The X-ray structure of the eukaryotic translation initiation factor 4E (eIF4E), bound to 7-methyl-GDP, has been determined at 2.2 A resolution. eIF4E recognizes 5' 7-methyl-G(5')ppp(5')N mRNA caps during the rate-limiting initiation step of translation. The protein resembles a cupped hand and consists of a curved, 8-stranded antiparallel beta sheet, backed by three long alpha helices. 7-methyl-GDP binds in a narrow cap-binding slot on the molecule's concave surface, where 7-methyl-guanine recognition is mediated by base sandwiching between two conserved tryptophans, plus formation of three hydrogen bonds and a van der Waals contact between its N7-methyl group and a third conserved tryptophan. The convex dorsal surface of the molecule displays a phylogenetically conserved hydrophobic/acidic portion, which may interact with other translation initiation factors and regulatory proteins.

Innate immune defenses are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs) 1. Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people 2. Infection is governed by hepatic immune defenses triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals IRF-3 activation to induce the expression of α/β interferon (IFN) and antiviral/interferon-stimulated genes (ISGs) that limit infection 3 -10. Here we identified the poly-uridine motif of the HCV genome 3' nontranslated region (NTR) as the PAMP substrate of RIG-I, and show that this and similar homopoly-uridine motifs present in the genome of RNA viruses is the chief feature of RIG-I recognition and immune triggering 8. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent upon homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signaling to induce a hepatic innate immune response in vivo, and triggered IFN and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by identifying homopoly-uridine motfis present in the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and define immunogenic features of the PAMP/RIG-I interaction that could be utilized as an immune adjuvant for vaccine and immunotherapy approaches.To determine the nature of the HCV PAMP RNA we conducted a functional screen to identify possible HCV PAMP RNA motifs. We assessed the ability of full length HCV genome RNA or contiguous HCV RNA segments to trigger the IFN-β promoter in transfected Huh7 cells. The full-length HCV genome RNA triggered innate immune signaling to induce the IFN-β promoter (Fig. 1a). Two regions of the HCV RNA, encoding nt 2406-3256 and nt 8872-9616, stimulated significant induction of the IFN-β promoter (Fig. 1b) with signaling activity respectively localized to nt 2406-2696 of the open reading frame and nt 9389-9619 encoding the 3' NTR (Fig.1c). Deletion of nt 9389-9619 but not nt 2408-2663 from the HCV genome significantly attenuated signaling to the IFN-β promoter (Fig 1d). PAMP motifs are typically conserved among strains of a pathogen 1 , and sequence comparison of multiple HCV genomes revealed global variability within nt 2406-3696 among virus strains but nt 9389-9616 encoded motifs of high conservation (Fig. S1) Thus, the viral 3' NTR might encode HCV PAMP motifs that trigger innate immune signaling in the host cell.The HCV 3' NTR is comprised of three regions: a variable region (VR) with potential secondary structure, a nonstructured poly-U/UC region containing polyuridine with interspersed ribocytidine, and the terminal X region containing three conserved stem-loop structures (Fig. 1e) 12 . We evaluated the ability of RNA encoding the HCV 3' NTR or each of its regions to trigger intracellular si...

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Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population 1 . Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multisubunit membrane-associated complex. The non-structural protein NS5A is an active HCV replicase component 2,3 , a pivotal regulator of replication 2,4 , and a modulator of cellular processes spanning from innate immunity to dysregulated cell growth 5,6 . NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic α-helix at its N-terminus that promotes membrane association 7-9 . Following this helix, NS5A is organized into three domains (Fig. 1a) 10 . The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule 10 . Mutations disrupting either the membrane anchor 7,16 or zinc binding 10 are lethal for RNA replication. Probing the role of NS5A in replication has been hampered by the lack of structural information for this enigmatic multifunctional protein.Herein we report the structure of domain I at 2.5 Å resolution, revealing a novel fold, a new zinccoordination motif, and a disulfide bond. Molecular surface analysis suggests the location of protein, RNA, and membrane interaction sites.The structure of domain I (amino acids 36-198) reveals two identical monomers per asymmetric unit packed as a dimer via contacts near the N-terminal ends of the molecules. For ease of discussion, the molecule is divided into two subdomains; the N-terminal subdomain IA and the C-terminal subdomain IB (figures 1b-e). A more schematic view of the fold is shown in Figure 1e. The DALI 11 server was unsuccessful at identifying structures related to domain I or either subdomain, indicating this protein represents a novel fold. Subdomain IA consists of an N-terminal extended loop lying adjacent to a 3-stranded anti-parallel β-sheet (strands B1, B2 and B3), with α-helix H2 (designated H2 to allow numbering of the N-terminal membrane anchoring helix H1 8 ) at the C-terminus of the third β-strand. These elements comprise the structural scaffold for a four-cysteine zinc atom coordination site at one end of the β-sheet. A view of subdomain IA (aa 36-100) highlighting the cysteine residues involved in zinc coordination is shown in Figure 2a and 2b. The anti-parallel β-sheet, composed of strands B1, B2, and B3, positioning Cys 57, Cys 59, and Cys 80 near the zinc-binding site is essentially as predicted in our previous model. 10 The long random coil region positioning Cys 39 and connecting the N-terminus to the β-sheet was incorrectly predicted to be a β-strand, but the arrangement of this region in relation to the other strands matches the original model. The distances of the cysteine side chain sulfur groups to the zinc atom for Cys 39 (2.36 Å), Cys 57 (2.47 Å), Cys 59 (2.42 Å) and Cys 80 (2.45 Å) observed in domain I are close to the ideal 2.35 (+/− 0.09) Å distances for structural metal zinc coordination sites 12 . Similarly, the side chain * corresponding authors; Correspondence and requests for materials shoul...

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.