Poliovirus RNA polymerase mutation 3D-M394T results in a temperature-sensitive defect in RNA synthesis

Virology
D J BartonJ B Flanegan

Abstract

Mutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3D pol was purified from infected cells and was shown to be rapidly heat-inactivated at 45 degrees when compared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA revealed a U to C transition at nt 7167 resulting in an amino acid change of methionine 394 of 3D pol to threonine. The 3D-M394T mutation was engineered into a wild-type infectious clone of poliovirus type 1. The resultant mutant virus, 3D-105, had a temperature-sensitive phenotype in plaque assays. The translation and replication of wild-type, ts10, and 3D-105 virion RNAs were all characterized in HeLa S10 translation-RNA replication reactions in vitro. The optimum temperatures for the replication of the wild-type and mutant viral RNAs in the HeLa S10 translation-replication reactions were 37 and 34 degrees, respectively. To characterize the temperature-sensitive defect in the replication of the mutant RNA, we used preinitiation RNA replication complexes which were formed in HeLa S10 in vitro reactions containing guanidine HCl. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replication complexes was normal at 34 degrees but was rapidly an...Continue Reading

Citations

Jun 11, 2010·Journal of Virology·Sarah E HobdeyOlve B Peersen
May 14, 2004·Journal of Virology·Sunny S Choe, Karla Kirkegaard
Mar 28, 2009·Virology·Carlos Sandoval-Jaime, Ana Lorena Gutiérrez-Escolano
Jul 3, 2007·Virus Research·Christopher L WashenbergerDavid J Barton
Mar 6, 2002·The Journal of Biological Chemistry·John M LyleKarla Kirkegaard
Aug 8, 2014·Journal of Virology·Cheng-Lin DengBo Zhang

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