Rapid full-length cloning of nonpolyadenylated RNA virus genomes

Abstract

Access to a full-length infectious clone of a viral genome is a virtual necessity for research aimed at understanding virus replication and gene expression mechanisms. While construction of a full-length clone may be straightforward, obtaining one that is infectious is by no means routine. Here the authors describe methods to maximize the likelihood of obtaining a full-length infectious clone. These include protocols to (1) sequence the ends of nonpolyadenylated RNA genomes, (2) obtain a full-length PCR product in a single reaction directly from viral RNA, and (3) efficiently clone the PCR product into a vector that allows in vitro transcription of viral RNA containing perfect or near-perfect termini. Given the traditional difficulty of obtaining infectious clones of RNA genomes (especially of nonpolyadenylated RNA viruses), this unit should be valuable to all virologists working with nonpolyadenylated as well as polyadenylated viruses of plants and animals.