Investigation of the Mechanism of Translation Initiation on Divergent Type I Cadicivirus-A 5’UTR IRES and the two members of Iflavirus family

Abstract

Picornaviruses are an important class of small single-stranded positive-sense RNA viruses. They were the first viruses to be identified as using an internal ribosomal entry site (IRES) in their 5’untranslated region (5’UTR), which promotes end-independent initiation of translation by a non-canonical mechanism and enables viral translation to continue during infection when host translation is shut off. IRESs are relatively large, modular RNA structures: individual IRES domains are responsible for interaction with different components of the translation apparatus (including eukaryotic initiation factors (eIFs), IRES trans-acting factors (ITAFs) and the ribosomal 40S subunit), and they cooperate to manipulate it to promote initiation on the viral mRNA. The IRESs from different picornaviruses differ in terms of sequence and structure, and have been classified into five major Types (I - V). Different IRESs may thus have different requirements for eIFs and ITAFs, use different mechanisms for initiation, and be active in different cell types, thereby influencing the tissue and species tropism of the virus. I report here the characterization of the structure and mechanism of initiation on IRESs from a recently identified picornavirus and two picorna-like viruses. The divergent Type I IRES in the 5’UTR of Cadicivirus-A (CDV) differs in its factor requirements from typical Type I IRESs and engages in direct interactions with eIF2 and 40S/eIF3 complex that have not previously been identified in this class of IRES. The study of the CDV IRES also yielded new insights into the roles of the ITAF Poly-C binding protein 2 (PCBP2) and of a GNRA tetraloop-containing IRES subdomain that will contribute to understanding of the function of several classes of IRES. Characterization of the IRESs of Ectropis obliqua picorna-like virus (EoPV) and Perina nuda picorna-like virus (PnPV), members of the family Iflavirus of Picornaviridae, provided insights into a novel mechanism of IRES-mediated initiation, into the plasticity of the eukaryotic translational apparatus which enables it adapt to different modes of initiation, and provided evidence that these insect virus IRESs are adapted to function specifically in insect but not other eukaryotic cells, thus indicating that the IRES is a determinant of host range for these viruses. Analysis of 5’-untranslated regions from novel picornaviruses derived from diverse organisms (mammals, birds and fish) identified a large number that contain Type IV IRES sequences that are related to the IRES of Hepatitis C virus (HCV), a member of the Hepacivirus genus of the family Flaviviridae. Some peripheral elements in these Type IV IRESs differ from corresponding HCV IRES elements, but the core functional elements are conserved. These findings provide further evidence that many picornaviruses have acquired HCV-like IRESs from the unrelated Flaviviridae virus family by recombination. It is well-established that coding regions can be exchanged between the genomes of RNA viruses by recombination, and these observations support the hypothesis that noncoding elements in RNA virus genomes are modular and can exchange and evolve independently of associated coding regions.