Enhanced measles virus cDNA rescue and gene expression after heat shock

Rescue of negative-stranded RNA viruses from full-length genomic cDNA clone s is an essential technology for genetic analysis of this class of viruses. Using this technology in our studies of measles virus (MV), we found that the efficiency of the measles virus rescue procedure (F. Radecke et al., EM BO J. 14:5773-5784, 1995) could be improved by modifying the procedure in t wo ways. First, we found that coculture of transfected 293-3-46 cells with a monolayer of Vero cells increased the number of virus-producing cultures about 20-fold. Second, we determined that heat shock treatment increased th e average number of transfected cultures that produced virus another two- t o threefold. In addition, heat shock increased the number of plaques produc ed by positive cultures. The effect of heat shock on rescue led us to test the effect on transient Expression from an AN minireplicon. Heat shock incr eased the level of reporter gene expression vr hen either minireplicon DNA or RNA was used regardless of whether complementation Has provided by cotra nsfection with expression plasmids or infection with MV helper virus. In ad dition, we found that MV minireplicon gene expression could be stimulated b y cotransfection with an Hsp72 expression plasmid, indicating that hsp72 li kely plays a role in the effect of heat shock.