Sanchez-David, R. Y.; Le, H. D.; Nemegeer, J.; Goncalves, A.; Merits, A.; Maillard, P. V.

Self-amplifying RNA (sa-RNA) technology is a promising strategy for vaccine design, as its intracellular replication boosts transgene expression and provides self-adjuvanticity. However, sa-RNA efficiency is limited by innate immune responses triggered by the presence of intracellular double-stranded RNA (dsRNA). In vertebrates, differentiated cells mainly use type I interferon (IFN) system for protection against viruses, while stem cells rely on IFN-independent mechanisms such as antiviral RNA interference (RNAi). Here, we found that the efficiency of sa-RNAs based on chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) genomes is enhanced when co-expressed in cis with the Nodamura virus (NoV) B2 protein, a viral suppressor of RNAi. In stem cells, NoV B2 prevents Dicer-mediated processing of dsRNA, while in somatic cells, it blocks the translation shutdown caused by protein kinase R (PKR), a key effector of the IFN system. Notably, NoV B2 does not interfere with IFN induction and signalling, preserving sa-RNA\'s self-adjuvant properties. Mechanistically, NoV B2 sequesters replication-derived dsRNA at the cell periphery, offering a novel strategy to boost sa-RNA efficiency without compromising its immune stimulatory properties.