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, 33 (5), 1016-26

Ribosomal Protein S25 Dependency Reveals a Common Mechanism for Diverse Internal Ribosome Entry Sites and Ribosome Shunting

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Ribosomal Protein S25 Dependency Reveals a Common Mechanism for Diverse Internal Ribosome Entry Sites and Ribosome Shunting

Marla I Hertz et al. Mol Cell Biol.

Abstract

During viral infection or cellular stress, cap-dependent translation is shut down. Proteins that are synthesized under these conditions use alternative mechanisms to initiate translation. This study demonstrates that at least two alternative translation initiation routes, internal ribosome entry site (IRES) initiation and ribosome shunting, rely on ribosomal protein S25 (RPS25). This suggests that they share a mechanism for initiation that is not employed by cap-dependent translation, since cap-dependent translation is not affected by the loss of RPS25. Furthermore, we demonstrate that viruses that utilize an IRES or a ribosome shunt, such as hepatitis C virus, poliovirus, or adenovirus, have impaired amplification in cells depleted of RPS25. In contrast, viral amplification of a virus that relies solely on cap-dependent translation, herpes simplex virus, is not hindered. We present a model that explains how RPS25 can be a nexus for multiple alternative translation initiation pathways.

Figures

Fig 1
Fig 1
A stable knockdown of RPS25 in HeLa cells is viable, and the cells do not have significant defects in growth or global translation. (A) Light (left) and florescence (right) microscopy of HeLashV and HeLashS25 cells at a ×100 magnification. (B) Northern and quantitative Western analyses of RPS25 RNA and protein in HeLashV and HeLashS25 cells. (C) Proliferation assay for HeLashV (black) and HeLashS25 (gray) cells in 1% or 10% serum. Standard errors for 3 experiments are shown. (D) Relative cap-dependent translation levels in HeLashV (black) and HeLashS25 (gray) cells. Cells were transfected with plasmids encoding one of two reporter proteins, Renilla luciferase or β-galactosidase (β-gal), and the enzyme activity was measured after 48 h. (E) Protein synthesis rate in 10% serum determined by [35S]methionine incorporation into HeLashV and HeLashS25 cells. The counts per minute were measured for HeLashV and HeLashS25 cells and expressed relative to HeLashV cells at 100%. Standard errors for 4 experiments are shown.
Fig 2
Fig 2
HeLashS25 cells exhibit a specific defect in IRES-mediated translation, which can be complemented by exogenous expression of the shRNA-resistant RPS25, hS25. (A) Schematic representation of the dual-luciferase reporter and the hS25 rescue construct. Transcription of the bicistronic dual-luciferase reporters was from the simian virus 40 promoter (pSV40) or cytomegalovirus promoter (pCMV). Cap-dependent translation drives expression of the Renilla luciferase, while the firefly luciferase is under the control of the CrPV IGR IRES located in the intercistronic region. A diagram of the hS25 rescue plasmid indicates the synonymous mutations made to confer resistance to the siRNA. (B) CrPV IGR IRES activity in cells cotransfected with the dual-luciferase reporter and either the hS25 plasmid or empty pcDNA3 vector. IRES activity (firefly luciferase) was normalized to the cap-dependent translation of β-galactosidase and expressed as a percentage of the activity in HeLashV cells with the empty vector (black), HeLashS25 cells with the empty vector (dark gray), HeLashV cells with hS25 (light gray), and HeLashS25 cells with hS25 (white). Standard errors for 3 experiments are shown. (C) RPS25 Western analysis of cells 24, 48, and 72 h following transfection with the hS25 rescue plasmid.
Fig 3
Fig 3
Viral IRESs that are structurally and functionally different rely on RPS25. (A) Normalized activity of several viral IRESs in HeLashS25 cells expressed as a percentage of the activity for each IRES in the control cells. Raw luciferase and β-galactosidase values are presented in Table S2 in the supplemental material. (B) Representative poliovirus plaque assay images and quantification of titers after one round of infection in HeLashV or HeLashS25 cells. (C) Northern analysis of RPS25 mRNA levels in Huh7.13 cells 72 h after siRNA knockdown. Quantifications of RPS25 levels are indicated. (D) Replication efficiency of HCV (JHF1 strain) in control and RPS25 knockdown Huh7.13 cells was assessed by a quantitative Western analysis for the HCV NS5A protein normalized to β-actin at 72 h postinfection. (E) Representative herpes simplex virus 1 plaque assay images and quantification of virus titers following one round of infection in HeLashV or HeLashS25 cells. Standard errors for 3 experiments are shown.
Fig 4
Fig 4
RPS25 aids in the translation of cellular IRESs. (A) Cellular IRES activity was measured 48 h after cotransfection with the bicistronic IRES reporter and the monocistronic β-galactosidase reporter (gray bars) or also with the hS25 rescue plasmid (white bars) and expressed as a percentage of the activity in HeLashV cells (wild-type [WT] cells) (dotted line) for each IRES. Raw luciferase and β-galactosidase values are presented in Table S3 in the supplemental material. (B) Polysome analysis of the HeLashV and HeLashS25 cells. P/M, polysome-to-monosome ratio. (C) RNA isolated from the HeLashV and HeLashS25 polysome fractions was separated on a denaturing agarose gel. 18S and 28S rRNAs are indicated on the ethidium bromide-stained gel. The RNA was probed by Northern analysis for p53 and β-actin mRNAs.
Fig 5
Fig 5
RPS25 is used for ribosome shunting during adenovirus infection. (A) Diagram of the Ad-hp-Luc adenovirus shunting reporter (35). A stable stem-loop at the 3′ end of the tripartite shunting sequence inhibits normal scanning of the 40S ribosome, allowing only shunting to proceed, as indicated by the arrow. (B) The relative shunting rate was determined in HeLashV (black bars) and HeLashS25 (gray bars) cells cotransfected with the Ad-hp-Luc shunting reporter and β-galactosidase reporter as a control for cap-dependent translation. After one round of Ad5 infection, ribosome shunting activity (firefly luciferase) was normalized to β-galactosidase activity and expressed as a percentage of the shunting activity in the mock-infected HeLashV cells. (C) Representative plaque assay images and titers following one round of infection with Ad5 in HeLa cells. Three independent replicates of each assay were performed, and error bars indicate standard errors for three experiments.
Fig 6
Fig 6
Model for how RPS25 plays a common role in initiation by IRESs and ribosome shunts. (A) Cap-dependent translation does not require RPS25 for any of the steps in initiation. (B) HCV or picornaviral IRESs bind to the 40S subunit independently of RPS25. Next, RPS25 likely functions in a downstream step following 40S subunit recruitment, such as loading of the mRNA into the mRNA binding channel of the 40S subunit, start codon recognition, or 60S subunit joining. (C) The CrPV IGR IRES depends on RPS25 for binding to the 40S subunit (17). (D) In ribosomal shunting, the 40S subunit is recruited in a cap-dependent manner. RPS25 may be involved in transferring the ribosome from the donor to the acceptor site on the mRNA during ribosome shunting or in the events following shunting. Tan, 40S ribosome subunit; green, RPS25; black lines, IRES; black rectangles, coding region; blue, cap binding complex (eIF4F [composed of eIF4E, eIF4G, and eIF4A]); purple, eIF3; brown, ternary complex; red, eIF1A; yellow, eIF1.

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