A simple, rapid and efficient way to obtain infectious clones of potyviruses

• Published in: Journal of virological methods Vol. 183; no. 1; pp. 94 - 97
• Main Authors: Desbiez, C., Chandeysson, C., Lecoq, H., Moury, B.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.07.2012; Elsevier
• Subjects: Bacteria; Biological and medical sciences; Carica - virology; Citrullus - virology; Citrullus lanatus; Cloning in yeast; Cucurbita - virology; DNA, Complementary - genetics; Fundamental and applied biological sciences. Psychology; homologous recombination; Infectious clone; Intron; Introns; Life Sciences; Microbiology; Microbiology and Parasitology; Molecular Biology - methods; Mutagenesis; Papaya ringspot virus; Phytopathology and phytopharmacy; Phytopathology. Animal pests. Plant and forest protection; Plant ssRNA virus; Plant viruses and viroids; Polymerase chain reaction; Potyvirus; Potyvirus - genetics; Potyvirus - isolation & purification; Potyvirus - pathogenicity; Recombination, Genetic; RNA viruses; RNA, Viral - genetics; Techniques used in virology; Toxicity; Vegetal Biology; Virology; Virology - methods; Yeasts - genetics; Zucchini yellow mosaic virus; Infectious clone; Plant ssRNA virus; Potyvirus; Intron; Cloning in yeast; Virus; Yeast; Plant pathogen; Method; Potyviridae; plant ssRNA virus; potyvirus; infectious clone; intron; cloning in yeast
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2012.03.035

The availability of an infectious cDNA clone is a prerequisite for genetic studies on RNA viruses. However, despite important improvement in molecular biology techniques during the last decades, obtaining such clones often remains tedious, time-consuming and rather unpredictable. In the case of potyviruses, cDNA clones are frequently unstable due to the toxicity of some viral proteins for bacteria. The problem can be overcome by inserting introns into the viral sequence but this requires additional steps in the cloning process and depends on the availability of suitable restriction sites in the viral sequence or adjunction of such sites by mutagenesis. Homologous recombination in yeast rather than in vitro restriction and ligation can be used to build infectious clones or other viral constructs. This paper describes how, by using recombination in yeast and fusion PCR, infectious intron-containing clones were obtained within a few weeks for two strains of watermelon mosaic virus (WMV, Potyvirus), whereas previous attempts using “classical” cloning techniques had failed repeatedly. Using the same approach, intronless infectious clones of two other potyviruses, zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV), were obtained in less than two weeks.