A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA.

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses. The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

[1]  J. H. Strauss,et al.  Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Ishikawa,et al.  In vitro mutagenesis of the putative replicase genes of tobacco mosaic virus. , 1986, Nucleic acids research.

[3]  A. Varshavsky,et al.  In vivo half-life of a protein is a function of its amino-terminal residue. , 1986, Science.

[4]  M. Kozak.  Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Ahlquist,et al.  High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cdna and effects of 5' extensions on transcript infectivity. , 1987, Virology.

[6]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[7]  M. Zuker.  On finding all suboptimal foldings of an RNA molecule. , 1989, Science.

[8]  P. Leroy,et al.  The protein encoded by a murine male germ cell-specific transcript is a putative ATP-dependent RNA helicase , 1989, Cell.

[9]  C. Rice,et al.  Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication , 1989, Journal of virology.

[10]  M. Kozak.  The scanning model for translation: an update , 1989, The Journal of cell biology.

[11]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[12]  K. Lehto,et al.  Regulation of Tobamovirus Gene Expression , 1990, Advances in Virus Research.

[13]  Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae. , 1991, Nucleic acids research.

[14]  T. Hall,et al.  The requirement for a 5' stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation , 1992, Journal of virology.

[15]  T. Hall,et al.  Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA. , 1992, Virology.

[16]  P. Ahlquist,et al.  Bromovirus RNA replication and transcription require compatibility between the polymerase- and helicase-like viral RNA synthesis proteins , 1993, Journal of virology.

[17]  P. Ahlquist,et al.  RNA-dependent replication, transcription, and persistence of brome mosaic virus RNA replicons in S. cerevisiae , 1993, Cell.

[18]  M. Hoekstra,et al.  Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination , 1994, Molecular and Cellular Biology.

[19]  C. Guthrie,et al.  Mutagenesis of the yeast gene PRP8 reveals domains governing the specificity and fidelity of 3' splice site selection. , 1996, Genetics.

[20]  J. de la Cruz,et al.  The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Chuang,et al.  Requirement of the DEAD-Box Protein Ded1p for Messenger RNA Translation , 1997, Science.

[22]  P. Ahlquist,et al.  Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  P. Ahlquist,et al.  cis-Acting Signals in Bromovirus RNA Replication and Gene Expression: Networking with Viral Proteins and Host Factors , 1997 .

[24]  C. Berset,et al.  A novel inhibitor of cap‐dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E , 1997, The EMBO journal.

[25]  P. Ahlquist,et al.  In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae , 1997, Journal of virology.

[26]  J. McCarthy,et al.  Posttranscriptional Control of Gene Expression in Yeast , 1998, Microbiology and Molecular Biology Reviews.

[27]  P. Ahlquist,et al.  Brome mosaic virus RNA replication protein 1a dramatically increases in vivo stability but not translation of viral genomic RNA3. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[28]  P. Ahlquist,et al.  RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[29]  P. Ahlquist,et al.  Putative RNA Capping Activities Encoded by Brome Mosaic Virus: Methylation and Covalent Binding of Guanylate by Replicase Protein 1a , 1999, Journal of Virology.

[30]  P. Ahlquist,et al.  Brome Mosaic Virus RNA Replication Proteins 1a and 2a Colocalize and 1a Independently Localizes on the Yeast Endoplasmic Reticulum , 1999, Journal of Virology.

[31]  C. Kao,et al.  The N-terminal half of the brome mosaic virus 1a protein has RNA capping-associated activities: specificity for GTP and S-adenosylmethionine. , 1999, Virology.

[32]  P. Ahlquist,et al.  A Brome Mosaic Virus Intergenic RNA3 Replication Signal Functions with Viral Replication Protein 1a To Dramatically Stabilize RNA In Vivo , 1999, Journal of Virology.

[33]  M. Schweizer,et al.  A High Affinity Binding Site for the Polypyrimidine Tract Binding Protein (PTB) Is Located in the 5'-Untranslated Region of the Rat Proteinase α1-Inhibitor 3 Variant I Gene , 1999, Biological chemistry.

[34]  M. Dreyfus,et al.  Ded1p, a DEAD-box Protein Required for Translation Initiation in Saccharomyces cerevisiae, Is an RNA Helicase* , 1999, The Journal of Biological Chemistry.

[35]  S. Lemon,et al.  Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo , 2000, Molecular and Cellular Biology.

[36]  P. Ahlquist,et al.  Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping , 2000, Journal of Virology.

[37]  P. Ahlquist,et al.  Identification and characterization of a host protein required for efficient template selection in viral RNA replication. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Jianbo Chen,et al.  Brome Mosaic Virus Polymerase-Like Protein 2a Is Directed to the Endoplasmic Reticulum by Helicase-Like Viral Protein 1a , 2000, Journal of Virology.

[39]  M. Ruiz-Echevarría,et al.  The RNA Binding Protein Pub1 Modulates the Stability of Transcripts Containing Upstream Open Reading Frames , 2000, Cell.