-cleavage reaction. Therefore, hepatitis delta antigen peptide acts as an RNA chaperone. The RNA chaperone domain of hepatitis delta antigen overlaps with the coiled-coil domain that is rich in lysine residues. The RNA binding domains of hepatitis delta antigen previously identified are not required for the RNA chaperone activity identified herein. The RNA chaperone activity of hepatitis delta antigen may be important for the regulation of HDV replicationin vivo. hepatitis delta virus nucleotide(s) amino acid(s) small delta antigen large delta antigen C-terminal domain. Hepatitis delta virus (HDV)1 is a subviral pathogen that requires hepatitis B virus (HBV) to supply envelope protein for completion of package, secretion, and infection (1Rizzetto M. Hoyer B. Canese M.G. Shih J.W.K. Purcell R.H. Gerin J.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6124-6128Crossref PubMed Scopus (344) Google Scholar, 2Bonino F. Hoyer B. Shih W.K. Rizzeto M. Purcell R.H. Gerin J.L. Infect. Immun. 1984; 43: 1000-1005Crossref PubMed Google Scholar, 3Bonino F. Heermann K.H. Rizzeto M. Gerlich W.H. J. Virol. 1986; 58: 945-950Crossref PubMed Google Scholar). The genome of HDV is a single-stranded circular RNA of ∼1700 nt and HDV RNA and is replicated through a rolling circle mechanism (4Wang K.S. Choo Q.L. Weiner A.J. Ou J.H. Najarian R.A. Thayer R.M. Mullenbach G.T. Denniston K.L. Gerin J.L. Houghton M. Nature. 1986; 323: 508-515Crossref PubMed Scopus (515) Google Scholar). HDV codes one protein of two forms during infection: the small delta antigen (SdAg) contains 195 aa and the large delta antigen (LdAg) has an extra 19 aa at the C terminus (5Weiner A.J. Choo Q.L. Wang K.S. Govindarajan S. Redeker A.G. Gerin J.L. Houghton M. J. Virol. 1988; 62: 594-599Crossref PubMed Google Scholar). Transfection studies with HDV cDNA elucidated that the two protein forms have distinct functions. SdAg initiates genome replication (6Chao M. Hsieh S.Y. Taylor J. J. Virol. 1990; 64: 5066-5069Crossref PubMed Google Scholar) and LdAg promotes package (7Glenn J.S. Watson J.A. Havel C.M. White J.M. Science. 1992; 256: 1331-1334Crossref PubMed Scopus (237) Google Scholar). There are two RNA binding domains in each protein form. The first is the arginine-rich sequence near the N terminus, and the second is the arginine-rich motifs (ARMs) located at the middle one-third of the protein (8Poisson F. Roingeard P. Baillou A. Dubois F. Bonelli F. Calogero R.A. Gouderau A. J. Gen. Virol. 1993; 74: 2473-2477Crossref PubMed Scopus (28) Google Scholar, 9Lin J.H. Chang M.F. Baker S.C. Govindarajan S. Lai M.M.C. J. Virol. 1990; 64: 4051-4058Crossref PubMed Google Scholar). The RNA binding activity is important for the function of the two protein forms: the second RNA binding domain of SdAg is required to initiate genome replication (9Lin J.H. Chang M.F. Baker S.C. Govindarajan S. Lai M.M.C. J. Virol. 1990; 64: 4051-4058Crossref PubMed Google Scholar, 10Chang M.F. Sun C.Y. Chen C.J. Chang S.C. J. Virol. 1993; 67: 2529-2536Crossref PubMed Google Scholar, 11Lazinski D.W. Taylor J.M. J. Virol. 1993; 67: 2672-2680Crossref PubMed Google Scholar, 12Lee C.Z. Lin J.H. Mcknight K. Lai M.M.C. J. Virol. 1993; 67: 2221-2227Crossref PubMed Google Scholar), and the first RNA binding domain of LdAg is responsible for potent inhibition of replication (13Lee C.Z. Chen P.J. Chen D.S. J. Virol. 1995; 69: 5332-5336Crossref PubMed Google Scholar). The specific interactions between the hepatitis delta antigen and HDV RNA appear to be involved in the regulation of virus replication although a molecular mechanism has not yet been elucidated.HDV RNAs of genomic and antigenomic senses cis-cleaved in the absence of protein factors in vitro (14Kuo M.Y.