Cleavage‐site preferences of Sindbis virus polyproteins containing the non‐structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo
The non‐structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2‐containing polyproteins. To generate poly...
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| Veröffentlicht in: | The EMBO journal 1990-08, Vol.9 (8), p.2631-2638 |
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| description | The non‐structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2‐containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2‐containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis. |
| doi_str_mv | 10.1002/j.1460-2075.1990.tb07445.x |
| format | Article |
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Evidence for temporal regulation of polyprotein processing in vivo</title><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>PubMed Central</source><source>Alma/SFX Local Collection</source><source>Free Full-Text Journals in Chemistry</source><creator>Groot, R. J. ; Hardy, W. R. ; Shirako, Y. ; Strauss, J. H.</creator><creatorcontrib>Groot, R. J. ; Hardy, W. R. ; Shirako, Y. ; Strauss, J. H.</creatorcontrib><description>The non‐structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2‐containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2‐containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.</description><identifier>ISSN: 0261-4189</identifier><identifier>EISSN: 1460-2075</identifier><identifier>DOI: 10.1002/j.1460-2075.1990.tb07445.x</identifier><identifier>PMID: 2142454</identifier><identifier>CODEN: EMJODG</identifier><language>eng</language><publisher>London: Nature Publishing Group</publisher><subject>Animals ; Base Sequence ; Biological and medical sciences ; Capsid - genetics ; Capsid - metabolism ; Cells, Cultured ; Chick Embryo ; Codon - genetics ; Endopeptidases - metabolism ; Fibroblasts - enzymology ; Fundamental and applied biological sciences. Psychology ; Gene Expression Regulation, Viral ; Molecular and cellular biology ; Molecular genetics ; Molecular Sequence Data ; Mutation ; Oligonucleotide Probes ; Plasmids ; Polymerase Chain Reaction ; Protein Biosynthesis ; Protein Processing, Post-Translational ; RNA, Viral - genetics ; Sindbis Virus - enzymology ; Sindbis Virus - genetics ; Transcription, Genetic ; Translation. Translation factors. 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J.</creatorcontrib><creatorcontrib>Hardy, W. R.</creatorcontrib><creatorcontrib>Shirako, Y.</creatorcontrib><creatorcontrib>Strauss, J. H.</creatorcontrib><title>Cleavage‐site preferences of Sindbis virus polyproteins containing the non‐structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo</title><title>The EMBO journal</title><addtitle>EMBO J</addtitle><description>The non‐structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2‐containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2‐containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.</description><subject>Animals</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Capsid - genetics</subject><subject>Capsid - metabolism</subject><subject>Cells, Cultured</subject><subject>Chick Embryo</subject><subject>Codon - genetics</subject><subject>Endopeptidases - metabolism</subject><subject>Fibroblasts - enzymology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Regulation, Viral</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>Oligonucleotide Probes</subject><subject>Plasmids</subject><subject>Polymerase Chain Reaction</subject><subject>Protein Biosynthesis</subject><subject>Protein Processing, Post-Translational</subject><subject>RNA, Viral - genetics</subject><subject>Sindbis Virus - enzymology</subject><subject>Sindbis Virus - genetics</subject><subject>Transcription, Genetic</subject><subject>Translation. Translation factors. Protein processing</subject><subject>Viral Core Proteins - genetics</subject><subject>Viral Core Proteins - metabolism</subject><subject>Viral Nonstructural Proteins</subject><issn>0261-4189</issn><issn>1460-2075</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVUUuO0zAYjhBoGAaOgGQhwa7htx0nMRKLoSovDWIBrC03-dtxldrBdsJ0xxE4BufiJMQ0KsOSlW19T_nLsicUcgrAnu9yWpSwYFCJnEoJeVxDVRQiv7mTnZ-gu9k5sJIuClrL-9mDEHYAIOqKnmVnjBasEMV59nPZoR71Fn99_xFMRNJ73KBH22AgbkM-GduuTSCj8UMgvesOvXcRjQ2kcTZqY43dkniNxDqbPKIfmjh43ZGZqAPmZDWaNnmSjfMk4r53ieFxO3Q6GmdT1C3zpJ0KhOQ9vUYzuofZvY3uAj6az4vsy-vV5-XbxdXHN--Wl1eLRjAQC16D4C1jAigta1kBcsASJW9Q0hZ5JaGsREFBF3UtKs4Yr9Z0LVsOGoSU_CJ7efTth_Ue2wZtnKqq3pu99gfltFH_ItZcq60blRCMyXLSP5v13n0dMES1N6HBrtMW3RAUFVODGlLQiyOx8S6E6ddPGRRUmlntVNpSpS1VmlnNM6ubSfz4dsuTdN51wp_OuA6N7jZe28aEvwmSQyF5NfEuj7xvpsPDfzRQqw-v3v-5898dH8x1</recordid><startdate>199008</startdate><enddate>199008</enddate><creator>Groot, R. 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H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5205-38053d22501168970e30e6e93ce91de3790675410a4885732237b1b9d30a05993</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Animals</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Capsid - genetics</topic><topic>Capsid - metabolism</topic><topic>Cells, Cultured</topic><topic>Chick Embryo</topic><topic>Codon - genetics</topic><topic>Endopeptidases - metabolism</topic><topic>Fibroblasts - enzymology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Regulation, Viral</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>Oligonucleotide Probes</topic><topic>Plasmids</topic><topic>Polymerase Chain Reaction</topic><topic>Protein Biosynthesis</topic><topic>Protein Processing, Post-Translational</topic><topic>RNA, Viral - genetics</topic><topic>Sindbis Virus - enzymology</topic><topic>Sindbis Virus - genetics</topic><topic>Transcription, Genetic</topic><topic>Translation. Translation factors. Protein processing</topic><topic>Viral Core Proteins - genetics</topic><topic>Viral Core Proteins - metabolism</topic><topic>Viral Nonstructural Proteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Groot, R. J.</creatorcontrib><creatorcontrib>Hardy, W. R.</creatorcontrib><creatorcontrib>Shirako, Y.</creatorcontrib><creatorcontrib>Strauss, J. 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H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cleavage‐site preferences of Sindbis virus polyproteins containing the non‐structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo</atitle><jtitle>The EMBO journal</jtitle><addtitle>EMBO J</addtitle><date>1990-08</date><risdate>1990</risdate><volume>9</volume><issue>8</issue><spage>2631</spage><epage>2638</epage><pages>2631-2638</pages><issn>0261-4189</issn><eissn>1460-2075</eissn><coden>EMJODG</coden><abstract>The non‐structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2‐containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. 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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Animals Base Sequence Biological and medical sciences Capsid - genetics Capsid - metabolism Cells, Cultured Chick Embryo Codon - genetics Endopeptidases - metabolism Fibroblasts - enzymology Fundamental and applied biological sciences. Psychology Gene Expression Regulation, Viral Molecular and cellular biology Molecular genetics Molecular Sequence Data Mutation Oligonucleotide Probes Plasmids Polymerase Chain Reaction Protein Biosynthesis Protein Processing, Post-Translational RNA, Viral - genetics Sindbis Virus - enzymology Sindbis Virus - genetics Transcription, Genetic Translation. Translation factors. Protein processing Viral Core Proteins - genetics Viral Core Proteins - metabolism Viral Nonstructural Proteins |
| title | Cleavage‐site preferences of Sindbis virus polyproteins containing the non‐structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo |
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