Specificity analysis of the conserved hexanucleotides for the replication of bamboo mosaic potexvirus RNA

In order to investigate the possible function of the potexviral conserved hexanucleotide sequence (ACc/uUAA) found in the 3′ untranslated region of bamboo mosaic potexvirus (BaMV) genomic RNA, each nucleotide in the hexamer motif was substituted. Transcripts derived from wild-type and mutants with a loop deletion or a single-nucleotide substitution were inoculated into protoplasts. The accumulation levels of viral coat protein and RNAs detected from Western and Northern blots of each inoculation were examined after a 48-h incubation. Our data revealed that the nucleotides at positions 4–6 of the hexamer motif cannot be replaced by other nucleotides; the first position of this hexamer is purine specific, and the second position is restricted to pyrimidine. Substitution at the third position has less effect on viral accumulation in protoplasts. In addition to the results reported previously that the E. coli over-expressed BaMV RNA-dependent RNA polymerase could specifically interact with the hexamer motif, the hexanucleotides were thought to serve as a recognition site of viral replicase and the specificity may be derived from the functional groups of each nucleotide of this hexamer motif.