Source:http://linkedlifedata.com/resource/pubmed/id/20937889
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
43
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| pubmed:dateCreated |
2010-10-27
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| pubmed:abstractText |
Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ?10(4) pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this "two-hit" reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.
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| pubmed:grant | |
| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Viral,
http://linkedlifedata.com/resource/pubmed/chemical/NS35 protein, rotavirus,
http://linkedlifedata.com/resource/pubmed/chemical/RNA-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Viral Nonstructural Proteins
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| pubmed:status |
MEDLINE
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| pubmed:month |
Oct
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| pubmed:issn |
1091-6490
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| pubmed:author | |
| pubmed:issnType |
Electronic
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| pubmed:day |
26
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| pubmed:volume |
107
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| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
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| pubmed:pagination |
18652-7
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| pubmed:meshHeading |
pubmed-meshheading:20937889-Amino Acid Sequence,
pubmed-meshheading:20937889-Base Sequence,
pubmed-meshheading:20937889-Cell Line,
pubmed-meshheading:20937889-DNA, Viral,
pubmed-meshheading:20937889-Genes, Viral,
pubmed-meshheading:20937889-Genetic Engineering,
pubmed-meshheading:20937889-Humans,
pubmed-meshheading:20937889-Molecular Sequence Data,
pubmed-meshheading:20937889-Mutation,
pubmed-meshheading:20937889-RNA Interference,
pubmed-meshheading:20937889-RNA-Binding Proteins,
pubmed-meshheading:20937889-Recombinant Proteins,
pubmed-meshheading:20937889-Recombination, Genetic,
pubmed-meshheading:20937889-Rotavirus,
pubmed-meshheading:20937889-Selection, Genetic,
pubmed-meshheading:20937889-Sequence Homology, Amino Acid,
pubmed-meshheading:20937889-Sequence Homology, Nucleic Acid,
pubmed-meshheading:20937889-Temperature,
pubmed-meshheading:20937889-Viral Nonstructural Proteins
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| pubmed:year |
2010
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| pubmed:articleTitle |
Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.
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| pubmed:affiliation |
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8026, USA.
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| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't,
Research Support, N.I.H., Extramural,
Research Support, N.I.H., Intramural
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