This work was supported by funding from the Medical and Health Research grant from the City of Paris, the French National grant ANR-09-JCJC-0118-1, and the ERC Starting Grant RNAvirusPopDivNVax Project no. 242719.

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No conflicts of interest declared.

Choice of cell line. The efficacy of base analogs as RNA mutagens correlates with their relative uptake by different cell types 11. If the cell line that is normally used for virus passage proves to be refractory to mutagen uptake or too sensitive (high cell toxicity), it may be necessary to use another cell line that meets these requirements and is still permissive to viral replication. Once the mutagen resistance variant is isolated, the remainder of the characterization can be performed in the original, preferred cell line. In our experience, HeLa cells readily take up mutagen; BHK cells require up to 10-fold higher concentrations and Vero cells are refractory to mutagen uptake.
Choice of mutagen. In trying to isolate fidelity variants by mutagen treatment, the likelihood of success is increased if more than one type of mutagen is used. Base analog mutagens of different structure, that are erroneously incorporated into genomes during replication will predominantly induce result in a specific subset of mutations in subsequent replication cycles: ribavirin treatment favors GtoA and CtoU transition mutations 12; 5-azacytidine has a similar bias, with the addition of CtoG and GtoC transversions 13; 5-fluorouracil preferentially induces AtoG and UtoC transitions 14. Alternatively, higher concentrations of Mg2+ or Mn2+ can be supplemented to the medium to increase the overall mutation frequency of RNA viruses without the bias described above 12. Depending on the virus' codon sequences, and the codon changes required to generate a fidelity variant, some of these conditions will favor the emergence of this variant over others. For the higher fidelity poliovirus G64S and Coxsackie virus A372V, ribavirin treatment most readily selected for the variants because the required AtoG transition at the codon site corresponded to the mutations predominantly generated by this ribavirin.
MOI vs. population size. In virology, protocols for tissue culture infection pay particular attention to the multiplicity of infection (MOI), to avoid the accumulation of defective interfering particles (low MOI) or to promote recombination between viruses (high MOI), for example. To select for emergence events over serial passaging, it is also important to consider virus population size. Since the resistant mutant initially exists at low frequency, it is best to transfer as large a population size as possible from one passage to the next (105-106 viruses, e.g.) to avoid losing these emerging variants at each passage. Scaling up the size of well or flask (number of cells infected) may help to minimize the increase in MOI if this is of concern. On the other hand, for experiments in which the sensitivity of a virus to mutagen is being tested, low MOI infection is performed in order to increase the number of replication cycles occurring in the experiment and to avoid rescue of mutagenized genomes by higher fitness genomes through complementation in co-infected cells. This is important since the mutations generated on progeny genomes during the first round of replication will not be immediately detected. Most of these mutagenized RNAs will still be packaged into virion. It is in the next round of infection that lethal mutations present in these genomes will result in an aborted replication cycle, and reduction in virus titer. It may be necessary to allow for several rounds of accumulation of mutations before a significant effect of lethal mutagenesis is observed. Finally, if over the passage series in the presence of mutagen, the virus titers continue to drop until extinction, then the researcher should try passaging virus in gradually increasing amounts of mutagen (starting from a very low concentration).
Isolation and generation of the RNA mutagen resistant clone from the RNA mutagen-resistant population. RNA mutagens introduce multiple random mutations to each genome, but selection for resistance will only enrich (and fix to consensus sequence) the resistance mutation. To identify this mutation, we sequence the mutagen resistant population (consensus of the population) and not individual viruses. Hence, the single, random mutations created by the mutagen are not detected in the sequence; only the mutations that result in consensus changes following selection, are found. In our experience we only identify one or two such consensus sequence changes. Once the mutagen resistant population is obtained and the resistance mutation is identified, it is necessary to generate a more pure stock of this variant. Above, we described a plaque purification procedure. Alternatively, if the virus of interest does not produce easily identifiable plaques, the desired variant may be purified by limiting dilution. This approach is essentially a TCID50 in 96-well format, where the virus stock is diluted such that less than 50% of wells are infected. Using this dilution, the same approach as above is taken, in isolating up to 10 individual variants and confirming their sequences. As mentioned, in the best cases, an infectious cDNA clone of the virus strain is available. Isolation of the variant would thus not be necessary. In our experience, fidelity variants are the result of single amino acid substitutions and can thus be generated using simple, commercialized mutagenesis kits such as Quikchange (Agilent). A secondary option is to use a cDNA clone of a closely related strain. However, if a related strain is used, we strongly suggest using both this approach and virus isolation (e.g. plaque purification) because we have found that the same fidelity altering mutation on two closely related viruses will not necessarily have the same effect.
Fidelity and replication. Selection of RNA mutagen resistant variants have resulted in the isolation of both higher and lower fidelity variants with growth characteristics that are similar to their wild type counterparts 4,12,15. Currently, the link between polymerase activity rates and fidelity is not fully understood. In vitro biochemical studies using purified RNA polymerase have shown that higher fidelity variants have slower processing rates, while lower fidelity variants tend to have faster processing 1-3,12. In tissue culture, these differences are not usually evident, suggesting that availability of resources, rather than intrinsic polymerase activity kinetics, is the rate-limiting step. If the fidelity variant replicates with kinetics that are not significantly different from wild type, then a comparison of their mutation frequencies can be directly made. If a very significant change in replication kinetics exists, then the data should be normalized to account for kinetic differences, for example by comparing viruses that have undergone the same number of replication cycles. In our experience, although no significant differences in one-step growth kinetics were observed between wild type and high fidelity variants, we observed that higher fidelity variants consistently titer higher (within 1 log) compared to wild type but they make slightly less RNA (within the same order of magnitude), further suggesting that the genomes they produce contain fewer mutations and are thus, more infectious.
Sample preparation and sequencing. For all steps in these protocols, it is imperative that high-fidelity, proof-reading enzymes are used for PCR and RT-PCR to limit introducing additional mutations since they cannot be distinguished from biologically relevant mutations. It is critical that the virus populations to be compared have been prepared in the same conditions (passage history, tissue culture medium, temperature, RNA extraction method, RT-PCR protocols, etc.) It is also important to ensure that enough starting material was obtained from the RNA extraction such that a strong band is generated by RT-PCR. A 1/100 dilution of the RNA sample should also give a detectable RT-PCR band, indicating that the sample contains sufficient numbers of RNA molecules to avoid representation bias (amplifying the same genome repeatedly). Since mutation frequency is a distribution, one would expect that similar values will be obtained regardless of population size, provided the aforementioned bias is not occurring. As Figure 6 shows, a 105-fold dilution of a virus stock gives a mutation frequency that is not significantly different from the parental stock.
Until the optimal conditions for TopoTA cloning are found, confirm the presence of inserts after blue/white screening, by colony PCR before sequencing. As a control for mutational noise (mutations introduced by RT-PCR and sequencing), clone a PCR product from a plasmid bearing the same viral sequence and/or clone and sequence RT-PCR products of in vitro transcribed RNA corresponding to virus genome (be aware that different in vitro transcription enzymes have different error rates and may not give useful information as to the real background error in your procedure). Some virus sequences may be toxic to bacteria, so it is important to verify this before deciding on the region of viral genome to be sequenced for mutation frequency. In analyzing sequences obtained by TopoTA, note that each clone should contain only one insert/sequence. If a double peak is observed, suggesting a mixed population, it is possible that two neighboring bacterial colonies were selected. It is also possible, although highly unlikely given the low mutation frequencies in bacterial replication, that the mutation was introduced during amplification of plasmid in the bacterial culture. In plaque purified populations, a double peak may represent overlapping plaques, or a virus that is acquiring a new mutation or reverting a mutation during plaque development. Be consistent and decide on whether to count or not count these mutations.
Finally, keep in mind that the mutation frequencies used here are relative values. They are valid only in comparing virus populations grown under the same condition, and sequenced over the same region! They should not be taken as absolute values of mutation rate, or the mutation frequency of the genome as a whole. However, when conditions are controlled, they do permit reproducible, quantitative comparisons of differences in mutation distribution and frequency.

