The double-stranded RNA binding protein 76:NF45 heterodimer inhibits translation initiation at the rhinovirus type 2 internal ribosome entry site

Abstract

Poliovirus (PV) plus-strand RNA genomes initiate translation in a cap-independent manner via an internal ribosome entry site (IRES) in their 5' untranslated region. Viral translation is codetermined by cellular IRES trans-acting factors, which can influence viral propagation in a cell-type-specific manner. Engineering of a poliovirus recombinant devoid of neuropathogenic properties but highly lytic in malignant glioma cells was accomplished by exchange of the cognate poliovirus IRES with its counterpart from human rhinovirus type 2 (HRV2), generating PV-RIPO. Neuroblast:glioma heterokaryon analyses revealed that loss of neurovirulence is due to trans-dominant repression of PV-RIPO propagation in neuronal cells. The double-stranded RNA binding protein 76 (DRBP76) was previously identified to bind to the HRV2 IRES in neuronal cells and to inhibit PV-RIPO translation and propagation (M. Merrill, E. Dobrikova, and M. Gromeier, J. Virol. 80:3347-3356, 2006). The results of size exclusion chromatography indicate that DRBP76 heterodimerizes with nuclear factor of activated T cells, 45 kDa (NF45), in neuronal but not in glioma cells. The DRBP76:NF45 heterodimer binds to the HRV2 IRES in neuronal but not in glioma cells. Ribosomal profile analyses show that the heterodimer preferentially associates with the translation apparatus in neuronal cells and arrests translation at the HRV2 IRES, preventing PV-RIPO RNA assembly into polysomes. Results of this study suggest that the DRBP76:NF45 heterodimer selectively blocks HRV2 IRES-driven translation initiation in neuron-derived cells.

FIG. 1.

Molecular size exclusion chromatography. (A) HEK-293 and HTB-14 S10 lysates were applied to a Sepharose matrix under constant pressure. After collection of void volume, 1-ml fractions were collected and protein content was analyzed by SDS-PAGE and Western blotting using α-RHA, α-DRBP76, and α-NF45 antibodies. The column was calibrated with known standards of various molecular sizes, as indicated by arrows. Asterisks denote ILF3, a larger isoform of DRBP76. (B) Coimmunoprecipitation of DRBP76 and NF45 from HEK-293 cells. Immunoprecipitates (IP) were generated with α-DRBP76, α-NF45, and mouse immunoglobulin G (IgG), as indicated. Coprecipitating proteins were analyzed by SDS-PAGE and Western blotting with α-DRBP76 and α-NF45 antibodies.

FIG. 2.

Comparative RNA affinity chromatography. HEK-293 (A), HTB-14 (B), and DRBP76-immunodepleted HEK-293 (D) S10 lysates were applied to RNA affinity columns. After collection of flowthrough (FT; lane 1), the column was washed with H200 (lanes 2 and 3) and eluted with a 400 to 1,000 mM KCl gradient (lanes 4 to 11). Column fractions were analyzed by SDS-PAGE and silver staining or Western blotting with α-DRBP76 and α-NF45 antibodies, as indicated. (C) Western blot analysis of HEK-293 S10 lysate (lane 1) and DRBP76-immunodepleted HEK-293 S10 lysate (lane 2) by use of α-DRBP76 and α-tubulin antibodies, as indicated. M, molecular size markers in kilodaltons.

FIG. 3.

Ribosomal sedimentation profiles of HEK-293 and HTB-14 cells. (Top panels) A₂₅₄ absorption spectra of 10 to 50% sucrose gradients containing lysates generated from mock-infected HEK-293 cells (A), lysates generated from HTB-14 cells (F), or HEK-293 lysates treated with EDTA (D). (Middle panels) A₂₅₄ absorption spectra of lysates generated from PV-RIPO-infected HEK-293 (B), PV-RIPO-infected HTB-14 (G), or PV-infected HEK-293 (E) cells. (C) A₂₅₄ absorption spectra of a 5 to 20% sucrose gradient containing free protein and 40S ribosomal subunit components from a PV-RIPO-infected HEK-293 cell lysate. Lysates from infected cells were generated at 4 hpi. Western blot analyses were performed with α-DRBP76, α-NF45, α-RpS6, and α-tubulin antibodies, as indicated. Viral cDNA corresponding to the IRES region was amplified by RT-PCR, and rRNA was analyzed from total RNA. M, monosome; P, polysome.

FIG. 4.

Ribosomal sedimentation profiles of shDRBP76 cells. A₂₅₄ absorption spectra of 10 to 50% sucrose gradients containing lysates generated from mock-infected (A) or PV-RIPO-infected (B) shDRBP76 cells. Lysates from infected cells were generated 4 hpi and analyzed as described in the legend for Fig. 3. M, monosome; P, polysome.