Long-distance RNA–RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5′ terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3′ NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3′ terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3′ NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3′ hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication

Restoration of a Stem-Loop Structure Required for Potato Virus X RNA Accumulation Indicates Selection for a Mismatch and a GNRA Tetraloop

AbstractThe 5′ region of potato virus X (PVX) RNA contains a stem-loop structure, stem-loop 1 (SL1), that is required for efficient plus-strand RNA accumulation. To determine how changes to individual elements in SL1 are accommodated by the virus, we inoculated PVX transcripts containing modifications in the terminal tetraloop (TL), stem C (SC), and stem D (SD) regions ontoNicotiana benthamianaplants and analyzed progeny RNAs over a series of passages. Several progeny RNAs isolated from plants inoculated with the TL mutants containing changes to the first nucleotide of the GAAA motif or deletion of the entire TL sequence were found to contain multiple A insertions within the terminal loop region. The wild-type TL motif, GAAA, was recovered for all TL mutants by the second passage, suggesting that the sequence and potential structure of this element are crucial for PVX infection. Revertant RNAs isolated from plants inoculated with mutants in SD and the central region of SC indicated that increased stem length is tolerated. Restoration of SD length to the 4 bp typical of the wild-type PVX RNA was accompanied by A insertion into loop C. Mutants with a conversion of the C55–C78 mismatch to a G–C pair, relocation of this mismatch within the central region of SC, or deletion of C55–C78 were unable to infect protoplasts and plants. In contrast, the mutant with a conversion of the C55–C78 mismatch to an A–C mismatch, which exhibited low levels of PVX plus-strand RNA in protoplasts, was able to infect plants and quickly reverted to the wild-type C–C mismatch. These data indicate that important sequence and secondary structural elements within SL1 are required for efficient viral infection and that multiple A insertions within the TL and loop C regions, potentially by polymerase stuttering, accompany restoration of SL1 structure

Cellular Protein Binds to Sequences near the 5′ Terminus of Potato virus X RNA That Are Important for Virus Replication

AbstractThe sequences in the 5′-nontranslated region (NTR) of Potato virus X (PVX) genomic RNA were previously reported to contain several regulatory elements that are required for genomic and subgenomic RNA accumulation. To investigate whether cellular proteins bind to these elements, we conducted electrophoretic mobility shift assays (EMSA) with protoplast protein extracts and RNA sequences from the PVX 5′-NTR. These analyses showed that the 5′ region of PVX positive-strand RNA formed complexes with cellular proteins. UV cross-linking studies of complexes formed with various deletions of the PVX RNA indicated that a 54-kDa cellular protein (p54) was bound to nt 1–46 at the 5′ terminus of PVX RNA. Site-directed mutations introduced within this 46-nt region further indicated that an ACCA sequence element located at nt 10–13 was important for optimal binding. In addition, mutations that decreased the affinity of the template RNA for the cellular factor decreased PVX plus-strand RNA accumulation in protoplasts. These studies suggest that the p54 may function in PVX RNA replication by binding to the 5′ terminus of the viral genomic RNA

Using Videoconferencing for Elementary/Secondary Teacher Professional Development

Working within a large geographic area, McDonald Observatory lacks the resources to send staff to present professional development workshops to teachers – and only limited numbers of teachers have the resources to attend workshops at our observatory. Our solution is the development of a new program to bring the workshop to teachers in their own communities through videoconferencing. Each workshop location has a co-facilitator who prepared for his/her duties through an orientation/training session held at McDonald Observatory. At the observatory, they experienced a variety of activities and selected the ones most suitable for the grade-levels of the teachers in their region; they recruit the teachers for the local workshop. Each videoconference session includes pre/post assessment of the participants, an interactive videoconference with an expert presenter, and assistance from the co-facilitator who manages the materials and assists with the activities. Through use of this technology, we expect to reach 500 teachers. An independent evaluator is preparing formative and summative evaluation for the project