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.

Fig. 1.

The SVV IRES extends into the viral coding sequence. (A) Structures of the dicistronic plasmids used in this study. The SVV 5′ UTR plus either 31, 55, or 79 nt of coding sequence were amplified by PCR, including added BamHI sites. These products were digested and inserted into a similarly digested plasmid containing the CAT and LUC ORFs, between the two ORFs at a unique BamHI site. (B) In vitro coupled transcription and translation reactions (RRL) containing [³⁵S]methionine were programmed with the dicistronic plasmids. Reactions were analyzed by SDS-PAGE and autoradiography. The CAT and LUC protein products are indicated. (C) Transient expression assay in 293T cells. The dicistronic plasmids were transfected into 293T cells that had been previously infected with vTF7-3. After 20 h, cell lysates were prepared and analyzed for CAT and LUC expression as outlined in Materials and Methods. Extracts were also subjected to SDS-PAGE and immunoblotting. The LUC expression data were obtained from three separate transfections and the results standardized to the LUC expression value from the EMCV IRES, which was set at 100%. LUC activities were normalized against CAT expression values. Mean LUC values (plus standard errors of the means) are shown.

Fig. 2.

The SVV IRES functions in the presence of cleaved eIF4G and an eIF4A inhibitor. (A) Dicistronic plasmids (2 μg) of the form CAT/IRES/LUC were transfected into 293T cells in the absence (−) or presence (+) of the plasmid pAΔ802 (0.5 μg), which expresses the PV 2A protease. After 20 h, cell extracts were prepared and analyzed for CAT and LUC expression as described in Fig. 1. LUC and CAT assays were also performed on extracts from three separate experiments, and the mean LUC values are shown, standardized to the LUC expression values from the EMCV IRES-containing construct, which was set at 100%. LUC activities were normalized to CAT expression as described in the legend to Fig. 1. (B) The same dicistronic plasmids were transfected into 293T cells in the absence (−) or presence (+) of the eIF4A inhibitor, hippuristanol (Hipp.). Cells were harvested 20 h after transfection, and the hippuristanol (0.5 μM) was added for the last 10 h of the incubation. Cell extracts were analyzed for CAT and LUC expression as described in the legend to Fig. 1. The data shown are representative of results from two independent experiments.

Fig. 3.

The SVV IRES is an HCV-like picornavirus IRES. (A) Alignment of the SVV, CSFV, and HCV 5′ UTR sequences that comprise domain II and domain III. Sequences were aligned with ClustalW and edited manually; the nucleotides which are shared by all three IRES elements are marked with an asterisk. Gaps introduced to maximize alignment are indicated by dashes. Shaded areas highlight specific stem-loop structures. The individual SVV IRES domains are indicated above the sequence. The overall sequence identity between SVV and HCV IRES sequences is 52.4%, but some regions, e.g., domains IIIa and IIIe, are highly conserved among all three viruses. (B) Proposed secondary structure of the SVV 5′ UTR. Domains are labeled by analogy to the domains in the HCV IRES. The structure was derived from comparative sequence analyses and using Mfold (50) to predict the most thermodynamically favorable structures. Outlines of the HCV and CSFV IRES element structures are also shown for comparison. Note that in the SVV IRES (as in the CSFV IRES) there is an additional IIId₂ stem-loop structure. In addition, a model of the 55-nt sequence from the coding sequence is included. Secondary structure probing of the IIId₂ domain is also indicated. Nucleotides affected by RNase V1, CMCT, or DMS are noted by arrows, circles, and squares, respectively.

Fig. 4.

Mutational analysis of the domain III region of the SVV IRES. (A) Predicted secondary structure of the domain IIId, IIIe, and IIIf regions of the SVV IRES. The stems that form the pseudoknot (stem 1 and stem 2) and the IIId₁ and IIId₂ stem-loops are shown. The nucleotides that were modified in the mutational analysis experiments are shown in bold type. Suffix “c” indicates compensatory mutations. Areas indicated with dashed lines (as in Mut7) indicate that the whole IIId2 region was deleted. (B) Dicistronic plasmids of the form CAT/IRES/LUC, containing the indicated mutations within the domain III region of the SVV+55 construct, were transfected into 293T cells as described in the legend to Fig. 1. Cell extracts were analyzed for CAT and LUC expression as described in the legend to Fig. 1. The results are derived from three independent experiments, and the mean values plus standard errors are shown. LUC activities from the mutant IRES elements are expressed relative to the activity from the wt SVV+55 IRES, which was set at 100%. LUC activities were normalized against CAT expression as described in the legend to Fig. 1.

Fig. 5.

The SVV IRES directly binds to 40S subunits and eIF3. (A) Binding curves of ³²P-labeled wild-type and IIId₂ deletion mutant RNAs to purified 40S subunits. Labeled RNAs were incubated with 40S subunits and binding assessed by filter binding assay. (B) Binding curves of ³²P-labeled wild-type and IIId₂ deletion mutant RNAs to purified eIF3. Labeled RNAs were incubated with eIF3, and binding was assessed by filter binding assay. Reported values are the average of results from three repetitions with standard errors. All calculations were performed with GraphPad Prism 5.