The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

Abstract

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotidelong 5 untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3 end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.Translation initiation on the majority of cellular mRNAs is mediated by a cap-dependent mechanism. The cap structure (m 7 GpppN) found on all cytoplasmic mRNAs is recognized by the translation initiation factor complex eIF4F (reviewed in reference 28). This complex contains three proteins: eIF4E, which is the cap-binding protein; eIF4A, which has RNA helicase activity; and eIF4G, which acts as a protein scaffold between the mRNA and the 40S ribosomal subunit via its interaction with eIF3 (reviewed in reference 14). In contrast, initiation of protein synthesis on some viral mRNAs, for example, from the picornaviruses, occurs by a cap-independent mechanism termed internal initiation. In this case, translation initiation is directed by an internal ribosome entry site (IRES) element located within the 5Ј untranslated region (5ЈUTR) of the viral genome (reviewed in references 2 and 11). These IRES elements are large, typically 450 nucleotides (nt) in length, and contain extensive secondary structure; they have been shown to interact with a variety of cellular proteins (2). Most of these elements work without any requirement for eIF4E and hence can continue to function when cap-dependent protein synthesis is inhibited.The picornavirus IRES elements are divided into several groups which display distinct secondary structures and biological properties. One group (class I) contains IRES elements from the entero-and rhinoviruses (e.g., poliovirus [PV]), while the second group contains the cardio-and aphthovirus IRES elements (e.g., encephalomyocarditis virus [EMCV]). The cardio-/aphthovirus IRES elements function efficiently in the rabbit reticulocyte lysate (RRL) translation system. However, the PV and rhinovirus IRES ele...

Picornavirus IRES elements: RNA structure and host protein interactions

“…The requirements for 48S complex formation on the simian picornavirus SPV9 IRES were similar to those for PTV-1 IRES, but in contrast to HCV IRES, eIF4F and eIF4B stimulated the formation of 48S complexes (de Breyne et al, 2008b). It is interesting to note that the AEV IRES, like the SPV9 IRES, showed partial requirement for eIF4F components (Bakhshesh et al, 2008). These results suggest mechanistic differences with the HCV IRES that need to be analyzed in further studies.…”

“…These elements are cis-acting RNA regulatory sequences endowed with the capacity to govern cap-independent translation initiation in mRNAs that are translated when cap-dependent translation is compromised. Subsequent studies demonstrated that IRES elements drive internal initiation of translation in the RNA of all members of the Picornaviridae family (Bakhshesh et al, 2008;Borman and Jackson, 1992;Brown et al, 1991;Hinton and Crabb, 2001;Hinton et al, 2000;Kaku et al, 2002;Kuhn et al, 1990;Nateri et al, 2000;Sweeney et al, 2012;Willcocks et al, 2011;Yu et al, 2011b). The genome of other RNA viruses also contains IRES elements, as illustrated by hepacivirus, pestivirus, dicistrovirus, retrovirus Tsukiyama-Kohara et al, 1992;Vallejos et al, 2010;Wilson et al, 2000), as well as some RNA viruses infecting plants and protozoa (reviewed in Martinez-Salas et al, 2012).…”

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

“…), and a seal picornavirus (Kapoor et al 2008), also contain this class of IRES element (see Hellen and de Breyne 2007). A distinctive feature of this class of IRES elements is their resistance to the action of hippuristanol (Bordeleau et al 2006;Chard et al 2006b;Bakhshesh et al 2008), a specific inhibitor of eIF4A, which is consistent with their lack of requirement for any eIF4 initiation factors for 48S initiation complex assembly in vitro (Pestova et al 1998;Pisarev et al 2004).…”

“…The cap dependence of these transcripts is readily demonstrated by the coexpression of an enterovirus 2A protease, which induces the cleavage of eIF4G and inhibits cap-dependent protein synthesis. In the following experiments, we have used the plasmid pGEM3Z/J1, which expresses the 1D-2A region of the swine vesicular disease virus (SVDV) polyprotein, for this purpose as described previously (Sakoda et al 2001;Chard et al 2006a;Bakhshesh et al 2008). Derivatives of the pRBRluc vector were produced containing the wildtype and defective forms of the EMCV IRES (Fig.…”

“…Initially this class of picornavirus IRES elements was identified within the PTV-1 RNA (Kaku et al 2002;Pisarev et al 2004;Chard et al 2006a). However, a number of other picornaviruses have now also been shown to contain this type of IRES, including porcine enterovirus-8 (Chard et al 2006b), certain simian picornaviruses (e.g., SV2, Chard et al 2006b, andSPV9, de Breyne et al 2008), and avian encephalomyelitis virus (AEV, Bakhshesh et al 2008). Sequence alignments also suggest that other picornaviruses, including the duck hepatitis virus-1 (Ding and Zhang, 2007), Seneca Valley virus (M. Willcocks, M. Bakhshesh, Z. Gomwalk, G.J.…”