Recombinant VSV lacking sites for UTP incorporation permit stalling of transcription.
(A) Schematic of the VSV genome containing an (A-) leader region and a 60-nt long gene inserted between the leader region and the N gene. The complete sequence of the 60-nt gene is shown, and positions at which G residues were engineered to replace the original A residues in the gene-start sequence are underlined. The sites (11, 21, 31, 41 and 51) at which 3 sequential adenylates were inserted within the 60-nt gene are indicated together with the anticipated sequence of the corresponding stalled transcript. (B) The plaque morphology is shown at 48 h postinoculation for rVSV(A-)-10, -20, -30, -40 and -50 and at 24 hpi for rVSV. (C) A sequence trace of the genomic RNA derived from the 60-nt gene of rVSV(A-)-50 is shown to illustrate the three introduced sites of UTP incorporation. (D) The indicated recombinant VSV were used in in vitro transcription (IVT) reaction lacking UTP, but containing [α-32P]-GTP. The products of transcription were analyzed on a 6% polyacrylamide gel and visualized using a phosphorimager. Where indicated, the products of transcription were first digested with tobacco acid pyrophosphatase (TAP) which hydrolyzes the cap-structure, or with an exonuclease (Exo) that digests uncapped RNA. The identity of the purified viruses used for the IVT reactions are shown at the top of the gel. The uncapped leader RNA and the 30, 40 and 50-nt long transcripts are indicated. A representative gel of two independent experiments is shown.
