“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.”
I am a sucker for good techniques like this one: long ago I helped invent a technique for idiot-proof cDNA cloning of the 3′ of the genome (Pappu et a., J Virol Methods. 1993 Jan;41(1):9-20), and have kept a watchful eye on potyvirus genome cloning ever since – and it is a challenge, because they are >10kb in length. This is an elegant solution to an old problem.
See on www.sciencedirect.com