-P. Sharmeen L. Dinter-Gottlieb Taylor J.M. J. Virol. 1988; 62: 4439-4444Crossref PubMed Google Scholar). The ribozyme activity of HDV RNA, which requires a pseudoknot-like structure of the RNA molecule (15Perrotta A.T. Been M.D. Nature. 1991; 350: 434-436Crossref PubMed Scopus (304) Google Scholar, 16Rosenstein S.P. Been M.D. Nucleic Acids Res. 1991; 19: 5409-5416Crossref PubMed Scopus (81) Google Scholar) and the catalysis of divalent cations (17Wu H.N. Lin Y.J. Lin F.P. Makino S. Chang M.F. Lai M.M.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1831-1835Crossref PubMed Scopus (248) Google Scholar), is essential for generating monomeric size RNA molecules during replication (18Macnaughton T.B. Wang Y.J. Lai M.M.C. J. Virol. 1993; 67: 2228-2234Crossref PubMed Google Scholar). Recently, Jeng et al. (19Jeng K.S. Su P.Y. Lai M.M.C. J. Virol. 1996; 70: 4205-4209Crossref PubMed Google Scholar) illustrated that hepatitis delta antigen may enhance, though is not required for, the processing of multiple length HDV RNA in vivo. Conceivably, hepatitis delta antigen per se or together with some other factor(s) acts as an RNA chaperone that modulates the ribozyme activity of HDV RNA.RNA chaperones are proteins that aid in the process of RNA folding by preventing misfolding or by resolving misfolded species (20Herschlag D. J. Biol. Chem. 1995; 270: 20871-20874Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). The RNA chaperone activities of several proteins that bind RNA with broad specificity have been explored through their effects on hammerhead ribozyme reactions and group I intron reactions. These proteins, including the nucleocapsid protein (NC) of human immunodeficiency virus (HIV), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 (A1 CTD), and Escherichia coli ribosomal proteins, can overcome the general limitations of ribozyme reactions, such as the formation/dissociation of base pairs and the adoption of functional structure, and facilitate ribozyme catalysis (21Tsuchihashi Z. Khosla M. Herschlag D. Science. 1993; 262: 99-102Crossref PubMed Scopus (190) Google Scholar, 22Bertrand E.L. Rossi J.J. EMBO J. 1994; 13: 2904-2912Crossref PubMed Scopus (147) Google Scholar, 23Coetzee T. Herschlag D. Belfort M. Genes Dev. 1994; 8: 1575-1588Crossref PubMed Scopus (146) Google Scholar, 24Herschlag D. Khosla M. Tsuchihashi Z. Kapel R.L. EMBO J. 1994; 13: 2913-2924Crossref PubMed Scopus (216) Google Scholar).Here we analyze the putative RNA chaperone activity of hepatitis delta antigen peptides in vitro. Using the facilitation of trans-cleavage reactions of the previously characterized hammerhead ribozyme HH16 and its 17-nucleotide substrate S (25Hertel K.L. Herschlag D. Uhlenbeck O.C. Biochemistry. 1994; 33: 3374-3385Crossref PubMed Scopus (252) Google Scholar) as the initial assay, we identify the strand-annealing and strand-dissociation activities of hepatitis delta antigen peptides. We then show that the functional hepatitis delta antigen peptides promote RNA unfolding that stimulates interstranded duplex formation. This activity is able to activate an antisense RNA as well as facilitate trans-acting hammerhead ribozymes to find their targets in cognate substrate RNAs. In addition, hepatitis delta antigen peptides can modulate the cis-cleaving activity of HDV genomic RNA fragments. Hepatitis delta antigen acts as an RNA chaperone and the RNA chaperone domain locates at the N-terminal domain of the protein that