The 3' end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase.

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3' translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3' end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3' end, which causes structural changes that potentiate the shift between translation and replication.

[1]  J. Flanegan,et al.  5′ cloverleaf in poliovirus RNA is a cis‐acting replication element required for negative‐strand synthesis , 2001, The EMBO journal.

[2]  K. White,et al.  Structure and prevalence of replication silencer-3' terminus RNA interactions in Tombusviridae. , 2006, Virology.

[3]  M. Lai,et al.  Viral and Cellular Proteins Involved in Coronavirus Replication , 2005, Current topics in microbiology and immunology.

[4]  A. Gamarnik,et al.  A 5' RNA element promotes dengue virus RNA synthesis on a circular genome. , 2006, Genes & development.

[5]  T. Panavas,et al.  Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase , 2005, Journal of Virology.

[6]  W. Miller,et al.  Cap-independent translation of plant viral RNAs. , 2006, Virus research.

[7]  K. Lehto,et al.  Translation mechanisms involving long-distance base pairing interactions between the 5' and 3' non-translated regions and internal ribosomal entry are conserved for both genomic RNAs of Blackcurrant reversion nepovirus. , 2008, Virology.

[8]  T. Dreher,et al.  Control of Translation by the 5′- and 3′-Terminal Regions of the Dengue Virus Genome , 2005, Journal of Virology.

[9]  Z. Wang,et al.  The amazing diversity of cap-independent translation elements in the 3'-untranslated regions of plant viral RNAs. , 2007, Biochemical Society transactions.

[10]  C. Kao,et al.  cis- and trans-Acting Functions of Brome Mosaic Virus Protein 1a in Genomic RNA1 Replication , 2007, Journal of Virology.

[11]  F. Grosse,et al.  Members of the NF90/NFAR protein group are involved in the life cycle of a positive‐strand RNA virus , 2003, The EMBO journal.

[12]  T. Dreher.  FUNCTIONS OF THE 3'-UNTRANSLATED REGIONS OF POSITIVE STRAND RNA VIRAL GENOMES. , 1999, Annual review of phytopathology.

[13]  W. Miller,et al.  Long-distance RNA-RNA interactions in plant virus gene expression and replication. , 2006, Annual review of phytopathology.

[14]  J. Feigon,et al.  H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification , 2007, Proceedings of the National Academy of Sciences.

[15]  Bruce A. Shapiro,et al.  Structural Domains within the 3′ Untranslated Region of Turnip Crinkle Virus , 2008, Journal of Virology.

[16]  E. Dobrikova,et al.  Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3′ nontranslated region , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Wagner,et al.  Translation initiation: structures, mechanisms and evolution , 2004, Quarterly Reviews of Biophysics.

[18]  E. Harris,et al.  End-to-end communication in the modulation of translation by mammalian RNA viruses , 2005, Virus Research.

[19]  Masayuki Ishikawa,et al.  The Arabidopsis Cucumovirus Multiplication 1 and 2 Loci Encode Translation Initiation Factors 4E and 4G , 2004, Journal of Virology.

[20]  P. D. Nagy,et al.  Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants , 2002, Journal of Virology.

[21]  F. Sobrino,et al.  IRES-driven translation is stimulated separately by the FMDV 3'-NCR and poly(A) sequences. , 2002, Nucleic acids research.

[22]  R. Bartenschlager,et al.  The Hepatitis C Virus RNA 3′-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site , 2006, Journal of Virology.

[23]  A. Simon,et al.  Evolution of virus-derived sequences for high-level replication of a subviral RNA. , 2006, Virology.

[24]  F. Qu,et al.  Cap-Independent Translational Enhancement of Turnip Crinkle Virus Genomic and Subgenomic RNAs , 2000, Journal of Virology.

[25]  Haiying Yu,et al.  Complex signals in the genomic 3' nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA. , 2004, RNA.

[26]  C. Song,et al.  Requirement of a 3'-terminal stem-loop in in vitro transcription by an RNA-dependent RNA polymerase. , 1995, Journal of molecular biology.

[27]  J. Flanegan,et al.  Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis , 1999, Journal of Virology.

[28]  W. Merrick.  Cap-dependent and cap-independent translation in eukaryotic systems. , 2004, Gene.

[29]  Paul Ahlquist,et al.  Host Factors in Positive-Strand RNA Virus Genome Replication , 2003, Journal of Virology.

[30]  S. Brenner,et al.  RNA structural motifs: building blocks of a modular biomolecule , 2005, Quarterly Reviews of Biophysics.

[31]  R. Breaker,et al.  An mRNA structure that controls gene expression by binding FMN , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[32]  C. Rice,et al.  Cis-acting RNA elements at the 5' end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis. , 2001, RNA.

[33]  P. Sarnow,et al.  Internal ribosome entry sites in eukaryotic mRNA molecules. , 2001, Genes & development.

[34]  M. Gromeier,et al.  The hepatitis C virus 3′-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase , 2006, Nucleic acids research.

[35]  M. Kozak.  Initiation of translation in prokaryotes and eukaryotes. , 1999, Gene.

[36]  E. Westhof,et al.  Topology of three-way junctions in folded RNAs. , 2006, RNA.

[37]  A. Simon,et al.  Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon , 2004, Journal of Virology.

[38]  A. Simon,et al.  Satellite RNA-mediated resistance to turnip crinkle virus in Arabidopsis involves a reduction in virus movement. , 1997, The Plant cell.

[39]  Xiaoping Sun,et al.  A cis-replication element functions in both orientations to enhance replication of Turnip crinkle virus. , 2006, Virology.

[40]  W. Miller,et al.  Translational control in positive strand RNA plant viruses. , 2006, Virology.

[41]  P. D. Nagy,et al.  A replication silencer element in a plus‐strand RNA virus , 2003, The EMBO journal.

[42]  Bruce A Shapiro,et al.  The 3' proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits. , 2008, RNA.

[43]  P. D. Nagy,et al.  Yeast as a model host to dissect functions of viral and host factors in tombusvirus replication. , 2006, Virology.

[44]  Christian M T Spahn,et al.  Codon-Anticodon Interaction at the P Site Is a Prerequisite for tRNA Interaction with the Small Ribosomal Subunit* , 2002, The Journal of Biological Chemistry.

[45]  R. Andino,et al.  Switch from translation to RNA replication in a positive-stranded RNA virus. , 1998, Genes & development.

[46]  Thomas Preiss,et al.  Starting the protein synthesis machine: eukaryotic translation initiation. , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[47]  J. Dinman,et al.  Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis , 2005, Molecular and Cellular Biology.