SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses

Abstract

Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infectious clone technology. As anticipated, the ISA method resulted in greater genetic diversity of the viral populations, but no significant difference in viral fitness in vitro was observed. On the basis of these results, a new ISA-derived reverse genetics procedure was developed. This method, designated 'SuPReMe' (Subgenomic Plasmids Recombination Method), in which digested plasmids containing subgenomic DNA fragments were directly transfected into permissive cells, retains the following major advantages of the ISA method: it is rapid, flexible and does not require the cloning of complete genomes. Moreover, SuPReMe has been shown to produce virus populations with genetic diversity and replicative fitness similar to those obtained using conventional infectious clone technology. SuPReMe, therefore, represents an effective and promising option for the rapid generation of clonal recombinant populations of single-stranded positive-sense RNA viruses.

Fig. 1. Impact of the ISA method on genetic diversity of viral populations.

To investigate the impact of reverse genetics on viral population heterogeneity, the complete genome sequence of each virus was determined in triplicate. a Represents the number of mutations detected. Mutation characteristics are represented in b (Nonsynonymous/Synonymous mutations) and c (Transition/Transversion). d–f Represent the number of low-, mid- and high-frequency mutations, respectively. In a, d–f, the bottom and top of the box represent the first and third quartiles, the band inside the box represents the median value and the ends of the error bars represent the minimum and maximum values. In b, c, the average number of mutations is shown, and the error bars represent the standard deviation

Fig. 2

Impact of the ISA method on replicative fitness. Viral loads in cell supernatant media, after one passage in Vero ATCC cells, were estimated using a real-time RT-PCR assay (molecular viral loads, a and a TCID₅₀ assay (infectious titers), b. We then performed competition experiments c, d: WT and *WT viruses were mixed together to infect Vero ATCC cells. Cell supernatant media were passaged 10 times. The relative proportion of each virus was evaluated by sequencing a genomic fragment encompassing the differentiating position 4420. In a, b, the bottom and top of the box represent the first and third quartiles, the band inside the box represents the median value and error bars represent the minimum and maximum values. In c, d, the error bars represent the standard deviation

Fig. 3. Impact of the ISA method on evolution of a low replicative fitness virus (ΔWT virus).

Evolution of cell supernatant infectious virus titers (TCID₅₀ assay) during passage in Vero ATCC cells (a). b, c represent the number of mutations detected at the first and fourth passage, respectively. In a, the average number of TCID₅₀/ml is shown, and error bars represent the standard deviation. In b, c, the bottom and top of the box represent the first and third quartiles, the band inside the box represents the median value, and the error bars represent the minimum and maximum values

Fig. 4. General overview of reverse genetics methods presented in this study.

The SuPReMe comprises the following steps: -Cloning subgenomic overlapping DNA fragments flanked by two unique restriction sites. -Digesting each cloned subgenomic overlapping DNA fragments by restriction enzymes. -Preparing an equimolar mix of digested subgenomic overlapping DNA fragments. -Using that mix for cell transfection

Fig. 5. Impact of SuPReMe on genetic diversity of viral populations.

To investigate the impact of the reverse genetics method on the genetic heterogeneity of the viral populations, the complete genome sequence of each virus was established in triplicate. a represents the number of mutations detected. Mutation characteristics are represented in b (nonsynonymous/synonymous mutations) and c (transition/transversion). d–f represent the number of low-, mid- and high-frequency mutations, respectively. In a, d–f, the bottom and top of the box are the first and third quartiles, the band inside the box represents the median value and error bars represent the minimum and maximum values. In b, c, the average number of mutations is shown, and error bars represent the standard deviation

Fig. 6. Impact of the SuPReMe on replicative fitness.

Cell supernatant media after one passage on Vero ATCC cells were used to measure viral loads using a real-time RT-PCR assay (molecular viral loads, a) and a TCID₅₀ assay (infectious titers, b). The bottom and top of the box represent the first and third quartiles, the band inside the box represents the median value, and error bars represent the minimum and maximum values