Construction of a dengue virus type 4 reporter replicon and analysis of temperature-sensitive mutations in non-structural proteins 3 and 5.
J Gen Virol
; 91(Pt 11): 2713-8, 2010 Nov.
Article
em En
| MEDLINE
| ID: mdl-20631089
ABSTRACT
Replicon systems have been useful to study mechanisms of translation and replication of flavivirus RNAs. In this study, we constructed a dengue virus 4 replicon encoding a Renilla luciferase (R(luc)) reporter, and six single-residue substitution mutants were generated L128F and S158P in the non-structural protein (NS) 3 protease domain gene, and N96I, N390A, K437R and M805I in the NS5 gene. The effects of these substitutions on viral RNA translation and/or replication were examined by measuring R(luc) activities in wild-type and mutant replicon RNA-transfected Vero cells incubated at 35, 37 and 39 °C. Our results show that none of the mutations affected translation of replicon RNAs; however, L128F and S158P of NS3 at 39°C, and N96I of NS5 at 37 and 39°C, presented temperature-sensitive (ts) phenotypes for replication. Furthermore, using in vitro methyltransferase assays, we identified that the N96I mutation in NS5 exhibited a ts phenotype for N7-methylation, but not for 2'-O-methylation.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Replicon
/
Temperatura
/
Biossíntese de Proteínas
/
Replicação Viral
/
Proteínas não Estruturais Virais
/
Mutação de Sentido Incorreto
/
Vírus da Dengue
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Gen Virol
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos