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. 2005 May;79(9):5676-83.
doi: 10.1128/JVI.79.9.5676-5683.2005.

Regulation of translation by ribosome shunting through phosphotyrosine-dependent coupling of adenovirus protein 100k to viral mRNAs

Qiaoran Xi  1 , Rafael Cuesta, Robert J Schneider
Affiliations

Regulation of translation by ribosome shunting through phosphotyrosine-dependent coupling of adenovirus protein 100k to viral mRNAs

Qiaoran Xi et al. J Virol. 2005 May.

Abstract

Adenovirus simultaneously inhibits cap-dependent host cell mRNA translation while promoting the translation of its late viral mRNAs during infection. Studies previously demonstrated that tyrosine kinase activity plays a central role in the control of late adenovirus protein synthesis. The tyrosine kinase inhibitor genistein decreases late viral mRNA translation and prevents viral inhibition of cellular protein synthesis. Adenovirus protein 100k blocks cellular mRNA translation by disrupting the cap-initiation complex and promotes viral mRNA translation through an alternate mechanism known as ribosome shunting. 100k protein interaction with initiation factor eIF4G and the viral 5' noncoding region on viral late mRNAs, known as the tripartite leader, are both essential for ribosome shunting. We show that adenovirus protein 100k promotes ribosome shunting in a tyrosine phosphorylation-dependent manner. The primary sites of phosphorylated tyrosine on protein 100k were mapped and mutated, and two key sites are shown to be essential for protein 100k to promote ribosome shunting. Mutation of the two tyrosine phosphorylation sites in 100k protein does not impair interaction with initiation factor 4G, but it severely reduces association of 100k with tripartite leader mRNAs. 100k protein therefore promotes ribosome shunting and selective translation of viral mRNAs by binding specifically to the adenovirus tripartite leader in a phosphotyrosine-dependent manner.