contains a high density of basic amino acids. Our findings suggest that the RNA binding domains of hepatitis delta antigen identified previously are, therefore, not required for RNA chaperone activity. Hepatitis delta virus (HDV)1 is a subviral pathogen that requires hepatitis B virus (HBV) to supply envelope protein for completion of package, secretion, and infection (1Rizzetto M. Hoyer B. Canese M.G. Shih J.W.K. Purcell R.H. Gerin J.L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6124-6128Crossref PubMed Scopus (344) Google Scholar, 2Bonino F. Hoyer B. Shih W.K. Rizzeto M. Purcell R.H. Gerin J.L. Infect. Immun. 1984; 43: 1000-1005Crossref PubMed Google Scholar, 3Bonino F. Heermann K.H. Rizzeto M. Gerlich W.H. J. Virol. 1986; 58: 945-950Crossref PubMed Google Scholar). The genome of HDV is a single-stranded circular RNA of ∼1700 nt and HDV RNA and is replicated through a rolling circle mechanism (4Wang K.S. Choo Q.L. Weiner A.J. Ou J.H. Najarian R.A. Thayer R.M. Mullenbach G.T. Denniston K.L. Gerin J.L. Houghton M. Nature. 1986; 323: 508-515Crossref PubMed Scopus (515) Google Scholar). HDV codes one protein of two forms during infection: the small delta antigen (SdAg) contains 195 aa and the large delta antigen (LdAg) has an extra 19 aa at the C terminus (5Weiner A.J. Choo Q.L. Wang K.S. Govindarajan S. Redeker A.G. Gerin J.L. Houghton M. J. Virol. 1988; 62: 594-599Crossref PubMed Google Scholar). Transfection studies with HDV cDNA elucidated that the two protein forms have distinct functions. SdAg initiates genome replication (6Chao M. Hsieh S.Y. Taylor J. J. Virol. 1990; 64: 5066-5069Crossref PubMed Google Scholar) and LdAg promotes package (7Glenn J.S. Watson J.A. Havel C.M. White J.M. Science. 1992; 256: 1331-1334Crossref PubMed Scopus (237) Google Scholar). There are two RNA binding domains in each protein form. The first is the arginine-rich sequence near the N terminus, and the second is the arginine-rich motifs (ARMs) located at the middle one-third of the protein (8Poisson F. Roingeard P. Baillou A. Dubois F. Bonelli F. Calogero R.A. Gouderau A. J. Gen. Virol. 1993; 74: 2473-2477Crossref PubMed Scopus (28) Google Scholar, 9Lin J.H. Chang M.F. Baker S.C. Govindarajan S. Lai M.M.C. J. Virol. 1990; 64: 4051-4058Crossref PubMed Google Scholar). The RNA binding activity is important for the function of the two protein forms: the second RNA binding domain of SdAg is required to initiate genome replication (9Lin J.H. Chang M.F. Baker S.C. Govindarajan S. Lai M.M.C. J. Virol. 1990; 64: 4051-4058Crossref PubMed Google Scholar, 10Chang M.F. Sun C.Y. Chen C.J. Chang S.C. J. Virol. 1993; 67: 2529-2536Crossref PubMed Google Scholar, 11Lazinski D.W. Taylor J.M. J. Virol. 1993; 67: 2672-2680Crossref PubMed Google Scholar, 12Lee C.Z. Lin J.H. Mcknight K. Lai M.M.C. J. Virol. 1993; 67: 2221-2227Crossref PubMed Google Scholar), and the first RNA binding domain of LdAg is responsible for potent inhibition of replication (13Lee C.Z. Chen P.J. Chen D.S. J. Virol. 1995; 69: 5332-5336Crossref PubMed Google Scholar). The specific interactions between the hepatitis delta antigen and HDV RNA appear to be involved in the regulation of virus replication although a molecular mechanism has not yet been elucidated. HDV RNAs of genomic and antigenomic senses cis-cleaved in the absence of protein factors in vitro (14Kuo M.Y.-P. Sharmeen L. Dinter-Gottlieb Taylor J.M. J. Virol. 1988; 62: 4439-4444Crossref PubMed Google Scholar). The ribozyme activity of HDV RNA, which requires a pseudoknot-like structure of the RNA molecule (15Perrotta A.T. Been M.D. Nature. 