Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome with a 5'-cap structure and a 3' poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3' untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3' UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.