Fig. 4.

Z blocks an early step of RNA synthesis initiation. (A) Gel shift assay of complex formation between L, RNA, and Z. Radiolabeled 3′ RNA was incubated with buffer alone (−) or the indicated combinations of purified L (L), GST-tagged MACV Z (Z^M), and GST-tagged LCMV Z (Z^L). The resulting complexes were separated by nondenaturing gel electrophoresis. (B) In vitro RNA synthesis reactions in the absence of GpC primer were labeled with [α-³²P]-GTP or (C) 5′ end-labeled [α-³²P]-GpC and supplemented with untagged MACV Z (Z^M). (D) Analysis of MACV in vitro transcripts subsequently labeled after RNA synthesis. Transcripts from unlabeled RNA synthesis reactions in the absence of GpC primer were purified by phenol-chloroform extraction and ethanol precipitation and then subsequently labeled by capping the 5′ end with vaccinia virus guanyltransferase and [α-³²P]-GTP and separated as in B. (E) RNA products were analyzed and quantified as in Fig. 1 (*P < 0.001 or for [α-³²P]-GpC P = 0.0042). See also Fig. S5.