Poly(A)-Binding Protein Facilitates Translation of an Uncapped/Nonpolyadenylated Viral RNA by Binding to the 3′ Untranslated Region

Abstract

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5= cap and a 3= poly(A) tail in their genomic RNAs. Instead, capindependent translation enhancer elements (CITEs) located in the 3= untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3=CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3=CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3=CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3= UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3=CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3=CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3= UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3=CITE as substitutes for the 3= poly(A) tail and the 5= cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA. Initiation is a rate-limiting step in eukaryotic translation, and is tightly regulated. Eukaryotic mRNAs possess an m 7 GpppN cap structure at the 5= end and a poly(A) tail at the 3= end. These two structures cooperate to recruit eukaryotic initiation factors (eIFs) and the 40S ribosome subunit (57) and stimulate translation initiation (19). The m 7 GpppN cap serves as the binding site for eIF4F, which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is an m 7 GpppN-cap-binding protein, and eIF4G is a scaffold protein that binds eIF4E, eIF4A, the poly(A)-binding protein (PABP), and mRNA. eIF4A is an RNA helicase that unwinds RNA duplex structures in an ATP-dependent manner (57). In plants, eIF4F is thought to be composed of only eIF4E and eIF4G (7), because eIF4A is purified as a single polypeptide and is not copurified with eIF4F in wheat germ (35). Plants have a second form of eIF4F (eIFiso4F), which is composed of eIFiso4E and eIFiso4G (8). Both eIF4F and eIFiso4F enhance the translation of m 7 GpppN-capped mRNAs with an unstructured 5= untranslated region (UTR), whereas only eIF4F can stimulate the translation of capped mRNAs with a highly structured 5= UTR and uncapped mRNAs, including viral mRNAs (20). PABP binds to a poly(A) tail at the 3= end of eukaryotic mRNAs via four RNA recognition motifs (RRMs) located in its N-terminal portion and simultaneously interacts with eIF4F via direct binding to eIF4G. This ternary interaction circularize...

Tombusvirus Y-Shaped Translational Enhancer Forms a Complex with eIF4F and Can Be Functionally Replaced by Heterologous Translational Enhancers

“…These explanations could also apply to our results with the CIRV 3=CITE; thus, the possibility remains that this 3=CITE may interact directly with eIF4F in order to function. In terms of auxiliary factors, one has been identified for Red clover necrotic mosaic virus (RCNMV) (41). For the BTE 3=CITE in the dianthovirus RCNMV (Table 1), poly(A)-binding protein binds to a purine-rich tract upstream from the 3=CITE and mediates eIF4F binding to the 3=CITE (41).…”

“…In terms of auxiliary factors, one has been identified for Red clover necrotic mosaic virus (RCNMV) (41). For the BTE 3=CITE in the dianthovirus RCNMV (Table 1), poly(A)-binding protein binds to a purine-rich tract upstream from the 3=CITE and mediates eIF4F binding to the 3=CITE (41). In our case, careful inspection of silver-stained gels of the proteins eluted from columns with the wt and mutant CIRV 3=CITEs did not reveal any differentially bound proteins of potential interest (other than eIFiso4G, which was confirmed via mass spectrometry) (data not shown).…”

Mechanism of Cytoplasmic mRNA Translation

“…This second domain is absent in mammals and yeast PABP. PABP is also implicated in viral replication (Smith and Gray, 2010) and plant PABP was shown to interact with the reverse transcriptase of turnip mosaic virus (TuMV) (Dufresne et al, 2008) and with the 3'UTR of tobacco etch virus (TEV) to promote internal initiation (Khan et al, 2008;Khan et al, 2009;Yumak et al, 2010;Iwakawa et al, 2012).…”

Differential Roles of Hsp70 and Hsp90 in the Assembly of the Replicase Complex of a Positive-Strand RNA Plant Virus

“…The bipartite genomic RNAs, RNA1 and RNA2, possess neither a cap structure at the 5= end (58) nor a poly(A) tail at the 3= end (48,95). Instead, RNA1 and RNA2 use distinct cap/ poly(A)-independent mechanisms to produce all viral proteins (30,57,58,74). RNA1 encodes N-terminally overlapping replicase component proteins, a 27-kDa auxiliary protein (p27), and an 88-kDa protein with an RdRP motif (p88).…”