A conformational switch at the 3′ end of a plant virus RNA regulates viral replication

3′ untranslated regions of alfamo‐ and ilar‐virus RNAs fold into a series of stem–loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection (‘genome activation’) and has been thought to substitute for a tRNA‐like structure that is found at the 3′ termini of related plant viruses. We propose the existence of an alternative conformation of the 3′ ends of alfamo‐ and ilar‐virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA‐like structure of the related bromo‐ and cucumo‐viruses. A low but specific interaction with yeast CCA‐adding enzyme was found. The existence of two mutually exclusive conformations for the 3′ termini of alfamo‐ and ilar‐virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.

[1]  T. Dreher,et al.  Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase. , 1998, RNA.

[2]  E. Westhof,et al.  Transfer RNA identity rules and conformation of the tyrosine tRNA-like domain of BMV RNA imply additional charging by histidine and valine. , 1998, Biochemical and biophysical research communications.

[3]  P. Ahlquist,et al.  Near identity of 3′ RNA secondary structure in bromoviruses and cucumber mosaic virus , 1981, Cell.

[4]  C. Kao,et al.  A minimal RNA promoter for minus-strand RNA synthesis by the brome mosaic virus polymerase complex. , 1999, Journal of molecular biology.

[5]  L. Coscoy,et al.  Localization and biochemical characterization of alfalfa mosaic virus replication complexes. , 1993, Virology.

[6]  J. Skuzeski,et al.  Characterization of chimeric turnip yellow mosaic virus genomes that are infectious in the absence of aminoacylation. , 1997, Virology.

[7]  O. Uhlenbeck,et al.  Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. , 1987, Nucleic acids research.

[8]  C. Reusken,et al.  Mutations in coat protein binding sites of alfalfa mosaic virus RNA 3 affect subgenomic RNA 4 accumulation and encapsidation of viral RNAs , 1997, Journal of virology.

[9]  E C Koper-Zwarthoff,et al.  Nucleotide sequence of the putative recognition site for coat protein in the RNAs of alfalfa mosaic virus and tobacco streak virus. , 1980, Nucleic acids research.

[10]  T. Dreher,et al.  Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation. , 1989, Journal of molecular biology.

[11]  J. Bol,et al.  Early and late functions of alfalfa mosaic virus coat protein can be mutated separately. , 1994, Virology.

[12]  E. Jaspars,et al.  Protein binding sites in nucleation complexes of alfalfa mosaic virus RNA 4. , 1982, Biochemistry.

[13]  C. Pleij,et al.  tRNA-like structures. Structure, function and evolutionary significance. , 1991, European journal of biochemistry.

[14]  C. Pleij,et al.  Minimal Template Requirements for Initiation of Minus-Strand Synthesis In Vitro by the RNA-Dependent RNA Polymerase of Turnip Yellow Mosaic Virus , 1998, Journal of Virology.

[15]  L. Gehrke,et al.  Specific RNA binding by amino‐terminal peptides of alfalfa mosaic virus coat protein. , 1994, The EMBO journal.

[16]  J. Bol.  Alfalfa mosaic virus and ilarviruses: involvement of coat protein in multiple steps of the replication cycle. , 1999, The Journal of general virology.

[17]  V. Dolja,et al.  Nucleotide sequence of the 3'-terminal tRNA-like structure in barley stripe mosaic virus genome. , 1984, Nucleic acids research.

[18]  T. Hall.  Transfer RNA-like structures in viral genomes. , 1979, International review of cytology.

[19]  C. Reusken,et al.  Structural elements of the 3'-terminal coat protein binding site in alfalfa mosaic virus RNAs. , 1996, Nucleic acids research.

[20]  A. Weiner,et al.  A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme. , 1998, RNA.

[21]  J. Skuzeski,et al.  The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability , 1996, Journal of virology.

[22]  E. Jaspars.  Genome activation in alfamo- and ilarviruses , 1999, Archives of Virology.

