The SL1 stem-loop structure at the 5'-end of potato virus X RNA is required for efficient binding to host proteins and for viral infectivity.
The 5'-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus replication.
Kwon
Sun-Jung
SJ
School of Agricultural Biotechnology and Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-921, Korea.
Kim
Kook-Hyung
KH
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017863:Potexvirus / Q000235:genetics / Q000502:physiology*; D011523:Protoplasts / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D052156:SELEX Aptamer Technique; D014026:Nicotiana / Q000821:virology
D020121:5' Untranslated Regions; D020038:RNA, Spliced Leader; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012313:RNA