Cis-acting Elements Important for Potato Virus X Minus-strand RNA Synthesis In Vitro and In Vivo

Abstract

In order to identify cis-acting elements required for Potato virus X (PVX) minus-strand RNA synthesis, replication in in vitro and in vivo systems was compared. Specifically, RNA dependent RNA polymerase activity using a 850 nt template in PVX infected tobacco plant extract and using infectious full-length transcripts in tobacco protoplasts was analyzed. To facilitate quantitation of results from the in vitro system, the RdRp assay was optimized in several ways. Initial experiments showed that processing of extracts from fresh plant tissue was optimal for the in vitro RdRp assay. Purification of nuclease Bal31 with stable RNase activity was critical for consistency in making and assaying template-dependent plant extracts. Optimal salt concentrations, reaction volume, incubation time, and template concentration conditions were found to ensure template specificity and quantifiable product levels for PVX RdRp. Comparison of data obtained with this optimal extract and the protoplast system showed that the conserved hexanucleotide element and conformation of stem-loop 3 are required for minus-strand RNA synthesis both in vitro and in vivo. More strikingly, we found that long-distance RNA-RNA interactions between conserved internal elements and the hexanucleotide element are required for optimal minus-strand RNA synthesis both in vitro and in vivo. In addition, multiple internal elements can serve as interaction partners. Thus, similar to plus-strand RNA synthesis, PVX minus-strand RNA synthesis requires local elements and long-distance RNA-RNA interactions. A model for RNA synthesis is proposed in which both termini of the genome are paired with internal elements.