A new, simple system was developed for the direct manipulation of sequences of the genomic RNA (vRNA) of human parainfluenza virus type 3 (PIV3). Specifically, cDNA was constructed to encode a truncated synthetic vRNA analog in which all of the viral genes have been removed and replaced by the bacterial chloramphenicol acetyl transferase (CAT) gene. The cDNA is under the control of the promoter for T7 RNA polymerase and, at the distal end, contains an Hga1 site for linearization. Transcription of the cDNA in vitro yields a negative-sense RNA containing the exact, correct vRNA termini. The RNA was transfected into tissue culture cells which were infected with PIV3 to supply complementing proteins. Under these conditions, the RNA was rendered biologically active (""""""""rescued"""""""") as evidenced by the intracellular expression of CAT and by the production of a component (presumably infectious particles) that was released into the medium and could be passed productively onto fresh cells in the presence of helper virus. Together with a similar system developed by us for human respiratory syncytial virus (RSV) (accompanying report) and by others for Sendai virus, this represents the first experimental method by which one can synthesize, and thereby manipulate, biologically-active analogs of the vRNA of a nonsegmented negative strand virus. It will now be possible to directly identify and characterize cis-acting signals in vRNA. It should also be possible to modify the system such that rescue is complemented by cDNA-encoded viral proteins in place of PIV3. this would make it possible to characterize the roles of the proteins in replication, transcription and virion morphogenesis. Importantly, the success in rescuing PIV3-CAT supports the idea that it ultimately will be possible to rescue a complete, nondefective synthetic vRNA encoding infectious virus.
