RNA Recombination and Function of the Motif1-hairpin of satC

A major project in the lab concerns the mechanism of RNA recombination. The poor processivity of the TCV RdRp leads to the frequent generation of recombinants between the subviral RNAs, and between the subviral RNAs and the TCV genomic RNA. We have focused our studies on the 28 base motif1-hairpin (M1H) of satC, which in its minus-sense orientation is both a recombination hotspot and an enhancer of plus-strand synthesis.   The M1H contains three short motifs identical to sequence in TCV promoter-like elements and enhances transcription from one promoter by 10-fold in vitro.  Recent results using an in vivo functional SELEX (systematic evolution of ligands by exponential enrichment) assay, where 10 or 28 random bases replaced 28 bases of the M1H, resulted in the identification of winners that replicated to higher levels in protoplasts compared with satC containing non-selected sequences. As with the wild-type  M1H, most winning MIH replacement sequences contained one to three short motifs in their minus-sense orientation that were identical, or nearly identical to sequence from TCV or satC replication elements.  We have also shown  that, surprisingly, both 28-base and 10-base M1H replacement sequences fold into hairpins predicted to be more stable on plus-strands than minus-strands, and the presence of these hairpins correlate with reduced levels of TCV virions.  This suggests that ability to reduce virion levels contributed to the fitness of the satRNA.  Several of the most fit satRNAs contained inserts of adenylates and cytidylates at the base of the hairpins whose presence correlated with enhanced replication and reduction of virion levels.  These results shown that a sequence non-specific plus-strand hairpin flanked by CA-rich sequence in the M1H region confers fitness to satC by reducing virion assembly.   By reducing virion assembly, the presence of satC leads to enhanced levels of free coat protein, which is the virus's repressor of RNA silencing, an anti-viral defense mechanism in plants and other organisms.  

 Publications:

Zhang, G. and Simon, A. E. (2003)  A multifunctional turnip crinkle virus replication enhancer revealed by in vivo functional selex.  J. Mol. Biol. 326, 35-48.

Nagy, P. D., Pogany, J., and Simon, A. E.  (2001)  In vivo and in vitro characterization of an RNA replication enhancer in a satellite RNA associated with Turnip crinkle virus:  comparison of sequences and structures stimulating primer-dependent and primer-independent RNA synthesis.  Virology 288, 315-324.

Nagy, P. D., Pogany, J., and Simon, A. E. (1999) RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus. EMBO J. 18, 5653-5665.

Simon, A. E. Replication, Recombination, and Symptom-Modulation Properties of the Satellite RNAs of Turnip Crinkle Virus (Book) Satellites and Defective Viral RNAs, Vogt, Jackson

Nagy, P. D., and Simon, A. E. (1998) In vitro characterization of late steps of RNA recombination in turnip crinkle virus II: role of the priming stem and flanking sequences. Virology 249, 393-405.

Nagy, P. D., and Simon, A. E. (1998) In vitro characterization of late steps of RNA recombination in turnip crinkle virus I: role of the motif1-hairpin structure. Virology 249, 379-392.

Nagy, P. D., Zhang, C., and Simon, A. E. (1998) Dissecting RNA recombination in vitro: role of RNA sequences and the viral replicase. EMBO J. 17, 2392-2403.

Simon, A. E. and Nagy, P. D (1997) New insights into the mechanisms of RNA recombination. Virology 235, 1-9.

Simon, A. E. and Nagy, P. D (1997) Recombination in the turnip crinkle virus system. Sem. Virol. 7, 373-379 (Review)

Carpenter, C. D. and Simon, A. E. (1996) Changes in locations of crossover sites over time in de novo generated RNA recombinants. Virology 223, 165-173.

Carpenter, C.D., Oh, J.-W., Zhang, C., and Simon, A. E. (1995) Involvement of a stem-loop structure in the location of junction sites in viral RNA recombination. J. Mol. Biol. 245, 608-622.

Simon, A.E. and Bujarski, J.J. (1994) RNA-RNA recombination and evolution in infected plants. Annu. Rev. Phytopath. 32, 337-362.

Cascone, P.J., Haydar, T. and Simon, A.E. (1993) Sequences and structures required for RNA recombination between virus-associated RNAs Science 260, 801-805.

Zhang, C., Cascone, P.J. and Simon, A.E. (1991) Recombination between satellite and genomic RNAs of turnip crinkle virus. Virology 184, 791-794.

Cascone, P.J., Carpenter, D.C., Li, X.H. and Simon, A.E. (1990) Recombination between satellite RNAs of turnip crinkle virus. EMBO J. 9, 1709-1715.