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 luciferase ( ) 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 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 () phenotypes for replication. Furthermore, using methyltransferase assays, we identified that the N96I mutation in NS5 exhibited a phenotype for 7-methylation, but not for 2′--methylation.


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