Fig. 1. CA-terminated capped RNA fragments are preferentially used as primers to initiate influenza viral mRNA synthesis. (A) The ³²P-labeled capped RNA substrates containing the indicated initial 5′ terminal sequence were each incubated with a nuclear extract expressing the three P proteins in the absence of 5′ and 3′ vRNA (lanes 1 and 4), or in the presence of 5′ and 3′ vRNA in either the absence (lanes 2 and 5) or the presence (lanes 3 and 6) of the four NTPs. The RNA products were separated on a denaturing 20% polyacrylamide gel. Positions of the RNA markers 10–100 nucleotides long are denoted; elong denotes elongation product. (B) Left panel: the ³²P-labeled capped RNA substrate containing a CA site was incubated with the nuclear extract in the absence of 5′ and 3′ vRNA (lane 1), or in the presence of 5′ and 3′ vRNA in either the absence (lane 2) or presence (lane 3) of the four NTPs. Right panel: the unlabeled capped RNA substrate containing a CA site was incubated with the nuclear extract in the presence of both 5′ and 3′ vRNA in the presence of either [α-³²P]GTP alone (lane 4) or [α-³²P]GTP and unlabeled ATP, CTP and GTP (lane 5).