Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant’s defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction—a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics.
The 5-leader of tobacco etch virus (TEV) genomic RNA directs the efficient translation from the naturally uncapped viral RNA. The TEV 143-nt 5-leader folds into a structure that contains two domains, each of which contains RNA pseudoknots. The 5-proximal pseudoknot 1 (PK1) is necessary to promote cap-independent translation (Zeenko, V., and Gallie, D. R. (2005) J. Biol. Chem. 280, 26813-26824). During the translation initiation of cellular mRNAs, eIF4G functions as an adapter that recruits many of the factors involved in stimulating 40 S ribosomal subunit binding to an mRNA. Two related but highly distinct eIF4G proteins are expressed in plants, animals, and yeast. The two plant eIF4G isoforms, referred to as eIF4G and eIFiso4G, differ in size (165 and 86 kDa, respectively) and their functional differences are still unclear. Although eIF4G is required for the translation of TEV mRNA, it is not known if eIF4G binds directly to the TEV RNA itself or if other factors are required. To determine whether binding affinity and isoform preference correlates with translational efficiency, fluorescence spectroscopy was used to measure the binding of eIF4G, eIFiso4G, and their complexes (eIF4F and eIFiso4F, respectively) to the TEV 143-nt 5-leader (TEV1-143) and a shorter RNA that contained PK1. A mutant (i.e. S1-3) in which the stem of PK1 was disrupted resulting in impaired cap-independent translation, was also tested. These studies demonstrate that eIF4G binds TEV1-143 and PK1 RNA with ϳ22-30-fold stronger affinity than eIFiso4G. eIF4G and eIF4F bind TEV1-143 with similar affinity, whereas eIFiso4F binds with ϳ6-fold higher affinity than eIFiso4G. The binding affinity of eIF4G, eIF4F, and eIFiso4G to S1-3 was reduced by 3-5-fold, consistent with the reduction in the ability of this mutant to promote cap-independent translation. Temperature-dependent binding studies revealed that binding of the TEV 5-leader to these initiation factors has a large entropic contribution. Overall, these results demonstrate the first direct interaction of eIF4G with the TEV 5-leader in the absence of other initiation factors. These data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.The first committed step in protein synthesis is the binding of the 5Ј mRNA cap (m 7 GpppN, where N is any nucleotide) by the eukaryotic initiation factor (eIF) 2 4E, the small subunit of eIF4F (1-4). eIF4G, the large subunit of eIF4F, acts as a scaffold for the assembly of other components of the initiation machinery. The participation of numerous proteins is required for the formation of an 80 S initiation complex at the correct AUG initiation codon. eIF4G interacts with eIF4A to facilitate helicase unwinding activity that removes mRNA secondary structure and promotes ribosome scanning to the initiation codon. eIF3 facilitates ribosome binding to an mRNA and also interacts with eIF4G. eIF4G associates with the poly(A)-binding protein, which enhances cap binding and stabilizes...
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