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>ribavirin</td>
 <td>Sigma</td>
 <td>R9644-10MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5-fluorouracil</td>
 <td>Sigma</td>
 <td>F6627-1G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5-azacytidine</td>
 <td>Sigma</td>
 <td>A2385-100MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>Sigma</td>
 <td>M1028-100ML</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MnCl2</td>
 <td>Sigma</td>
 <td>M1787</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypan blue</td>
 <td>Sigma</td>
 <td>T8154-20ML</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TopoTA cloning kit</td>
 <td>Invitrogen</td>
 <td>10351021</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Quikchange mutagenesis kit</td>
 <td>Agilent</td>
 <td>200516</td>
 <td>If a cDNA infectious clone is available</td>
 </tr>
 <tr>
 <td>96-well miniprep kit</td>
 <td>Macherey-nagel</td>
 <td>740625</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lasergene, Sequencher</td>
 <td>DNAstar, Gene Codes Corporation</td>
 <td><a href="http://www.dnastar.com">www.dnastar.com</a> 
 <a href="http://www.genecodes.com">www.genecodes.com</a></td>
 <td>Or other alignment software</td>
 </tr>
 </tbody>
</table>

isolation of fidelity variants of rna viruses and characterization of virus mutation frequency

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.

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

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

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 ...

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Immunology and Infection

29.4K Views.  Institut Pasteur. The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.