Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.
Identifieur interne : 002383 ( PubMed/Corpus ); précédent : 002382; suivant : 002384Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.
Auteurs : Gail Rocheleau ; Jessica Petrillo ; Laura Guogas ; Lee GehrkeSource :
- Journal of virology [ 0022-538X ] ; 2004.
English descriptors
- KwdEn :
- 3' Untranslated Regions (chemistry), Alfalfa mosaic virus (genetics), Amino Acid Sequence, Base Sequence, Capsid Proteins (metabolism), Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Protein Binding, Protein Biosynthesis, RNA, Viral (biosynthesis), RNA, Viral (chemistry), RNA, Viral (metabolism).
- MESH :
- chemical , biosynthesis : RNA, Viral.
- chemical , chemistry : 3' Untranslated Regions, RNA, Viral.
- genetics : Alfalfa mosaic virus.
- chemical , metabolism : Capsid Proteins, RNA, Viral.
- Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Protein Binding, Protein Biosynthesis.
Abstract
The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.
DOI: 10.1128/JVI.78.15.8036-8046.2004
PubMed: 15254175
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pubmed:15254175Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.</title><author><name sortKey="Rocheleau, Gail" sort="Rocheleau, Gail" uniqKey="Rocheleau G" first="Gail" last="Rocheleau">Gail Rocheleau</name><affiliation><nlm:affiliation>Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Petrillo, Jessica" sort="Petrillo, Jessica" uniqKey="Petrillo J" first="Jessica" last="Petrillo">Jessica Petrillo</name></author><author><name sortKey="Guogas, Laura" sort="Guogas, Laura" uniqKey="Guogas L" first="Laura" last="Guogas">Laura Guogas</name></author><author><name sortKey="Gehrke, Lee" sort="Gehrke, Lee" uniqKey="Gehrke L" first="Lee" last="Gehrke">Lee Gehrke</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2004">2004</date><idno type="RBID">pubmed:15254175</idno><idno type="pmid">15254175</idno><idno type="doi">10.1128/JVI.78.15.8036-8046.2004</idno><idno type="wicri:Area/PubMed/Corpus">002383</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002383</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.</title><author><name sortKey="Rocheleau, Gail" sort="Rocheleau, Gail" uniqKey="Rocheleau G" first="Gail" last="Rocheleau">Gail Rocheleau</name><affiliation><nlm:affiliation>Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.</nlm:affiliation></affiliation></author><author><name sortKey="Petrillo, Jessica" sort="Petrillo, Jessica" uniqKey="Petrillo J" first="Jessica" last="Petrillo">Jessica Petrillo</name></author><author><name sortKey="Guogas, Laura" sort="Guogas, Laura" uniqKey="Guogas L" first="Laura" last="Guogas">Laura Guogas</name></author><author><name sortKey="Gehrke, Lee" sort="Gehrke, Lee" uniqKey="Gehrke L" first="Lee" last="Gehrke">Lee Gehrke</name></author></analytic><series><title level="j">Journal of virology</title><idno type="ISSN">0022-538X</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>3' Untranslated Regions (chemistry)</term><term>Alfalfa mosaic virus (genetics)</term><term>Amino Acid Sequence</term><term>Base Sequence</term><term>Capsid Proteins (metabolism)</term><term>Molecular Sequence Data</term><term>Mutation</term><term>Nucleic Acid Conformation</term><term>Protein Binding</term><term>Protein Biosynthesis</term><term>RNA, Viral (biosynthesis)</term><term>RNA, Viral (chemistry)</term><term>RNA, Viral (metabolism)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>RNA, Viral</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>3' Untranslated Regions</term><term>RNA, Viral</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Alfalfa mosaic virus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Capsid Proteins</term><term>RNA, Viral</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Base Sequence</term><term>Molecular Sequence Data</term><term>Mutation</term><term>Nucleic Acid Conformation</term><term>Protein Binding</term><term>Protein Biosynthesis</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15254175</PMID><DateCompleted><Year>2004</Year><Month>08</Month><Day>06</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0022-538X</ISSN><JournalIssue CitedMedium="Print"><Volume>78</Volume><Issue>15</Issue><PubDate><Year>2004</Year><Month>Aug</Month></PubDate></JournalIssue><Title>Journal of virology</Title><ISOAbbreviation>J. Virol.</ISOAbbreviation></Journal><ArticleTitle>Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.</ArticleTitle><Pagination><MedlinePgn>8036-46</MedlinePgn></Pagination><Abstract><AbstractText>The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Rocheleau</LastName><ForeName>Gail</ForeName><Initials>G</Initials><AffiliationInfo><Affiliation>Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Petrillo</LastName><ForeName>Jessica</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Guogas</LastName><ForeName>Laura</ForeName><Initials>L</Initials></Author><Author ValidYN="Y"><LastName>Gehrke</LastName><ForeName>Lee</ForeName><Initials>L</Initials></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>R01 GM042504</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>GM42504</GrantID><Acronym>GM</Acronym><Agency>NIGMS NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, P.H.S.</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>J Virol</MedlineTA><NlmUniqueID>0113724</NlmUniqueID><ISSNLinking>0022-538X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D020413">3' Untranslated Regions</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D036022">Capsid Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D012367">RNA, 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