Reverse Genetics of RNA Viruses

Shringkhala Bajimaya, Tsuyoshi Hayashi, Toru Takimoto, Bajimaya, Shringkhala, Hayashi, Tsuyoshi, Takimoto, Toru

Daniel R. Perez

Sendai virus (SeV) is a non-segment negative-sense RNA virus that naturally infects and causes pneumonia in mice. As a prototypic member of the family Paramyxoviridae, SeV has been characterized well, and these studies revealed numerous traits of paramyxovirus biology. The reverse genetics system to rescue SeV was first established in 1995. The virus was rescued from a cloned cDNA that contains full genome sequence flanked by T7 promoter and hepatitis delta virus ribozyme. To rescue SeV, it is necessary to infect cells with a recombinant vaccinia virus vTF7.3 that expresses T7 RNA polymerase, and transfect with the SeV full genome cDNAs together with supporting plasmids encoding NP, P, and L genes under the T7 promoter. Synthesized viral RNA by T7 RNA polymerase will be encapsidated with NP and associated with a polymerase complex composed of P and L. The polymerase complex transcribes and replicates the genome, and produces progeny virions. Rescued SeV needs to be plaque purified to exclude vTF7.3 from viral stock. Reverse genetics system of SeV is relatively efficient compared to other paramyxoviruses, but alternative approaches to rescue poorly growing mutant viruses are also available.