Dual tRNA mimicry in the Cricket Paralysis Virus IRES uncovers an unexpected similarity with the Hepatitis C Virus IRES

Co-opting the cellular machinery for protein production is a compulsory requirement for viruses. The Cricket Paralysis Virus employs an Internal Ribosomal Entry Site (CrPV-IRES) to express its structural genes in the late stage of infection. Ribosome hijacking is achieved by a sophisticated use of molecular mimicry to tRNA and mRNA, employed to manipulate intrinsically dynamic components of the ribosome. Binding and translocation through the ribosome is required for this IRES to initiate translation. We report two structures, solved by single particle electron cryo-microscopy (cryoEM), of a double translocated CrPV-IRES with aminoacyl-tRNA in the peptidyl site (P site) of the ribosome. CrPV-IRES adopts a previously unseen conformation, mimicking the acceptor stem of a canonical E site tRNA. The structures suggest a mechanism for the positioning of the first aminoacyl-tRNA shared with the distantly related Hepatitis C Virus IRES

A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate.

Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.Columbia University Start package Israel S Fernandez National Institute of General Medical Sciences GM097014 Andrey V Pisarev The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

Specific functional interactions of nucleotides at key (−)3 and (+)4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CC AUGG, in which the purines at (−)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon–anticodon base-pairing during 48S initiation complex formation, and that the role of (−)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C(1696) and less well to AA(1818–1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818–1819). U at (−)3 cross-linked to rpS5 and eIF2α, whereas G cross-linked only to eIF2α. Results of UV cross-linking experiments and of assays of 48S complex formation done using α-subunit-deficient eIF2 indicate that eIF2α’s interaction with the (−)3 purine is responsible for recognition of the (−)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818–1819) interaction might be responsible for recognizing the (+)4 position

Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning

During ribosomal scanning intact mRNA stem loops close to the start codon can pass the mRNA entry, but not the Exit channel, resulting in incorrect mRNA positioning and defective translation. The DExH-box helicase DHX29 and the initiation factor eIF1 resolve these aberrant translation initiation complexes