Structure of the Ribosome-Bound Kashmir Bee Virus IRES RNA

Internal ribosome entry sites (IRESs) are highly structured regions commonly encountered in the 5’-untranslated region (UTR) of viral messenger RNAs (mRNAs). They are capable of driving translation of the ensuing open reading frame (ORF) with a reduced set of canonical translation initiation factors. The Dicistrovirus family of positive-single stranded RNA viruses, features two ORFs that are both translated from IRESs and separated by a highly structured intergenic region (IGR). The IGR contains a unique IRES that circumvents translation initiation entirely by directly binding to the small ribosomal (40S) subunit, omitting the need for translation initiation factors to express the second ORF. In this thesis, the structure of the IGR-IRES from Kashmir bee virus (KBV) is reported. This IRES belongs to a subset of IGR-IRESs that initiates translation of two overlapping ORFs in the 0 and +1 reading frame and features several notable structural differences compared to the previously visualised members of the IGR-IRES family. Similar to other IGR-IRESs, KBV IRES folds into a structure composed of three domains. Domain 1 contacts the L1-stalk of the large ribosomal (60S) subunit and connects the other two domains of the IRES. Domain 2 binds to the 40S subunit near the ribosomal exit (E) site and inserts a stem loop (SL) into the cleft between the head and the body. This binding mode locks the head in a defined conformation and prevents domain closure, a rearrangement of the head and shoulder region of the 40S subunit, which usually follows transfer RNA (tRNA) binding in the aminoacyl (A) site. Domain 3 is accommodated in the ribosomal A site, where a pseudoknot (PK) contacts the decoding centre of the 40S subunit. These contacts are similar to cognate codon anti-codon helices in mRNA / tRNA complexes but differ in a number of key aspects. Domain 3 of KBV IRES features an extended SL (SL3) that was found to contact the A site finger (ASF) on the 60S subunit as well as ribosomal protein uL16. The structure additionally revealed the variable loop region (VLR), which connects both strands of the PK in the decoding centre, in a defined conformation. Previously, this region was reported to be disordered in the pre-translocated state of related IRESs but in the structure reported in this thesis the VLR binds to the ribosomal peptidyl-tRNA (P) site. It is possible that this contact facilitates accommodation of the IRES and stabilises the binding of domain 3 to the A site. The structure of KBV IRES provides detailed insights into the accommodation of domain 3 into the A site and for the first time reveals the functionally important VLR. It enables future experiments to better understand the function of the VLR and mechanistic studies of the as of yet enigmatic translation in the +1 frame