The Hepatitis Delta Virus Large Antigen Is Farnesylated Both in Vitro and in Animal Cells

The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbo...

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Veröffentlicht in:The Journal of biological chemistry 1996-03, Vol.271 (9), p.4569-4572
Hauptverfasser: Otto, J C, Casey, P J
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Casey, P J
description The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S -transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent K = 0.8 μM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.
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Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S -transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent K = 0.8 μM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. 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Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S -transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent K = 0.8 μM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Brain - enzymology</subject><subject>Cattle</subject><subject>Cell Line</subject><subject>Chlorocebus aethiops</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Cytosol - enzymology</subject><subject>Dimethylallyltranstransferase - metabolism</subject><subject>Glutathione Transferase - metabolism</subject><subject>Hepatitis Antigens - chemistry</subject><subject>Hepatitis Antigens - isolation &amp; purification</subject><subject>Hepatitis Antigens - metabolism</subject><subject>hepatitis D virus</subject><subject>Hepatitis delta Antigens</subject><subject>Hepatitis Delta Virus - metabolism</subject><subject>Kinetics</subject><subject>Molecular Sequence Data</subject><subject>Polyisoprenyl Phosphates - metabolism</subject><subject>Protein Prenylation</subject><subject>Recombinant Fusion Proteins - isolation &amp; 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Casey, P J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c388t-51b29366fa33f4f2e33d7ce1437ec405e563b95aaeffe25e74b1f330774f65053</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Brain - enzymology</topic><topic>Cattle</topic><topic>Cell Line</topic><topic>Chlorocebus aethiops</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Cytosol - enzymology</topic><topic>Dimethylallyltranstransferase - metabolism</topic><topic>Glutathione Transferase - metabolism</topic><topic>Hepatitis Antigens - chemistry</topic><topic>Hepatitis Antigens - isolation &amp; purification</topic><topic>Hepatitis Antigens - metabolism</topic><topic>hepatitis D virus</topic><topic>Hepatitis delta Antigens</topic><topic>Hepatitis Delta Virus - metabolism</topic><topic>Kinetics</topic><topic>Molecular Sequence Data</topic><topic>Polyisoprenyl Phosphates - metabolism</topic><topic>Protein Prenylation</topic><topic>Recombinant Fusion Proteins - isolation &amp; purification</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Sesquiterpenes</topic><topic>Spodoptera</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Otto, J C</creatorcontrib><creatorcontrib>Casey, P J</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Otto, J C</au><au>Casey, P J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The Hepatitis Delta Virus Large Antigen Is Farnesylated Both in Vitro and in Animal Cells</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1996-03-01</date><risdate>1996</risdate><volume>271</volume><issue>9</issue><spage>4569</spage><epage>4572</epage><pages>4569-4572</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S -transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent K = 0.8 μM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>8617711</pmid><doi>10.1074/jbc.271.9.4569</doi><tpages>4</tpages><oa>free_for_read</oa></addata></record>
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subjects Amino Acid Sequence
Animals
Brain - enzymology
Cattle
Cell Line
Chlorocebus aethiops
Chromatography, High Pressure Liquid
Cytosol - enzymology
Dimethylallyltranstransferase - metabolism
Glutathione Transferase - metabolism
Hepatitis Antigens - chemistry
Hepatitis Antigens - isolation & purification
Hepatitis Antigens - metabolism
hepatitis D virus
Hepatitis delta Antigens
Hepatitis Delta Virus - metabolism
Kinetics
Molecular Sequence Data
Polyisoprenyl Phosphates - metabolism
Protein Prenylation
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
Sesquiterpenes
Spodoptera
Transfection
title The Hepatitis Delta Virus Large Antigen Is Farnesylated Both in Vitro and in Animal Cells
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