Cis-acting Elements in the 3' Region of Potato virus X are Required for Host Protein Binding

Abstract

The 3' region of Potato virus X (PVX) has the 74 nt 3'-nontranslated region (NTR) that is conserved among all potexviruses and contains several cis-acting elements for minus-strand and plus-strand RNA accumulation. Three stem-loop structures (SL1-SL3), especially formation of SL3 and U-rich sequence of SL2, and near upstream elements in the 3' NTR were previously demonstrated as important cis-acting elements. To Investigate the binding of these cis-acting elements within 3' end with host protein, we used the electrophoretic mobility shift assays (EMSA) and UV-cross linking analysis. The EMSA with cellular extracts from tobacco and RNA transcripts corresponding to the 150 nt of the 3' end of PVX RNA showed that the 3' end of PVX formed complexes with cellular proteins. The specificity of protein binding was confirmed through competition assay by using with 50-fold excess of specific and non-specific probes. We also conducted EMSA with RNAs containing various mutants on those cis-acting elements (<TEX>${\Delta}10$</TEX>10, SL3B, SL2A and <TEX>${\Delta}21$</TEX>; J Mol Biol 326, 701-720) required for efficient PVX RNA accumulation. These analyses supported that these cis-acting elements are required for interaction with host protein(s). UV-cross linking analysis revealed that at least three major host proteins of about 28, 32, and 42 kDa in mass bound to these cis-elements. These results indicate that cis-acting elements from 3' end which are important for minus and plus-strand RNA accumulation are also required for host protein binding.