12 research outputs found

    Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

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    RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7

    Identification of a binding site for ASF/SF2 on an RNA fragment derived from the hepatitis delta virus genome.

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    The hepatitis delta virus (HDV) is a small (~1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used

    Arbovirus high fidelity variant loses fitness in mosquitoes and mice.

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    International audienceThe error rate of RNA-dependent RNA polymerases (RdRp) affects the mutation frequency in a population of viral RNAs. Using chikungunya virus (CHIKV), we describe a unique arbovirus fidelity variant with a single C483Y amino acid change in the nsP4 RdRp that increases replication fidelity and generates populations with reduced genetic diversity. In mosquitoes, high fidelity CHIKV presents lower infection and dissemination titers than wild type. In newborn mice, high fidelity CHIKV produces truncated viremias and lower organ titers. These results indicate that increased replication fidelity and reduced genetic diversity negatively impact arbovirus fitness in invertebrate and vertebrate hosts

    Conservation of the proposed ASF/SF2 binding site on HDV RNA.

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    <p>(<b>A</b>) Multiple alignment of sequences corresponding to the putative ASF/SF2 binding site in 92 HDV variants indexed in the Subviral RNA Database (<a href="http://subviral.med.uottawa.ca" target="_blank">http://subviral.med.uottawa.ca</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054832#pone.0054832-Rocheleau1" target="_blank">[34]</a>). (<b>B</b>) The percentage of purine and pyrimidine for each position was calculated. Blue and red indicate pyrimidine and purine, respectively. (<b>C</b>) Logo representation of the consensus sequence created from the sequence alignment of the putative ASF/SF2 binding site, which is indicated by a box in (A).</p

    Fluorescence properties of His-ASF/SF2 and quantification of the binding of His-ASF/SF2 to R199G.

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    <p>(<b>A</b>) Emission spectrum of His-ASF/SF2 (100 nM) at an excitation wavelength of 280 nm. Solid line, purified protein in 20 mM Tris-HCl, 100 mM KCl, pH 7.5; Dashed line, purified protein after a 2 h incubation in 20 mM Tris-HCl, 100 mM KCl, pH 7.5, and 8 M urea; Inset, SDS-PAGE (P) and Western blot (W) of purified His-ASF/SF2. (<b>B</b>) Molar fluorescence of His-ASF/SF2. His-ASF/SF2 was excited at 280 nm and fluorescence intensity at increasing concentration of His-ASF/SF2 was measured at an emission wavelength of 340 nm. (<b>C</b>) Binding curve generated by plotting the observed change in fluorescence of His-ASF/SF2 as a function of increasing concentration of R199G. The curve was fitted to a one-site binding model using non-linear regression and the apparent K<sub>D</sub> (appK<sub>D</sub>) was calculated. The inset represent typical ASF/SF2 emission spectra generated by the addition of increasing amount of R199G. (<b>D</b>) Variation of the observed change in fluorescence of His-ASF/SF2 as a function of increasing concentration of P11.60, a non-related RNA hairpin competitor. The insert represents the sequence and secondary structure of P11.60.</p

    Representation of the hepatitis <i>delta</i> virus RNA genome.

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    <p>Both genomic (G) and antigenomic (AG) polarities of HDV RNA are superimposed; location of <i>delta</i> ribozyme (<i>delta</i> Rz) on each polarity is shown. The HDAg (delta Ag) coding region is indicated by the black rectangle and the transcription start site is depicted by an arrow and an asterisk. The location of the R199G fragment is indicated.</p

    Effect of ASF/SF2 knock-down on HDV RNA accumulation.

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    <p>HDV-replicating 293 cells were transfected with plasmids harbouring different shRNAs against ASF/SF2 mRNA (sh1-4), a non-target shRNA (shNT), or an empty vector. (<b>A</b>) Representative of a Western blotting showing ASF/SF2 knock-down efficiency using ß-actin as a loading control. (<b>B</b>) HDV replication was induced by TET and HDV RNA accumulation was normalized to the amount of β-actin mRNA and quantified relative to non-induced cells by RT-qPCR analysis. A mean (+/− standard deviation) of three biological replicates is presented for each sample. Unpaired two-tailed t-test between HDV RNA accumulation from the sh1-4 treated cells and the shNT sample was performed: *, p-value <0.05; **, p-value <0.01.</p
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