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

The Journal of General Virology
Sofia L Alcaraz-EstradaR Padmanabhan

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.

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Citations

Mar 15, 2013·Journal of Virology·Shi-Hua LiPei-Yong Shi
Jan 10, 2014·Memórias do Instituto Oswaldo Cruz·Jefferson José da Silva SantosLaura Helena Vega Gonzales Gil
Sep 7, 2011·Journal of Virological Methods·Fei CaoCheng-Feng Qin
Aug 15, 2015·Methods : a Companion to Methods in Enzymology·Radhakrishnan PadmanabhanKyung H Choi
Apr 1, 2014·Antiviral Research·Siwaporn BoonyasuppayakornRadhakrishnan Padmanabhan
Aug 20, 2016·Antiviral Research·Anuradha BalasubramanianRadhakrishnan Padmanabhan
May 30, 2020·Viruses·Luis Adrián De Jesús-GonzálezRosa María Del Ángel

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Methods Mentioned

BETA
in vitro transcription

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