Poliovirus internal ribosome entry segment structure alterations that specifically affect function in neuronal cells: molecular genetic analysis

Abstract

Translation of poliovirus RNA is driven by an internal ribosome entry segment (IRES) present in the 5' noncoding region of the genomic RNA. This IRES is structured into several domains, including domain V, which contains a large lateral bulge-loop whose predicted secondary structure is unclear. The primary sequence of this bulge-loop is strongly conserved within enteroviruses and rhinoviruses: it encompasses two GNAA motifs which could participate in intrabulge base pairing or (in one case) could be presented as a GNRA tetraloop. We have begun to address the question of the significance of the sequence conservation observed among enterovirus reference strains and field isolates by using a comprehensive site-directed mutagenesis program targeted to these two GNAA motifs. Mutants were analyzed functionally in terms of (i) viability and growth kinetics in both HeLa and neuronal cell lines, (ii) structural analyses by biochemical probing of the RNA, and (iii) translation initiation efficiencies in vitro in rabbit reticulocyte lysates supplemented with HeLa or neuronal cell extracts. Phenotypic analyses showed that only viruses with both GNAA motifs destroyed were significantly affected in their growth capacities, which correlated with in vitro translation defects. The phenotypic defects were strongly exacerbated in neuronal cells, where a temperature-sensitive phenotype could be revealed at between 37 and 39.5 degrees C. Biochemical probing of mutated domain V, compared to the wild type, demonstrated that such mutations lead to significant structural perturbations. Interestingly, revertant viruses possessed compensatory mutations which were distant from the primary mutations in terms of sequence and secondary structure, suggesting that intradomain tertiary interactions could exist within domain V of the IRES.

FIG. 1.

Diagram of the PV 5′-UTR. (A) Line drawing of predicted secondary structure motifs (3, 15, 30). The first and last base-paired nucleotides in each stem-loop are numbered [numbering system for PV1(M)]. Stem-loops II to VI constitute the IRES. The genome-linked protein is shown as a speckled circle, and the lateral bulge-loop of stem-loop V targeted in this study is indicated by an arrow. (B) Sequence of stem-loop V of the PV1(M) IRES. Nucleotide conservation among all members of the Enterovirus genus is shown for the lateral bulge-loop as follows: bold, underlined, uppercase letters, absolute conservation; uppercase letters, purine-pyrimidine conservation; lowercase letters, variable nucleotides. The conservation pattern was last comprehensively checked against all sequences at our disposal at the end of 1998. Nucleotides mutated in this study are boxed.

FIG. 1.

Diagram of the PV 5′-UTR. (A) Line drawing of predicted secondary structure motifs (3, 15, 30). The first and last base-paired nucleotides in each stem-loop are numbered [numbering system for PV1(M)]. Stem-loops II to VI constitute the IRES. The genome-linked protein is shown as a speckled circle, and the lateral bulge-loop of stem-loop V targeted in this study is indicated by an arrow. (B) Sequence of stem-loop V of the PV1(M) IRES. Nucleotide conservation among all members of the Enterovirus genus is shown for the lateral bulge-loop as follows: bold, underlined, uppercase letters, absolute conservation; uppercase letters, purine-pyrimidine conservation; lowercase letters, variable nucleotides. The conservation pattern was last comprehensively checked against all sequences at our disposal at the end of 1998. Nucleotides mutated in this study are boxed.

FIG. 2.

Effects of site-directed mutagenesis of the two GNAA motifs in the lateral bulge-loop of PV1(M) stem-loop V on virus viability. The sequence of the entire loop is given with mutation sites underlined. For each mutant, changes from the wild-type (WT) sequence are indicated by lowercase letters. The specific infectivity of in vitro-derived transcripts (PFU per microgram of RNA) was determined by transfection of HeLa cell monolayers, which were then overlaid with semisolid medium. Plaque phenotypes of recovered viruses were determined after infection of HeLa cell monolayers for 48 h at the indicated temperatures.

FIG. 3.

One-step growth curves for PV1(M) IRES mutants in neuronal cells. IMR-32 cell monolayers were infected with various mutants at an MOI of 10 and incubated at 37°C (A) or 39.5°C (B). At the indicated times postinfection (post infect), intracellular virus was harvested and quantified (PFU per cell) by titration on HeLa cells. WT, wild type.

FIG. 4.

Structure probing of 5′-end-labeled mutant RNAs. (A) In vitro transcripts corresponding to stem-loop V of PV1(M) RNA (wt) or mutant derivatives (F2-5 and F2-24) were subjected to limited RNA hydrolysis. Reactions were performed such that each molecule would receive only a single hit (as evidenced by the fact that >90% of the radiolabeled probe remained intact [free RNA probe]) either with RNase T₁ (T₁ lanes,from left to right: 0, 0.1, and 0.4 U) or with lead acetate (Pb²⁺ lanes, from left to right: 0, 1, 2, and 4 mM [final concentrations]). Control reactions consisted of either pure RNA (0 lanes) or alkaline hydrolysis ladders (OH⁻ lanes). The autoradiogram of the 10.5% acrylamide-urea gel is shown (the first 30 nt were deliberately cropped, as no differences in profiles between wt and mutant RNAs were seen). Open circles in the OH⁻ lanes indicate intervals of 10 nt, as indicated on the left. Differences between the mutant profiles and the wild-type profiles are indicated by asterisks for RNase T₁ and black bars for lead acetate. (B, C, and D) Different probing profiles summarized for the wild type and mutants F2-5 and F2-24, respectively (mutations are indicated by lowercase letters). The predicted secondary structure of nt 478 to 527 of wild-type PV1(M) RNA (top of domain V of the IRES) is shown (30). RNase T₁ hits are shown as triangles; lines highlight lead acetate-sensitive regions. The intensity of the symbols is proportional to the intensity of the signals obtained in the probing experiments.

FIG. 5.

Translation efficiencies for PV1(M) IRES mutants in vitro. RRL-based translation reactions were programmed with 5 μg of uncapped in vitro transcripts synthesized from pKK-C2 derivatives/ml (see Materials and Methods). Reaction mixtures contained 95.5 mM added KCl (final concentration) and were supplemented with increasing concentrations (0, 5, 10, and 20%, by volume, from left to right) of S10 extracts from HeLa (A) or IMR-32 (B) cells. The different RNAs are indicated above the autoradiograms of the dried sodium dodecyl sulfate-23% polyacrylamide gels. The results of densitometric quantifications are shown below the autoradiograms (▪, wt; ♦, F2-5; •, F2-8; ▴, F2-24). Translation efficiency is expressed as a percentage of that of wild-type RNA in reaction mixtures supplemented with 20% (by volume) S10 extracts.