The 3' mRNA I-shaped structure of maize necrotic streak virus binds to eukaryotic translation factors for eIF4F-mediated translation initiation

Abstract

Unlike the mRNAs of their eukaryotic hosts, many RNAs of viruses lack a 5' m⁷GpppN cap and the 3' polyadenosine tail, and yet they are translated efficiently. Plant RNA viruses, in particular, have complex structures within their mRNA UTRs that allow them to bypass some cellular translation control steps. In the 3' UTR of maize necrotic streak virus (MNeSV), an I-shaped RNA structure (ISS) has been shown to bind eukaryotic initiation factor (eIF)4F and to mediate viral translation initiation. A 5'-3' RNA "kissing-loop" interaction is required for optimal translation. However, the details of how the 3' ISS mediates translation initiation are not well understood. Here, we studied the binding of the 3' ISS with eIFs. The eIF4A-eIF4B complex was found to increase binding affinity of eIF4F with the 3' ISS by 4-fold (from K_D = 173 ± 34 nm to K_D = 48 ± 11 nm). Pre-steady-state analysis indicated that the eIF4A-eIF4B complex increased the RNA association rate and decreased the dissociation rate in an ATP-independent manner. Furthermore, our findings suggest that eIF4F could promote binding of the 3' ISS with the MNeSV 5'UTR, enhancing the long-distance kissing-loop interaction. However, the association of the 5'UTR with the 3' ISS-eIF4F complex did not increase 40S ribosomal subunit binding affinity. These quantitative results suggest a stepwise model in which the first committed step is eIF4F binding to the 3' ISS, followed by an interaction with the 5'UTR and subsequent 40S ribosomal subunit binding.

Keywords: I-shaped RNA structure; RNA UTR; RNA structure; RNA virus; RNA–protein interaction; eukaryotic translation initiation; kissing loop; maize necrotic streak virus; plant virus; viral protein translation.

Figure 1.

Predicted mfold secondary structures of WT 3′ISS (A), 3′ISS-C1 (B), 3′ISS-iA/B2 (C), and 3′ISS-CA (D) by mfold. The relative in vitro translation efficiencies in wheat germ extract were reported elsewhere (21) and are shown below (normalized to a value of 100 for WT 3′ISS).

Figure 2.

A, normalized anisotropy values of 3′ISS (black box), 3′ISS-C1 (red circle), 3′ISS-iA/B2 (blue diamond), and 3′ISSCA (purple triangle) interactions with eIF4F. 50 nm of the fluorescein-labeled RNA was used for each reaction in titration buffer (20 mm HEPES-KOH (pH 7.6), 2.0 mm MgCl₂, 1.0 mm DTT, and 100 mm KCl) at 25 °C. Fluorescence anisotropy changes were monitored with increasing eIF4F concentrations. The excitation and emission wavelengths were 492 and 519 nm, respectively. B, van't Hoff plots for the interactions of 3′ISS (red circle), 3′ISS-iA/B2 (blue box), and 3′ISS-CA (black triangle) with eIF4F. K_eq values were determined at various temperatures as described under “Experimental procedures.”

Figure 3.

Kinetics of eIF4F binding with 3′ISS and 3′ISS-CA. A, time course of eIF4F protein fluorescence intensity decrease caused by binding with different concentrations, 1 μm (red trace), 1.5 μm (blue trace), 2.25 μm (orange trace), and 2.75 μm (black trace), of 3′ISS at 25 °C. Excitation wavelength was 280 nm. A 320-nm cut-on filter was used for monitoring emission fluorescence. The starting fluorescence intensities are offset for clarity. B, time course of eIF4F protein fluorescence intensity decrease caused by binding with different concentrations, 1 μm (red trace), 1.5 μm (blue trace), 2.1 μm (orange trace), and 3 μm (black trace), of 3′ISS-CA at 25 °C. C, observed rate constants were plotted versus concentrations of 3′ISS (♦) and 3′ISS-CA (●).

Figure 4.

Effects of eIF4A and eIF4B on eIF4F–3′ISS binding equilibrium. A, eIF4A and eIF4B separately do not affect eIF4F binding to 3′ISS. Normalized anisotropy values of 3′ISS interaction with eIF4F protein alone (black circle), eIF4F–eIF4A complex (red box), and eIF4F–eIF4B complex (blue diamond) are shown. B, eIF4A–eIF4B together increase eIF4F binding to 3′ISS. Normalized anisotropy change of 3′ISS interaction with eIF4F (blue diamond), eIF4A–eIF4B–eIF4F–ATP complex (red box), and eIF4A–eIF4B–eIF4F complex (black circle) is shown.

Figure 5.

Effects of eIF4A–eIF4B on kinetics of eIF4F binding to 3′ISS. A, time course of eIF4F–eIF4A–eIF4B protein complex fluorescence intensity decrease caused by binding with different concentrations, 1 μm (red trace), 1.5 μm (blue trace), 2.25 μm (orange trace), and 3 μm (black trace), of 3′ISS at 25 °C. Excitation wavelength was 280 nm. A 320-nm cutoff filter was used for monitoring emission light fluorescence. The starting fluorescence intensities were offset for clarity. B, observed rate constants of eIF4F–3′ISS binding in the presence (♦) and absence (●) of eIF4A–eIF4B were plotted versus concentration of ISS.

Figure 6.

A, long-distance kissing-loop interaction between MNeSV 5′UTR and 3′ISS. Complementary base pair interactions are shown in the red box. B, normalized anisotropy changes of fluorescein-labeled 5′UTR interactions with 3′ISS (purple box), in the presence of eIF4F (black triangle), and eIF4F–eIF4A–eIF4B complex (red circle). Excitation and emission wavelengths were 492 and 519 nm, respectively. C, thermodynamic cycle for formation of the 5′UTR–3′ISS–eIF4F complex.

Figure 7.

A, normalized anisotropy change for 3′ISS–eIF4F complex binding to 40S ribosomal subunits (red circle) and the effect of the 5′UTR (blue box) (excitation = 492 nm and emission = 519 nm). B, thermodynamic cycle for formation of 5′UTR–3′ISS–eIF4F–40S ribosomal subunit complex. *, K_D value was calculated based on the thermodynamic cycle.

Figure 8.

Proposed model for eIF4F- and 3′ ISS-mediated translation initiation. In the first step, eIF4A–eIF4B complex promotes the binding of 3′ISS to eIF4F. In the second step, eIF4F facilitates 5′UTR binding with the 3′ISS–eIF complex. In the third step, the 3′ISS–eIFs–5′UTR complex binds the 40S ribosomal subunit. The assembled complex is transferred to the 5′UTR for translation initiation.