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Figures

FIG. 1.
FIG. 1.
Genistein impairs promotion of ribosome shunting by Ad 100k protein. (A) Diagrammatic representation of the B202 form of the tripartite leader is shown. Insertion of a stable (Δ-70 kcal/mol) hairpin between the 3′ end of the tripartite leader and the reporter AUG initiation codon blocks initiation by 5′ to 3′ ribosome scanning and only allows initiation by ribosome shunting. (B and C) 293 cells were cotransfected with vector alone (pFLAG-CM2) or vectors expressing Flag-tagged wild-type 100k protein (pTLFLAG100), a luciferase reporter encoded by an mRNA containing the Ad B202 tripartite leader 5′ NCR mutant, and the GFP protein controlled by the EMCV IRES (pIRES-EGFP). Transfected cells were treated with genistein (300 μM) or PP2 (30 μM) posttransfection or were mock treated. At 36 h posttransfection, cells were collected and lysates and total RNAs were prepared. (B) Wild-type 100k was recovered from equal amounts of lysates by immunoprecipitation with anti-Flag antibody. 100k protein complexes were analyzed by immunoblotting using anti-FLAG and anti-phosphotyrosine (P-Tyr) antibodies. Immunoprecipitations were performed with anti-C-terminal eIF4G antibodies (c-eIF4G antibody). eIF4G immune complexes were analyzed by immunoblotting using anti-c-eIF4G and anti-Flag antibodies. V, vector alone control. (C) Luciferase activity was measured after normalizing to similar reporter mRNA levels by Northern blotting and protein lysate amounts. Autoradiograms were quantified by densitometry, and typical results are shown. Standard deviations were calculated from at least three independent experiments.
FIG. 2.
FIG. 2.
100k protein phosphotyrosine sites are located between aa 345 and aa 726. (A) Schematic representation of Ad 100k protein functional domains. The N terminus is responsible for Ad 100k protein inhibition of cellular mRNA translation, the middle region contains a virus-specific RNA-binding domain, and the C-terminal domain contains a general RNA-binding domain. (B) 293 cells were transfected with vector alone (pFLAG-CM2) or vector pFlag-TL100k, pFlag-TL100kN726, or pFlag-TL100kN345. At 32 h posttransfection, cells were treated with or without 150 μM genistein for 4 h before lysis. Wild-type or mutant 100k protein was recovered from equal amounts of lysate by immunoprecipitation with anti-Flag antibody. 100k protein complexes were analyzed by immunoblot using anti-Flag and anti-phosphotyrosine (P-Tyr) antibodies. Autoradiograms were quantified by densitometry and typical results are shown.
FIG. 3.
FIG. 3.
Mutations of tyrosines at aa 365 and aa 682 in 100k protein disable its ability to promote ribosome shunting. (A) 293 cells were cotransfected with plasmid pIRES-EGFP, vector alone, or pFlag-TL100k, pFlag-TL100kY365F, or pFlag-TL100kY682F, and a plasmid expressing luciferase reporter mRNA containing the mutant tripartite leader 5′ NCR (B202) that translates solely by ribosome shunting. Wild-type or mutant 100k protein was recovered from equal amounts of lysate by immunoprecipitation with anti-Flag antibody. 100k protein complexes were analyzed by immunoblotting using anti-Flag and anti-phosphotyrosine (P-Tyr) antibodies. (B) 293 cells were cotransfected with pIRES-EGFP, vector alone, pFlag-TL100k, or pFlag-TL100kYYF, and a plasmid expressing tripartite leader B202 luciferase mRNA. Wild-type or mutant 100k protein was recovered from equal amounts of lysate and processed as described above. (C) Luciferase activity was measured after normalization to similar reporter mRNA levels by Northern blotting. Autoradiograms were quantified by densitometry, and typical results are shown. Standard deviations were calculated from at least three independent experiments.
FIG. 4.
FIG. 4.
100k protein tyrosine mutants associate with eIF4G equally well as wild-type 100k. 293 cells were transfected with vector alone, pFlag-TL100k, pFlag-TL100kY365F, pFlag-TL100kY682F, or pFlag-TL100kYYF. eIF4G was recovered from equal amounts of RNase A-treated lysates by immunoprecipitation with anti-C-eIF4G antibody. eIF4G complexes were analyzed by immunoblotting using anti-Flag and anti-c-eIF4G antibodies.
FIG. 5.
FIG. 5.
Genistein treatment abolishes the ability of 100kN726 protein to preferentially bind tripartite leader mRNA. 293 cells were cotransfected with vector alone, pFlag-TL100k, pFlag-TL100kN726, pFlag-TL100kN726YYF, and plasmids expressing β-gal mRNA containing either the wild-type tripartite leader (3LDR) or an eIF4F-dependent 5′ NCR (CR3). Cells were treated with or without 300 μM genistein for 4 h and then lysed. Wild-type or mutant 100k proteins were recovered from equal amounts of lysate by immunoprecipitation with anti-Flag antibody. (A) Immunoprecipitated protein was analyzed by immunoblot with anti-p-Y and anti-Flag antibodies. (B) mRNA was extracted from the immunoprecipitated protein and identified by reverse transcription followed by semiquantitative PCR (RT-PCR). (C) Total RNA was isolated from lysates, and 3LDR mRNAs and CR3 mRNAs were determined by Northern blot analysis. Autoradiograms were quantified by densitometry, and typical results are shown.
FIG. 6.
FIG. 6.
Mutant 100kYYF fails to associate with viral mRNA. 293 cells were cotransfected with vector alone or pFlag-TL100kN726, pFlag-TL100kYYF, and plasmids expressing β-gal mRNA containing either the wild-type tripartite leader (3LDR) or an eIF4F-dependent 5′ NCR (CR3). Cells were treated with or without 300 μM genistein for 4 h before lysis. Mutant 100k proteins were recovered from equal amounts of lysate by immunoprecipitation with anti-Flag antibody. (A) The immunoprecipitated protein was analyzed by immunoblot with anti-p-Y and anti-Flag antibodies. (B) mRNA was extracted from the immunoprecipitated protein and identified by reverse transcription followed by quantitative PCR (RT-PCR). (C) Total RNA was isolated from lysates. 3LDR and CR3 mRNAs amounts were determined by Northern blot analysis. Autoradiograms were quantified by densitometry, and typical results are shown.
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