FIG 2.

Development of murine polymerase I-mediated HRTV minigenome system. (A) A diagram of the HRTV M segment minigenome plasmid (HRTV-MMG) and its replication, transcription, and translation mechanisms. A cDNA fragment containing the M 3′ UTR, Nluc ORF, and the M 5′ UTR in the genomic sense is inserted between the murine pol I promoter and the terminator of a pRF vector. The MMG plasmid is transcribed using RNA polymerase I to generate vRNA in BSRT7/5 cells. The vRNAs are then encapsidated with the N protein. The cRNAs are produced in the presence of the L protein. The encapsidated vRNA is transcribed into a reporter-gene mRNA. Finally, the transcribed mRNA is translated by the host to produce Nluc. (B) An HRTV minigenome system was established. BSRT7/5 cells were transfected with HRTV-MMG and pTM1-HRTV-N or pCAGGS, pTM1-HRTV-L or pCAGGS, and pT7-Fluc. Transfected cells were incubated for 2 days at 37°C. Nluc and Fluc activities were measured, and the RLUs of luciferase were determined. (C) BSRT7/5 cells were transfected with HRTV-MMG, and pTM1-HRTV-N or pKS-HRTV-N, pTM1-HRTV-L or pKS-HRTV-L, and pT7-Fluc. Control cells were transfected with HRTV-MMG, pCAGGS, and pT7-Fluc. Transfected cells were incubated for 2 days at 37°C. Nluc and Fluc activities were measured, and the RLUs of luciferase were determined. Error bars represent the SD. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P ≤ 0.05; ns, P > 0.05. Experiments shown in panels B and C were performed in quadruplicate and octuplicate, respectively.