Linked Life Data

20976195

Source:http://linkedlifedata.com/resource/pubmed/id/20976195

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2010-10-26
pubmed:abstractText
We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:status
MEDLINE
pubmed:issn
1553-7374
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
6
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
e1001146
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing.
pubmed:affiliation
The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S.