FIG. 5.

Negative-strand synthesis on P3 RNA in the presence of P2 or processed forms of the P2 proteins. (A) Negative-strand RNA synthesis was measured using PIRCs isolated from 150 μl of HeLa S10 translation-replication reaction mixtures containing each of the RNAs indicated at the top of each lane at a concentration of 25 nM (lane 1), 17 nM (lanes 2 to 4 and lane 6), and 13 nM (lane 5). The RNA replication reaction mixtures did not contain puromycin. ³²P-labeled RNA synthesized was analyzed by CH₃HgOH-agarose gel electrophoresis and autoradiography of the dried gel. (B) Negative-strand RNA synthesis was measured using PIRCs isolated from 150 μl of HeLa S10 translation-replication reaction mixtures that contained a 13 nM concentration of each of the P3, P2, 2AB, and 2BC RNAs as indicated. In lanes 3 and 5, 8 nM 2A RNA, 9 nM 2B RNA, and 7 nM 2C RNA were present in the reaction mixtures. Protein synthesis was monitored as described in Materials and Methods. ³²P-labeled RNA synthesized was analyzed by CH₃HgOH-agarose gel electrophoresis and autoradiography of the dried gel.