Construction of a dengue virus type 4 reporter replicon and analysis of temperature-sensitive mutations in non-structural proteins 3 and 5

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-str...

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Bibliographic Details
Published in:Journal of general virology 2010-11, Vol.91 (Pt 11), p.2713-2718
Main Authors: ALCARAZ-ESTRADA, Sofia L, MANZANO, Mark Irvin M, DEL ANGEL, Rosa M, LEVIS, Robin, PADMANABHAN, R
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Animal
Animals
Biological and medical sciences
Cercopithecus aethiops
Dengue Virus - genetics
Dengue Virus - growth & development
Dengue Virus - physiology
Dengue virus type 4
Flavivirus
Fundamental and applied biological sciences. Psychology
Genes, Essential
Genes, Reporter
Genes, Viral
Luciferases, Renilla - genetics
Luciferases, Renilla - metabolism
Microbiology
Miscellaneous
Mutation, Missense
Protein Biosynthesis
Protein Stability
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Renilla
Replicon
Temperature
Vero Cells
Viral Nonstructural Proteins - genetics
Virology
Virus Replication
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Summary: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.
ISSN:0022-1317
1465-2099
1465-2099
DOI:10.1099/vir.0.024083-0