Introducing Recombinant Picornaviral Genomes into Cells

Introduction Picornarviruses comprise a large group of small, nonenveloped, positive-sense, single-stranded RNA viruses. The picornavirus life cycle is usually rapid and exclusively cytoplasmic, without integration into the host cell’s genome or a nuclear phase. Due to their biology and genetic constraints, the utility of picornaviruses for general gene delivery purposes is limited. However, they may prove useful as vaccine vectors. Furthermore, picornavirus-driven expression of various reporter genes or foreign RNA elements is of interest to the picornavirus molecular virologist. Introduction of recombinant picornaviral genomes into the cell relies on the historic observation that the isolated virion RNA is infectious. This property extends to in-vitro-transcribed RNA as long as the authentic viral 5' end is preserved. That said, up to two additional 5'-terminal guanine residues (remnants from the T7 RNA polymerase-based in vitro transcription), although reducing infectivity, can be tolerated. Additional nucleotides at the 3' end are of far less consequence. Thus, any unique restriction site downstream from the poly(A) sequence (preferably as close as possible downstream) can be used to linearize the plasmid containing the viral genome before in vitro transcription.