Figure 4. In spite of strong enhancement of the MV DI-RNA production due to the insertion of the second copy of the N protein within the MV genome, the 5′ copy-back DI-RNA specifically interacts with the MV nucleocapsid. (A) Efficient production of the 1,212 nucleotide-long 5′ copy-back MV DI-RNA by rMV2/N-STrEP in four different cell lines. Total RNA (20 ng) was analyzed by RT-qPCR. Absolute quantification of DI-RNA was performed using serial dilutions of in vitro transcribed MV DI-RNA. Samples were analyzed in triplicates, with standard deviation represented on the figure, and two biological replicates were performed each time. (B) Production of the 1,212 nucleotide DI genome and the full-length genome by recombinant MV expressing the second copy of MV-N. 293T cells were infected with either rMV2/N-STrEP, or a low-passage Schwarz vaccine strain of MV, or a Schwarz vaccine strain grown for five consecutive passages at a MOI of 1. Total RNA was purified and 1 μg of each RNA sample was analyzed by RT-qPCR against either the 1,212 nucleotide-long DI-RNA or the full-length MV genome. Absolute quantification was performed using serial dilutions of the in vitro transcribed MV DI-RNA or the MV genome RNA fragment. Samples were analyzed in triplicates, with standard deviation represented on the figure, and two biological replicates were performed each time. (C) Affinity chromatography of 1-STrEP tagged viral proteins form infected cells is an efficient and specific approach to purify RNA partners of viral proteins. Results of RT-qPCR against the 1,212 nucleotide-long DI-RNA on 10 ng of RNA purified complexed with the MV-N protein were compared with data obtained for 10 ng of total RNA (before purification). Absolute quantification was performed as described in (A). As negative control one-step RT-qPCR against the β-actin mRNA was performed with absolute quantification using serial dilutions of in vitro transcribed RNA fragments encompassing the β-actin sequence. Samples were analyzed in triplicates and two biological replicates were performed.