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

• 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
• ISSN: 0022-1317; 1465-2099; 1465-2099
• DOI: 10.1099/vir.0.024083-0

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.