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Enhanced measles virus cDNA rescue and gene expression after heat shock - PubMed

Enhanced measles virus cDNA rescue and gene expression after heat shock

C L Parks et al. J Virol. 1999 May.

Abstract

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.

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Figures

FIG. 1
FIG. 1

Flow diagram of the modified cDNA rescue procedure. The use of a heat shock step and the coculture of transfected cells with Vero cells are the primary differences from the procedure described by Radecke et al. (42).

FIG. 2
FIG. 2

Effect of heat shock on minireplicon gene expression. 293-3-46 cells were transfected overnight with MV-CAT minireplicon plasmid DNA (1 μg). Some transfections (lanes 3 and 7) also received the MV L-gene expression plasmid (100 ng) to provide L complementation. About 14 h after transfection, the medium was replaced and the cells were infected with MV at an MOI of 5 per cell (lanes 4 and 8). After allowance of 2 h for infection, the indicated cell cultures (lanes 5 to 8) were heat shocked at 43 to 44°C for 3 h. Cells were harvested at 48 h after the start of transfection, and CAT assays were performed as described in Materials and Methods.

FIG. 3
FIG. 3

Minireplicon RNA transfection. RNA prepared in vitro was transfected by the calcium phosphate procedure (Materials and Methods). After the precipitate was added to the cells, MV (MOI of 5; lanes 3 and 6) was added to the culture medium to initiate the infection immediately to lessen the chance of degradation of intracellular RNA before it could be packaged into nucleocapsids. After a 5- to 6-h transfection-infection incubation, the media was replaced and the indicated cell cultures were heat shocked 2 h at 43 to 44°C. Cell extracts were prepared 24 to 28 h after the start of transfection-infection for CAT assays.

FIG. 4
FIG. 4

Stimulation of minireplicon gene expression by Hsp72. The hsp72 cDNA (17, 22, 29) was cloned into a CMV expression vector. The amino terminus coding region was fused to the influenza virus (flu) HA epitope tag (49). Whole-cell extracts prepared from transfected cells were analyzed by Western blotting using an antibody specific for the epitope tag (A). CAT assay results from cotransfection of 293-3-46 cells with the Hsp72 expression vector, minireplicon DNA, and L expression plasmid are also shown (B). Transfections were performed as described for Fig. 2.

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