Combinaison d’approches classiques et de génétique inverse en vue d'une meilleure compréhension du tropisme et de l'activité oncolytique du réovirus de mammifères
The mammalian reovirus preferentially replicates and kills cancer cells. It is therefore currently used in clinical trials to treat various types of cancers in humans. The main objective of this study was to get a better understanding of the various components involved in the reovirus replication cycle that could potentially be important for the optimization of its oncolytic properties using a combination of traditional approaches and the newly described reverse genetics technique. Classically, the viral persistence approach is undertaken to identify new reovirus variants. This has led to the selection of uncoating mutants in L929 cells. In this study, adapted viruses were recovered from Vero cells (VeroAV) and unlike other persistence mutants, VeroAV had amino acid substitutions on the mu1 and the sigma1 proteins. The reverse genetics approach demonstrated that VeroAV had a better binding ability onto sialic acids of Vero cells. Substitutions on sigma1 were found to be mainly responsible for this phenotype although mu1 substitutions may affect virus infectivity. In a second study, we noticed that the reverse genetics wild-type virus (T3DK) was more sensitive to interferon when compared with the wild-type virus from our laboratory (T3DS). After complete sequencing of the T3DS, we then constructed a T3DS by reverse genetics. This allowed us to proceed in our studies of a mutant virus previously recovered in our laboratory by chemical mutagenesis (P4L-12). It was previously established that P4L-12 had an enhanced replication in transformed cells while sparing the parental cells and this phenotype was correlated to an increased sensitivity to interferon. In this study, amino acid substitutions on the sigma3, mu1, muNS and lambda2 proteins were identified. By using the reverse genetics approach, we demonstrated that the substitution found on the lambda2 protein was primarily responsible for the increase in sensitivity to interferon of P4L-12. The combination of approaches based on persistence or selection of mutants sensitive to interferon followed by characterization with reverse genetics will certainly be useful to get a better understanding of reovirus in order to improve its oncolytic capacity.
Reovirus Reverse Genetics Viral persistence Interferon Binding Uncoating Viral oncolysis