La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro

Abstract

Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

FIG. 1.

La knockdown diminishes translation and replication of poliovirus. Transfection of siRNA in HeLa cells (CLL2) was performed in 24-well plates. Twenty-four hours after transfection, cells were infected with poliovirus (A) or adenovirus (B) or mock infected, and protein synthesis was examined by pulse-labeling at various times as described in Materials and Methods. Cells were transfected with a nonspecific siRNA (control siRNA [Ctlr-siRNA]), La85-siRNA, or siRNA against nucleolin (Nuc-siRNA). (C) Western blot analysis of La protein. (D) Poliovirus yield is affected by La knockdown. A plaque assay was performed on a confluent monolayer of HeLa cells using serial dilutions of samples obtained from the lysates shown in panel A (5 h postinfection.).

FIG. 2.

IRES-driven translation of poliovirus RNA is inhibited by La85-siRNA. A schematic diagram of the bicistronic construct pcDNA3-RLuc-PolioIRES-FLuc is shown at the top of the figure (CMV, cytomegalovirus). The bicistronic construct pcDNA3-RLuc-PolioIRES-FLuc was cotransfected into HeLa cells with either La85-siRNA or control siRNA (Ctlr-siRNA). Twenty hours after transfection, cells were analyzed for FLuc and RLuc activities. The ratios of RLuc/FLuc activity are presented by the bars in the histogram. Absolute levels of RLuc and FLuc activities (in relative light units) are presented below the histogram.

FIG. 3.

Effects of LaDN on translation of poliovirus RNA and infectious virus synthesis in HeLa S10 cell extracts. (A) Effects of LaDN on poliovirus RNA translation in vitro. Translation reactions were performed in a total volume of 30 μl and contained HeLa S10 cell extract, poliovirus RNA (0.45 μg), [³⁵S]methionine, and other components as described in Materials and Methods and previously (67). La(226-348) was added at the following final concentrations: 1.25 μM (lane 2), 2.5 μM (lane 3), 5 μM (lane 4), and 10 μM (lane 5). In lanes 1 and 6, reaction mixtures were supplemented with control buffer and GST (10 μM), respectively. Reaction mixtures were incubated at 34°C for 4 h, and the reactions were stopped by the addition of Laemmli sample buffer. Proteins were separated by SDS-15% PAGE, blotted onto a nitrocellulose membrane, and analyzed by autoradiography. The positions of the major virus proteins are indicated to the right of the gel. (B) The membrane from panel A was probed with antibodies against GST. The positions of GST-La(226-348) and GST are indicated at the sides of the gel. (C) TCA-insoluble radioactivity assay of 1-μl aliquots of the reaction mixtures from panel A. (D) Plaque assay for poliovirus infectivity. Reaction mixtures lacking [³⁵S]methionine were programmed with poliovirus RNA for 16 h as described above for panel A. Virus titers were determined as described in Materials and Methods.

FIG. 4.

LaDN reduces poliovirus replication in vivo. (A) The kinetics of synthesis of poliovirus proteins was determined by pulse-labeling as described in Materials and Methods. Twenty-four hours after transfection with pcDNA3-myc-La226-348 (mutant) (LaDN) and pcDNA3-myc (control), cells were infected with poliovirus or mock infected and labeled with [³⁵S]methionine. The positions of the major virus proteins are indicated to the right of the gel. (B) Kinetics of poliovirus RNA synthesis. At various times postinfection, control and LaDN-transfected cells were treated with 5 μg of actinomycin D per ml for 1 h and then labeled with [³H]uridine for 1 h. Noninfected cells (n.i) treated with actinomycin D are also shown. TCA-precipitated radioactivity was determined as described in Materials and Methods. (C) Plaque assays of lysates derived from LaDN- and control poliovirus-infected cells were performed as described in Materials and Methods.

FIG. 5.

LaDN inhibits the initiation of formation of 80S ribosome complexes. RNA-ribosome binding assay was performed in a cycloheximide (0.6 mM)-treated HeLa cell extract (40 μl) with poliovirus (∼10⁵ cpm) (A), HCV IRES-poliovirus chimera (∼10⁶ cpm) (B), and rabbit globin (∼10⁵ cpm) (C) 3′-end-labeled mRNAs. The extract was incubated with GST-La226-348 (4 μg) or GST (4 μg) at 34°C for 15 min and diluted with ice-cold high-salt buffer (66). Ribosome complexes were analyzed as described in Materials and Methods.

FIG. 6.

LaDN inhibits the initiation of formation of 48S ribosome complexes. HCV IRES-labeled mRNA (∼10⁶ cpm) was incubated for 10 min with a HeLa S10 cell extract and 4 μg of GST-La(226-348). GMP-PNP was included in the reaction mixtures where indicated. Samples were analyzed by sucrose gradient centrifugation (see Materials and Methods). Radioactivity was determined by scintillation counting.