Enhanced measles virus cDNA rescue and gene expression after heat shock.
Rescue of negative-stranded RNA viruses from full-length genomic cDNA clones 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., EMBO J. 14:5773-5784, 1995) could be improved by modifying the procedure in two 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 the average number of transfected cultures that produced virus another two- to threefold. In addition, heat shock increased the number of plaques produced by positive cultures. The effect of heat shock on rescue led us to test the effect on transient expression from an MV minireplicon. Heat shock increased the level of reporter gene expression when either minireplicon DNA or RNA was used regardless of whether complementation was provided by cotransfection with expression plasmids or infection with MV helper virus. In addition, we found that MV minireplicon gene expression could be stimulated by cotransfection with an Hsp72 expression plasmid, indicating that hsp72 likely plays a role in the effect of heat shock.
Parks
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CL
Department of Viral Vaccine Research, Wyeth-Lederle Vaccines and Pediatrics, Pearl River, New York 10965, USA.
Lerch
R A
RA
Walpita
P
P
Sidhu
M S
MS
Udem
S A
SA
D000818:Animals; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D050884:HSP72 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D008459:Measles virus / Q000235:genetics*; D012093:Replicon; D014709:Vero Cells
D004279:DNA, Viral; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins