Disease Virus Internal Ribosome Entry Site Research Articles

Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES-driven RNA localization.

Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here, we have combined proteomic and imaging analysis to study novel foot-and-mouth disease virus IRES interactors recognizing specific RNA structural subdomains. Besides known picornavirus IRES-binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, whereas Rab1b stimulated IRES function, ARF5 diminished IRES activity. RNA-FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctate distribution. Second, the IRES-driven RNA localized in close proximity with ARF5 and Rab1b, but not with the dominant-negative of Rab1b that disorganizes the Golgi. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.

Interconversion between Parallel and Antiparallel Conformations of a 4H RNA junction in Domain 3 of Foot-and-Mouth Disease Virus IRES Captured by Dynamics Simulations

RNA junctions are common secondary structural elements present in a wide range of RNA species. They play crucial roles in directing the overall folding of RNA molecules as well as in a variety of biological functions. In particular, there has been great interest in the dynamics of RNA junctions, including conformational pathways of fully base-paired 4-way (4H) RNA junctions. In such constructs, all nucleotides participate in one of the four double-stranded stem regions, with no connecting loops. Dynamical aspects of these 4H RNAs are interesting because frequent interchanges between parallel and antiparallel conformations are thought to occur without binding of other factors. Gel electrophoresis and single-molecule fluorescence resonance energy transfer experiments have suggested two possible pathways: one involves a helical rearrangement via disruption of coaxial stacking, and the other occurs by a rotation between the helical axes of coaxially stacked conformers. Employing molecular dynamics simulations, we explore this conformational variability in a 4H junction derived from domain 3 of the foot-and-mouth disease virus internal ribosome entry site (IRES); this junction contains highly conserved motifs for RNA-RNA and RNA-protein interactions, important for IRES activity. Our simulations capture transitions of the 4H junction between parallel and antiparallel conformations. The interconversion is virtually barrier-free and occurs via a rotation between the axes of coaxially stacked helices with a transient perpendicular intermediate. We characterize this transition, with various interhelical orientations, by pseudodihedral angle and interhelical distance measures. The high flexibility of the junction, as also demonstrated experimentally, is suitable for IRES activity. Because foot-and-mouth disease virus IRES structure depends on long-range interactions involving domain 3, the perpendicular intermediate, which maintains coaxial stacking of helices and thereby consensus primary and secondary structure information, may be beneficial for guiding the overall organization of the RNA system in domain 3.

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.

Trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements

A region of about 435 bases from the 5' noncoding region of foot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive secondary structure. Precise deletion of five predicted secondary structure features has been performed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of the modified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intact foot-and-mouth disease virus IRES complemented four of these defective elements to an efficiency of up to 80% of wild-type activity. No complementation was observed with the structurally analogous element from encephalomyocarditis virus. The role of RNA-RNA interactions in the function of the picornavirus IRES is discussed.