1991; 350: 434-436Crossref PubMed Scopus (304) Google Scholar, 16Rosenstein S.P. Been M.D. Nucleic Acids Res. 1991; 19: 5409-5416Crossref PubMed Scopus (81) Google Scholar) and the catalysis of divalent cations (17Wu H.N. Lin Y.J. Lin F.P. Makino S. Chang M.F. Lai M.M.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1831-1835Crossref PubMed Scopus (248) Google Scholar), is essential for generating monomeric size RNA molecules during replication (18Macnaughton T.B. Wang Y.J. Lai M.M.C. J. Virol. 1993; 67: 2228-2234Crossref PubMed Google Scholar). Recently, Jeng et al. (19Jeng K.S. Su P.Y. Lai M.M.C. J. Virol. 1996; 70: 4205-4209Crossref PubMed Google Scholar) illustrated that hepatitis delta antigen may enhance, though is not required for, the processing of multiple length HDV RNA in vivo. Conceivably, hepatitis delta antigen per se or together with some other factor(s) acts as an RNA chaperone that modulates the ribozyme activity of HDV RNA. RNA chaperones are proteins that aid in the process of RNA folding by preventing misfolding or by resolving misfolded species (20Herschlag D. J. Biol. Chem. 1995; 270: 20871-20874Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar). The RNA chaperone activities of several proteins that bind RNA with broad specificity have been explored through their effects on hammerhead ribozyme reactions and group I intron reactions. These proteins, including the nucleocapsid protein (NC) of human immunodeficiency virus (HIV), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 (A1 CTD), and Escherichia coli ribosomal proteins, can overcome the general limitations of ribozyme reactions, such as the formation/dissociation of base pairs and the adoption of functional structure, and facilitate ribozyme catalysis (21Tsuchihashi Z. Khosla M. Herschlag D. Science. 1993; 262: 99-102Crossref PubMed Scopus (190) Google Scholar, 22Bertrand E.L. Rossi J.J. EMBO J. 1994; 13: 2904-2912Crossref PubMed Scopus (147) Google Scholar, 23Coetzee T. Herschlag D. Belfort M. Genes Dev. 1994; 8: 1575-1588Crossref PubMed Scopus (146) Google Scholar, 24Herschlag D. Khosla M. Tsuchihashi Z. Kapel R.L. EMBO J. 1994; 13: 2913-2924Crossref PubMed Scopus (216) Google Scholar). Here we analyze the putative RNA chaperone activity of hepatitis delta antigen peptides in vitro. Using the facilitation of trans-cleavage reactions of the previously characterized hammerhead ribozyme HH16 and its 17-nucleotide substrate S (25Hertel K.L. Herschlag D. Uhlenbeck O.C. Biochemistry. 1994; 33: 3374-3385Crossref PubMed Scopus (252) Google Scholar) as the initial assay, we identify the strand-annealing and strand-dissociation activities of hepatitis delta antigen peptides. We then show that the functional hepatitis delta antigen peptides promote RNA unfolding that stimulates interstranded duplex formation. This activity is able to activate an antisense RNA as well as facilitate trans-acting hammerhead ribozymes to find their targets in cognate substrate RNAs. In addition, hepatitis delta antigen peptides can modulate the cis-cleaving activity of HDV genomic RNA fragments. Hepatitis delta antigen acts as an RNA chaperone and the RNA chaperone domain locates at the N-terminal domain of the protein that contains a high density of basic amino acids. Our findings suggest that the RNA binding domains of hepatitis delta antigen identified previously are, therefore, not required for RNA chaperone activity. We thank Huey-Wen Huang and Sing-Yen You for assistance in protein purification and D. Platt for English editing.

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