In order to probe the molecular mechanisms that account for the difference in infection outcomes between mammalian and insect cells, recently we have established a 'minireplicon' system in Aedes albopictus C6/36 cells (Fig. 4; A. Kohl and others, unpublished). C6/36 cells expressing bacteriophage T7 RNA polymerase (C6-IBT7/3 cells) were selected following transfection with a plasmid encoding the polymerase gene and a blasticidin resistance gene. Bunyamwera virus proteins could be expressed in these cells by transfecting plasmids containing the viral coding sequences under T7 promoter control and baculovirus translational enhancers (Scheper et al., 1997) to ensure efficient expression. The minireplicon plasmid contained a negative-sense Renilla luciferase gene flanked by Bunyamwera virus non-coding sequences under control of the T7 promoter. The 3'-end of the minireplicon transcript was generated by hepatitis delta virus ribozyme-mediated self cleavage.
C6-IBT7/3 cells were transfected with various combinations of L and N protein expressing plasmids and the minireplicon plasmid, were incubated at 28 °C for 24 h, and Renilla luciferase activity was measured. No Renilla luciferase activity was detected in cells transfected with only two of the three plasmids. However, in cells expressing both Bunyamwera virus N and L proteins together with the minireplicon, about 100-fold induction of Renilla luciferase activity was detected. This indicates that the minireplicon was transcribed (and by analogy with the mammalian cell system, probably replicated) by the expressed BUN proteins.
The non-coding regions at the 3'- and 5'-ends of bunyavirus genomes or antigenomes interact to form a panhandle in the ribonucleoprotein (RNP), and these non-coding sequences also contain promoter elements that regulate replication levels for each segment. Previous studies in mammalian cells, where minireplicon RNA levels were measured directly, indicated that promoter activity varied between the three segments in the order M>L>S (Barr et al, 2003). Measurement of Renilla luciferase activity in cell extracts does not allow discrimination between replication and transcription, but shows that a biologically active RNP is reconstituted and transcribed in the transfected cell. Therefore, we used this as a means to compare promoter activity in mosquito and mammalian cells. T7-expressing cells were transfected with plasmids encoding the L and N proteins, and minireplicons were derived from the L, M or S segments. In addition a minireplicon containing a U-to-G mutation in position 16 of the viral
S S-mut (-)Control Mosquito cells
S S-mut (-)Control Mosquito cells
S 20 cc
Fig. 5. Analysis of promoter strength in mammalian (BSR-T7/5) or mosquito (C6-IBT7/3) cells expressing T7 RNA polymerase. Cells were transfected with Bunyamwera virus L and N expression vectors and the appropriate minigenome plasmids with different segment promoters. (-) Control, negative control, showing background minigenome activity in the absence of functional L protein. Activities are indicated in light units or as fold induction of Renilla luciferase activity. Ren luc, Renilla luciferase.
genome end, which results in a strong increase in promoter activity in mammalian cells (Kohl et al., 2003a), was used. As shown in Fig. 5(a), Renilla luciferase activities from the different minireplicons in mammalian cells follow the replicative ability of the L, M and S promoters as deduced by RNA analysis (Barr et al., 2003), that is, Renilla luciferase activities were strongest in cells transfected with the M segment minireplicon, followed by that from the L and the S segment. As reported previously (Kohl et al., 2003a), the mutant S minireplicon gave about three times higher activity than the minireplicon containing the wild-type S sequence. When the same experiment was repeated in mosquito cells the strongest activity was mediated by the M-segment-derived minireplicon. The activities of the other minireplicons, including the mutantS-derived minireplicon, were similar to each other, giving on average four times less Renilla luciferase than the corresponding M sequences (Fig. 5b). Hence using a minireplicon system there appears to be a difference in promoter activities between mammalian and mosquito cells.
Previously it was shown that the NSs protein, whether it is expressed with N from an S-segment-like mRNA or from separate plasmids, strongly inhibits minireplicon activity in mammalian cells (Weber et al., 2001). Therefore, we investigated whether the same pertained in mosquito cells. As shown in Fig. 6, expression of both S segment proteins N and NSs compared to expression of N protein alone led to a reduction in Renilla luciferase activity in mammalian cells but not in mosquito cells. When expressed from a separate plasmid NSs also had no major effect on overall minireplicon activity in mosquito cells.
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