title: Construction of a dengue virus type 4 reporter replicon and analysis of temperature-sensitive mutations in non-structural proteins 3 and 5.
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.
authors.affiliation: Georgetown University Medical Center, Washington, DC, USA.
authors.forename: Sofia L
authors.initials: SL
authors.lastname: Alcaraz-Estrada
authors.forename: Mark Irvin M
authors.initials: MI
authors.lastname: Manzano
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Del Angel
authors.forename: Robin
authors.initials: R
authors.lastname: Levis
authors.forename: R
authors.initials: R
authors.lastname: Padmanabhan
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D020043:Genes, Essential; D017930:Genes, Reporter; D005814:Genes, Viral; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D014176:Protein Biosynthesis; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D013696:Temperature; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D049410:Luciferases, Renilla