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. 2011 Sep;85(17):8884-93.
doi: 10.1128/JVI.00792-11. Epub 2011 Jun 22.

Poliovirus switches to an eIF2-independent mode of translation during infection

James P White  1 , Lucas C Reineke, Richard E Lloyd
Affiliations

Poliovirus switches to an eIF2-independent mode of translation during infection

James P White et al. J Virol. 2011 Sep.

Abstract

Inhibition of translation is an integral component of the innate antiviral response and is largely accomplished via interferon-activated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). To successfully infect a host, a virus must overcome this blockage by either controlling eIF2α phosphorylation or by utilizing a noncanonical mode of translation initiation. Here we show that enterovirus RNA is sensitive to translation inhibition resulting from eIF2α phosphorylation, but it becomes resistant as infection progresses. Further, we show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral internal ribosome entry site, plays a role in mediating viral translation under conditions that are nonpermissive for host cell translation. Together, these results provide a mechanism by which enteroviruses evade the antiviral response and provide insight into a noncanonical mechanism of translation initiation.

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Figures

Fig. 1.
Fig. 1.
Poliovirus (PV) translation becomes resistant to sodium arsenite (Ars) stress. (A and C) HeLa cells infected with PV were stressed with 0.5 mM Ars (+) or not stressed with Ars (−) for 30 min prior to the time points indicated (hpi, hours postinfection). Cells were then pulse-labeled for 30 min with Tran35S-label before analysis by SDS-PAGE and autoradiography. The numbers below the gel in lanes 3 to 6 indicate the percentage of translation in Ars-stressed lanes compared to unstressed conditions as determined by densitometry as a function of time. The migration positions of molecular mass standards (in kilodaltons) are shown to the left of the gel, and the migration positions of PV proteins are shown to the right of the gel. (B) Western blot analysis of PV-infected cells to determine the cleavage of G3BP and phosphorylation of eIF2α using specific antibodies as indicated for each panel. Values indicate percentage of maximal eIF2α phosphorylation. G3BPcp, G3BP cleavage product.
Fig. 2.
Fig. 2.
Expression of cleavage-resistant G3BP does not cause resistance to Ars stress. (A) HeLa cells expressing hamster pREP-G3BP-GFP were infected, stressed, and pulse-labeled with Tran35S-label and analyzed as described in the legend to Fig. 1A. The migration positions of molecular mass standards (in kilodaltons) are indicated to the left of the gel. (B) Immunoblot analysis of lysates for total and phosphorylated eIF2α. (C) Immunoblot analysis for G3BP showing the cleavage of endogenous G3BP but not G3BP-GFP. (D) HeLa S10 lysates were treated with a constant concentration of Ars (1 mM) plus various concentrations (1 μg to 10 ng) of recombinant viral 2Apro or 3Cpro to determine the effects of Ars treatment on proteinase activity. The height of the triangle above the lanes indicates the relative amount of 2Apro or 3Cpro. 2A and 3C proteinase activities were monitored by the cleavage of eIF4GI and G3BP, respectively. (E) 293T cells transfected with control DNA or plasmids expressing His-G3BP or His-G3BPQ326E were infected with PV, translating proteins were pulse-labeled at 4 or 6 hpi and analyzed by SDS-PAGE/autoradiography and densitometry of the indicated viral polypeptide bands. Data are expressed as percent incorporation relative to untransfected control cells (% Cont).
Fig. 3.
Fig. 3.
Enterovirus translation is resistant to stressors. (A to C and E) SDS-PAGE/autoradiographs of cell proteins labeled with Tran35S-label. (A) PV-infected HeLa cells were infected with PV and stressed with heat shock 30 min prior to labeling at the indicated time points. (B) PV-infected 293T cells were treated with 20 μM Sal003 or dimethyl sulfoxide (DMSO) vehicle for 90 min prior to 30 min pulse-labeling. (C) HeLa cells infected with CVB3 were stressed as described in the legend to Fig. 1. (D) Immunoblots showing phospho-eIF2α in the panels. (E) PV-infected cells pretreated with alpha interferon (IFNα) (1,000 U) for 16 h. Control infection without IFN at 4 h is shown.
Fig. 4.
Fig. 4.
Enteroviral IRES reporter RNAs are inhibited by eIF2α phosphorylation. (A) HeLa TetON cells were transfected with luciferase (pTRE2-FL) reporters containing PV or coxsackievirus B3 (CVB3) IRES elements upstream of the firefly luciferase (FL) open reading frame (ORF). Expression of IRES-FL reporters and stress was coinduced for 3 h with constant exposure to tetracycline and Ars, respectively. Firefly luciferase (FL) activity is shown in relative light units (RLU). (B) In vitro-transcribed capped FL or uncapped IRES-FL reporter RNAs were electroporated into HeLa S3 cells prior to treatment with Ars for 90 min and analysis of FL expression. (C) Rabbit reticulocyte lysates supplemented with HeLa ribosomal salt wash initiation factors were pretreated with water or poly(I · C) for 30 min before being programmed with FL reporter RNAs. FL expression was measured after 45 min of translation at 34°C. (D) In vitro-transcribed capped FL or PV-FL reporter RNAs were electroporated into cells, which were then treated with 10 μm Sal003 for 90 min prior to analysis of FL expression. Results were compiled from three independent experiments, and error bars represent standard errors. (E) Immunoblots showing eIF2α or phospho-eIF2α (P-eIF2α) in the experiments above.
Fig. 5.
Fig. 5.
Expression of eIF5B modulates PV translation under stress. (A) (Top) Schematic depicting the 3Cpro cleavage site on eIF5B. (Bottom) Gel showing kinetics of cleavage of endogenous eIF5B during PV infection in the presence or absence of Ars. frag., fragment. (B) 293T cells expressing wild-type eIF5B or cleavage-resistant eIF5BQ478E were infected and stressed with 0.5 mM Ars before pulse-labeling translating proteins and analysis by SDS-PAGE and autoradiography. (C) Stimulation of PV translation by eIF5B expression was quantified by densitometry of autoradiographs. Results are an aggregate of three independent experiments, and error bars indicate standard errors. (D) Immunoblot analysis of eIF2α and phosphorylated eIF2α. (E) HA-specific immunoblot indicating expression of HA-eIF5B transgenes.
Fig. 6.
Fig. 6.
Expression of eIF5B479-1220 stimulates PV translation. (A) 293T cells expressing control GFP or eIF5B479-1220 were infected with PV and stressed with 0.5 mM Ars for 30 min, and then translating proteins were pulse-labeled and analyzed by SDS-PAGE/autoradiography. (B) Densitometric analysis of three independent experiments showing stimulation of translation of PV RNA in the presence of eIF5B479-1220 with and without Ars treatment relative to controls (Cont) expressing GFP. Error bars indicate the standard errors. (C) Immunoblot analysis of cell lysates for HA-tagged eIF5B479-1220 and phospho-eIF2α (p-eIF2α) as indicated.
Fig. 7.
Fig. 7.
Expression of eIF5B479-1220 rescues PV IRES translation activity. (A) 293T cells expressing GFP or eIF5B479-1220 were electroporated with FL reporter RNAs and treated with 50, 100, and 200 μM Ars for 90 min, and then lysates were analyzed for FL activity. Data are an aggregate of three experiments, and error bars represent the standard errors. (B) Data are expressed as fold stimulation from eIF5B expression versus GFP expression. (C) Immunoblot analysis of eIF5B479-1220 expression and phospho-eIF2α in 293T cells electroporated with FL reporters. Blots used eIF5B-specific antibody and phospho-eIF2α antibody as indicated.
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