The 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. Studies with the first RNA-dependent RNA polymerase (RdRp) fidelity variant demonstrated that poliovirus RdRp error rate is a major contributor to population mutation frequency (1) and that reduced genetic diversity negatively impacts dissemination and pathogenesis in vivo (refs. 2-4; although the implications of these findings were limited by a relatively artificial model). The explanation for this reduced fitness is based on the idea that a diverse RNA virus population contains, by chance, more variants with potentially advantageous adaptive mutations, whereas a less diverse population is not as likely to possess such variants. Based on these observations, we hypothesized that RdRp fidelity of other RNA viruses can be modulated with similar fitness costs.Nucleoside analogs including ribavirin and 5-fluorouracil (5-FU) are mutagenic compounds that are misincorporated into viral genomes during RNA synthesis, resulting in deleterious mutations by mispairing in the following replication cycle (5-7). The consequent increase in the number of mutations places more genomic RNAs beyond a hypothetical error threshold: a mutation frequency at which the majority of the population harbors low fitness or lethal mutations (5,8). In this theoretical context, RNA virus populations lie close to this threshold-even moderate increases in mutation frequency could severely diminish infectivity. Increasing polymerase fidelity would potentially create a population farther from this error threshold that could better tolerate mutational input. Solid evidence supports this logic: DNA polymerase (9, 10) and reverse transcriptase (11-13) virus variants with antimutator phenotypes are linked to mutations that increase fidelity and reduce base analog incorporation. These observations present an interesting paradox: If point mutations in RNA viruses can increase RdRp fidelity, thereby decreasing replication mistakes, why is higher fidelity in RNA viruses not selected in nature? Is there a tradeoff between being error-prone but adaptable or being less adaptable but possessing higher fidelity? Does higher fidelity necessarily incur a phenotypic disadvantage in hosts, where a reduced ability to produce adaptive mutations impairs fitness, as was suggested by poliovirus studies?Arthropod borne (arbo-) viruses are unique among RNA viruses in that they obligatel...

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 function [1][2][3] . Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution [4][5][6][7] . Video LinkThe video component of this article can be found at https://www.jove.com/video/2953/ Protocol Determine the range of mutagen concentrations that is minimally toxic to cellsThe purpose of this exercise is to determine what range of mutagen concentrations can be used during an infection without excess cell toxicity. Essentially, you will want to reproduce the conditions that will be required for virus infection. For most viruses, infections last between 2 and 7 days. Prepare enough plates to sample cells at each day. If non-adherent cells are used, modify the protocol accordingly.1. On the day before the experiment, seed 7 x 10 5 HeLa cells/well in a 6-well plate that achieve a sub-confluent (75%) monolayer the day of the experiment. Each well of the plate will be treated with a different concentration of mutagen, permitting a range of 6 concentrations. 2. On the day of the experiment, prepare mutagen dilutions in tissue culture medium. For HeLa cells, use a range of 0 to 1000 μM for base analogs (ribavirin, 5-fluorouracil, 5-azacytidine), 0 to 50 mM for MgCl 2 , and 0 to 5 mM for MnCl 2 . 3. Aspirate the medium from the wells and replace with 2 ml mutagen-supplemented medium and return to incubator. 4. Every 24 hours, use one plate of cells to check cell viability. This can be accomplished by performing trypan blue staining or using commercialized fluorescence/luminescence assays (e.g. Promega's CellTiter-Glo® Luminescent Cell Viability Assay). 5. For trypan blue exclusion staining, detach cells from one plate (treated with different concentrations of mutagen) and gently pellet cells by centrifugation. 6. Discard supernatant and resuspend cells in PBS (serum ...

The Hepatitis Delta Virus (HDV) relies mainly on host proteins for its replication. We previously identified that PSF and p54nrb associate with the HDV RNA genome during viral replication. Together with PSP1, these proteins are part of paraspeckles, which are subnuclear bodies nucleated by the long non-coding RNA NEAT1. In this work, we established the requirement for PSF, p54nrb and PSP1 in HDV replication using RNAi-mediated knockdown in HEK-293 cells replicating the HDV RNA genome. We determined that HDV replication induces the delocalization of PSP1 to cytoplasmic foci containing PABP and increases NEAT1 level causing an enlargement of NEAT1 foci. Overall, our data support a role for the main paraspeckles proteins in HDV life cycle and indicate that HDV replication causes a cellular stress and induces both a delocalization of the PSP1 to the cytoplasm and a disruption of paraspeckles.

Potato spindle tuber viroid (PSTVd) is a small, single-stranded, circular, non-coding RNA pathogen. Host DNA-dependent RNA polymerase II (RNAP II) was proposed to be critical for its replication, but no interaction site for RNAP II on the PSTVd RNA genome was identified. Using a co-immunoprecipitation strategy involving a mAb specific for the conserved heptapeptide (i.e. YSPTSPS) located at the carboxy-terminal domain of the largest subunit of RNAP II, we established the interaction of tomato RNAP II with PSTVd RNA and showed that RNAP II associates with the left terminal domain of PSTVd (+) RNA. RNAP II did not interact with any of several PSTVd (") RNAs tested. Deletion and site-directed mutagenesis of a shortened model PSTVd (+) RNA fragment were used to identify the role of specific nucleotides and structural motifs in this interaction. Our results provide evidence for the interaction of a RNAP II complex from a natural host with the rod-like conformation of the left terminal domain of PSTVd (+) RNA.

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.