Fig. 1.

Transfer of m⁷Gp from cap donor to the 5′ end of viral transcript. (A) Labeled 5-nt cap donor was incubated in a transcription reaction mixture with L-A virions (lane 1) or M1 virions with WT (lane 2) or mutant (Arg154, lane 3) Gag protein and the full-sized transcripts were separated in an agarose gel. Ethidium bromide staining and autoradiogram of the gel are shown. λ, lambda-HindIII markers. (B) L-A virions were incubated with 5-nt cap donor in a transcription mixture from which CTP and GTP were omitted. Transcription was done in the presence of GDP (lane 4) or in its absence (lane 3), and the products were analyzed on a 15% acrylamide gel. As mobility markers, GTP (lane 1), cap donor (lane 2), 16-mer (lane 5), and 17-mer primed with m⁷GpppG (lane 6) were run in parallel. The sequence of the cap donor and the position of ³²P (indicated by the asterisk) are shown (Left). Under the panel the 5′ end sequence of L-A transcript is shown. The first C appears at position 17. (C) The labeled product (marked by the arrowhead in lane 4 of B) was isolated from the gel, treated with the enzymes as indicated, and analyzed on polyethyleneimine-cellulose (PEI-cellulose) with 0.3 M (NH₄)₂SO₄. Cap donor was also processed in parallel as control. The mobility of nonlabeled nucleotides is indicated (Left). Deduced transfer reaction is shown under the panel along with TAP and S1 cleavage sites. (D) 7-methylated and nonmethylated cap donors were synthesized in the presence and in the absence of S-adenosyl methionine (SAM), respectively, and incubated with L-A virions as described in the legend to B. The product (17-mer) was separated in a 8-M urea/15% acrylamide gel.