Structural features of an Xrn1-resistant plant virus RNA

Xrn1 is a major 5ʹ-3ʹ exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3ʹ untranslated region of several beny- and cucumovirus RNAs harbors a so-called ‘coremin’ motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence to deduce putative structural features responsible for Xrn1 stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5ʹ proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3ʹ proximal hairpin loop sequence is variable. The sequence of the 10-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae, Potyviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn1-stalling motifs are more widespread among RNA viruses than previously realized.