RNAs transcribed from a full-length infectious cDNA clone of the bamboo mosaic potexvirus (strain O) genome, pBaMV-O, were infectious to Nicotiana benthamiana plants. Mutant genomes in which the poly(A) tail is absent or replaced by a 3' tRNA-like structure from turnip yellow mosaic virus RNA failed to amplify detectably in N. benthamiana protoplasts. No amplification was detected in protoplasts inoculated with transcripts containing 4, 7, or 10 adenylate residues at the 3' end, whereas transcript inocula with 15 adenylate residues resulted in coat protein accumulation to a level 26% of that resulting from inoculation with transcripts with 25 adenylate residues (designated as wild type). Coat protein accumulation levels of 69 and 98% relative to wild type were observed after inoculation of protoplasts with transcripts bearing poly(A) tails 18 and 22 nucleotides long, respectively. The presence of a putative 3' pseudoknot structure including at least 13 adenylate residues of the 3'-terminal poly(A) tail was supported by enzymatic and chemical structural analysis. The functional relevance of this putative pseudoknot was tested by mutations that affected basepairing within the pseudoknot. These results support the existence of functional 3' pseudoknot that includes part of the 3' poly(A) tail.