[23]  D. Draper,et al.  Strategies for RNA folding. , 1996, Trends in biochemical sciences.

[24]  E. Jaspars,et al.  Influence of a few coat protein subunits on the base-paired structure of the RNA species of alfalfa mosaic virus. , 1978, Biochimica et biophysica acta.

[25]  D. Draper,et al.  Folding of an mRNA pseudoknot required for stop codon readthrough: effects of mono- and divalent ions on stability. , 1997, Biochemistry.

[26]  T. Dreher,et al.  Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity. , 1988, Journal of molecular biology.

[27]  C. Pringle,et al.  The universal system of virus taxonomy of the International Committee on Virus Taxonomy (ICTV), including new proposals ratified since publication of the Sixth ICTV Report in 1995 , 2014, Archives of Virology.

[28]  C. Pleij,et al.  tRNA‐like structures , 1991 .

[29]  I. Tinoco,et al.  RNA pseudoknots. Stability and loop size requirements. , 1990, Journal of molecular biology.

[30]  P. Taschner,et al.  Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes. , 1991, Virology.

[31]  E. Jaspars,et al.  Alterations of the conformation of the RNAs of alfalfa mosaic virus upon binding of a few coat protein molecules. , 1982, Biochimica et biophysica acta.

[32]  T. Dreher,et al.  Transfer RNA mimicry in a new group of positive-strand RNA plant viruses, the furoviruses: differential aminoacylation between the RNA components of one genome. , 1998, Virology.

[33]  J P Abrahams,et al.  Five pseudoknots are present at the 204 nucleotides long 3' noncoding region of tobacco mosaic virus RNA. , 1985, Nucleic acids research.

[34]  A. Haenni,et al.  An enigma: the role of viral RNA aminoacylation. , 1997, Acta biochimica Polonica.

[35]  R. Quadt,et al.  Analysis of the protein composition of alfalfa mosaic virus RNA-dependent RNA polymerase. , 1991, Virology.

[36]  G. Martelli,et al.  The nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 and its relationships in the family Bromoviridae. , 1996, The Journal of general virology.

[37]  L. Ryabova,et al.  The 3′‐terminal untranslated region of alfalfa mosaic virus RNA 4 facilitates the RNA entry into translation in a cell‐free system , 1993, FEBS letters.

[38]  K. Asano,et al.  Copy number control of IncIα plasmid ColIb‐P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site , 1998, The EMBO journal.

[39]  C. W. Hilbers,et al.  NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA. , 1998, Science.

[40]  C. Pleij,et al.  Three‐dimensional models of the tRNA‐like 3′ termini of some plant viral RNAs. , 1983, The EMBO journal.

[41]  C. Pleij,et al.  Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability. , 1992, Journal of molecular biology.

[42]  A. C. Webb,et al.  Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA , 1997, Molecular and cellular biology.

[43]  T. Hall,et al.  Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation , 1993, Journal of virology.

[44]  H. Hinz,et al.  Energetics of the thermal transitions of RNA 1 and RNA 4 of alfalfa-mosaic virus in the presence and absence of coat protein. , 1979, European journal of biochemistry.

[45]  P. Walstra,et al.  Nucleotide sequence of the 3'-noncoding region of alfalfa mosaic virus RNA 4 and its homology with the genomic RNAs. , 1979, Nucleic acids research.

[46]  L. Gehrke,et al.  RNA determinants of a specific RNA-coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs. , 1998, Journal of molecular biology.

[47]  R. Giegé,et al.  Interplay of tRNA-like structures from plant viral RNAs with partners of the translation and replication machineries. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[48]  D. A. Roth,et al.  Computer-aided nucleic acid secondary structure modeling incorporating enzymatic digestion data , 1984, Nucleic Acids Res..

[49]  B. Berkhout,et al.  Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2. , 1987, Journal of molecular biology.

[50]  L. Gehrke,et al.  In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs , 1997, Journal of virology.