Stem-loop structure in the 5 ' region of potato virus X genome required for plus-strand RNA accumulation

Computer-generated thermodynamic predictions and solution structure probing indicated two stem-loop structures, stem-loop 1 (SL1; nt 32-106) and stem- loop 2 (SL2; nt 143-183), within the 5' 230 nt of potato virus X (PVX) RNA. Because the existence of SL1 was further supported by covariation analysis of several PVX strains, the functional significance of this structure was investigated by site-directed mutational analysis in a tobacco protoplast s ystem. In general, mutations that reduced genomic plus-strand RNA accumulat ion similarly affected coat protein accumulation, indicating that subgenomi c plus-strand RNA was also affected. In contrast, minus-strand RNA levels r emained relatively unchanged. Mutational analysis of the stem C (SC) region of SL1 indicated that pairing was more important than sequence, which was consistent with the covariation analysis. Alterations that increased length and stability of either SC or stem D (SD) were deleterious to plus-strand RNA accumulation. The formation of internal loop C between SC and SD, as we ll as specific nucleotides within this loop, were also required. Several mo difications were made to the terminal GAAA tetraloop, a motif known for enh anced RNA stability. Both GANA and GAAG motifs resulted in wild-type levels of RNA accumulation. However, a UUCG tetraloop was detrimental, indicating that the sequence of this element was important beyond just providing stab ilization of the structure. These data indicate that multiple features of S L1 are critical for accumulation of PVX plus-strand RNA. (C) 1998 Academic Press.