Structural Features of the Seneca Valley Virus Internal Ribosome Entry Site (IRES) Element: a Picornavirus with a Pestivirus-Like IRES

Abstract

The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId 2 domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.Seneca Valley virus (SVV) is a recently discovered member of the picornavirus family. It was found as a contaminant in PER.C6 cell cultures, and its natural host has not yet been definitively identified, but a number of closely related viruses have been isolated from pigs (16). The complete genome sequence of SVV-001 (16) and the crystal structure of the virus capsid (52) have now been determined. The virus is most closely related to the cardioviruses, but there are some significant differences (see below), and hence it has been recommended that the virus is classified as a new species within a new genus (Senecavirus) of the Picornaviridae.SVV-001 and two of the related viruses (isolates 1278 and 66289) were inoculated into pigs; evidence of viral replication was obtained for all three viruses and for transmission of isolate 66289. However, in none of the experiments was any sign of illness observed (unpublished data and personal communication from J. Landgraf, USDA). An important feature of SVV is its ability to replicate selectively within human tumor cells. Owing to this novel activity and lack of observed pathogenicity in animals and humans, there is interest in using SVV as an oncolytic virus against neuroendocrine cancers (39), for which it is currently in clinical trials.All picornaviruses have a positive-sense, single-stranded RNA genome that is infectious and has to act both as an mRNA and as a template for RNA replication (32). Picornavirus RNA includes a single large open reading frame (ORF), encoding a polyprotein, which is flanked by a long 5Ј untranslated region (UTR) of approximately 600 to 1,300 nucleotides (nt) (depending on the virus) plus a shorter 3Ј UTR (Ͻ100 nt) with a poly(A) tail. The viral RNA lacks the 5Ј m 7 GpppN... cap structure found on all eukaryotic cytoplasmic mRNAs. Instead, a small virus-enco...

Picornavirus IRES elements: RNA structure and host protein interactions

“…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).…”

Functional analysis of Kaposi's sarcoma–associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation

“…Therefore, eIFs and ribosomal subunits were purified from HeLa cells according to described procedures (Pisarev et al 2007). Then, individual purified components were incubated with 32 P-labeled vFLIP IRES before conducting filter-binding assays to analyze the affinity and specificity of putative interactions (Willcocks et al 2011). We determined that the vFLIP IRES binds the 40S subunit with an apparent equilibrium dissociation constant (K d ) of 28 nM, while the CSFV IRES (as a control) bound with an affinity of 8 nM (Fig.…”

“…The c-myc IRES was kindly provided by Anne E. Willis (MRC Toxicology Unit, University of Leicester) and the IRES sequence from encephalomyocarditis (EMCV, nucleotides 406-930) and hepatitis C virus (HCV; nucleotides 1-426) were described previously (Easton et al 2009). For in vitro translation, the IRES fragments were cloned into the pGEM-CAT/LUC plasmid described previously which encodes chloramphenicol acetyltransferase (CAT) and firefly luciferase (fLUC) under the control of a T7 promoter (Willcocks et al 2011). The sequence of the stem-loop sequence inserted upstream of the IRES is 5 ′ -CAGATCTACGCGGTTCGCCGCGTAGATCTG-3 ′ .…”

“…The filter-binding assays were performed as described previously without modification (Willcocks et al 2011). Control RNA encoding the CSFV IRES (1-427; Paderborn strain) was generated by standard molecular cloning using a bicistronic plasmid containing the CSFV IRES (kind gift of Graham Belsham, National Veterinary Institute).…”

“…The secondary structure of the KSHV IRES was probed using dimethyl sulfate (DMS), N-cyclohexyl-N ′ -[N-methylmorpholinoethyl]-carbodiimid-4-toluenesulfonate (CMCT) and RNAse V1 and analyzed by reverse extension as described previously (James and Sargueil 2008;Weill et al 2010;Willcocks et al 2011). RNA Selective 2 ′ Hydroxyl Acylation analysis by Primer Extension (SHAPE) analysis was conducted using 1-methyl-7-nitroisatoic anhydride (1M7) as a modifying agent as previously described vFLIP IRES activity requires eIF4G and eIF4E www.rnajournal.org 1811…”