Packaging of the Rotavirus Genome. (360G-Wellcome-082127_Z_07_Z)

This project focuses on the genome segment selection/packaging process in rotaviruses. Building on the recent report from Taniguchi and colleagues of the first gene rescue in rotaviruses (20) it will seek to develop this technology in the context of mapping the segment selection/packaging signals in rotavirus RNAs. A hybrid cDNA of viral genome segment 5 carrying an inserted bacterial xanthine-guanine phosphribosyl transferase gene (XGPRT) expression cassette will be constructed. The hybrid cDN A has been manipulated by the addition of a T7 promoter and a hepatitis virus ribozyme at its 5 and 3 termini respectively such that when transcribed by T7 RNA polymerase a transcript with authentic viral gene termini will be generated. XGPRT expression will be used to provide a positive selection screen for rescue of the hybrid into infectious virus. The hybrid will used to develop first a helper dependent gene rescue protocol in which hybrid gene 5 transcripts will be rescued into infectio us virus by a helper rotavirus infection of transfected cells. Building on the helper dependent system a wholly plasmid based gene rescue protocol will be developed with which to probe for segment selection/packing signals in the other viral genes