High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing.
PLoS Pathog
; 6(10): e1001146, 2010 Oct 14.
Article
em En
| MEDLINE
| ID: mdl-20976195
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Mutagênese Insercional
/
Mapeamento Cromossômico
/
Genoma Viral
/
Análise de Sequência de DNA
/
Vírus da Encefalite Equina Venezuelana
Limite:
Animals
País/Região como assunto:
America do sul
/
Venezuela
Idioma:
En
Revista:
PLoS Pathog
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos