FIG. 6.

(A) The 5′ end sequences of in vitro transcripts synthesized from pU5′Eco-5′rzm, pU5′Eco-5′rzm-8bp, and pU5′Eco. pU5′Eco have the T7 promoter followed by the 5′-end 391 nucleotides of the Aichi virus sequence. pU5′Eco-5′rzm and pU5′Eco-5′rzm-8bp were constructed by introducing the cis-active hammerhead ribozyme sequence between the T7 promoter and the virus sequence. In pU5′Eco-5′rzm and pU5′Eco-5′rzm-8bp, stem I segments of the ribozyme were 22 bp and 8 bp in length, respectively. The Aichi virus sequences and nonviral nucleotides are represented by uppercase letters and lowercase letters, respectively. (B) Cleavage by the hammerhead ribozyme. In vitro transcripts synthesized from pU5′Eco-5′rzm, pU5′Eco-5′rzm-8bp, and pU5′Eco were electrophoresed on a 3.5% polyacrylamide-7 M urea gel, and then the gel was stained with ethidium bromide. (C) Schematic diagrams of in vitro transcripts derived from full-length cDNA clones. The in vitro transcripts synthesized from plasmids containing the hammerhead ribozyme have a precise 5′ end, while those from plasmids without the hammerhead ribozyme have three nonviral nucleotides, GGU, at the 5′ end. In pAV-FL-3Dmut, the GDD motif within the 3D RNA polymerase-coding sequence was changed to Gly-Ala-Ala to inactivate the RNA polymerase. (D) Protein synthesis in a cell-free reaction programmed with AV-FL-5′rzm RNA, AV-FL RNA, or AV-FL-3Dmut RNA. Reactions were performed in the presence of [³⁵S]methionine-cysteine for 4 h, and then proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In lane represented as Cont, viral proteins that were metabolically labeled with [³⁵S]methionine-cysteine in transfected cells were loaded. The positions of capsid proteins are indicated. (E) RNA products in a cell-freereaction programmed with AV-FL-5′rzm RNA, AV-FL RNA, or AV-FL-3Dmut RNA. RNA was labeled with [α-³²P]CTP at 3 to 5 h after the start of incubation, and then total RNA was extracted. The extracted RNA was analyzed by nondenaturing agarose gel electrophoresis after being either treated with RNase A and RNase T₁ (lanes 4 to 6) or untreated (lanes 1 to 3). The positions of double-stranded RF and ssRNA are indicated. (F) RPA. Labeled viral RNA products of a cell-free reaction were subjected to RPA using an unlabeled probe that hybridizes with the Aichi virus positive-strand RNA or negative-strand RNA. To detect the negative-strand RNA, two-cycle RPA was carried out. As a positive control (Cont), in vitro-labeled full-length positive-strand or negative-strand RNA was used. Protected RNAs were analyzed by 3.5% polyacrylamide-7 M urea gel electrophoresis. (G) Effect of guanidine HCl on viral RNA replication in cell extracts. AV-FL-Luc-5′rzm RNA was used for a cell-free reaction with or without 2 mM guanidine HCl. Labeled RNA products were analyzed by nondenaturing agarose gel electrophoresis. The positions of RF and ssRNA are indicated.