Construction of Infectious Clones for RNA Viruses: TMV

The generation of infectious clones is routinely the first step for reverse genetic studies of RNA plant virus gene and sequence function. The procedure given here, details the creation of cDNA clones of tobacco mosaic virus, from which infectious transcripts can be generated in vitro with T7 RNA polymerase. The procedure describes methods for virion purification, viral RNA extraction, reverse transcription, PCR amplification of genomic cDNA fragments, generation of a full-length cDNA clone under the control of a T7 promoter, in vitro transcription, and infectivity testing.