Cloning of Large Positive‐Strand RNA Viruses

AbstractFull‐length, biologically active cDNA clones of the positive‐strand RNA plant viruses are indispensable for investigating the functions of viral genes and control elements as well as generating virus‐derived gene expression and silencing vectors. Even though engineering of such clones for 4‐ to 10‐kb viral RNAs has become routine, it remains a challenging task for 15‐ to 20‐kb RNA genomes of the monopartite viruses in a family Closteroviridae. This unit describes strategic considerations and techniques used to generate an infectious cDNA clone of a closterovirus. The use of agroinfection to improve specific infectivity of the resulting clone is also explained. Curr. Protoc. Microbiol. 7:16F.1.1‐16F.1.26. © 2007 by John Wiley & Sons, Inc.