Secondary Structures in the Capsid Protein Coding Sequence and 3′ Nontranslated Region Involved in Amplification of the Tobacco Etch Virus Genome

The 3′-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3′ nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis -active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370–4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis -active region and the 3′ NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (β-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis -active sequence and at several distinct locations within the 3′ NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis -active sequence, a base-paired structure between two segments of the CP-coding cis -active sequence and a contiguous 14-nucleotide segment of the 3′ NTR, and a base-paired structure near the 3′ terminus of the 3′ NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3′ terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3′ NTR are coadapted for genome amplification function through a requirement for base pair interactions.