The ability of alphavirus replicases to synthesize non-viral type I interferon-inducing RNAs correlates with viral RNA synthesis and has a diverse impact on virus replication and pathogenicity

Alphaviruses have positive-strand RNA genomes that mimic cellular mRNAs, and their translation results in the synthesis of nonstructural (ns) polyprotein, the precursor of viral replicase. The ns polyprotein is processed by its protease activity to form an early replicase complex, responsible for the synthesis of negative-strand RNA that forms a double-stranded RNA (dsRNA) replication intermediate with the RNA genome. The following processing results in the formation of a late replicase complex responsible for the synthesis of positive-strand RNAs. Replication complexes are anchored to membranes, and dsRNA is shielded from cellular pattern recognition receptors. Nevertheless, alphavirus infection triggers a type I interferon response; this is partly due to the ability of replicases to utilize cellular RNAs as templates for synthesis of specific dsRNAs (rPAMPs). Here, we demonstrate that replicases of 11 alphaviruses, representing most of the antigenic complexes of alphaviruses, are all capable of rPAMP synthesis in human cells and that some replicases also do the same in mosquito cells. The levels of rPAMPs generally correlate with the efficiency of viral RNA synthesis and are increased by mutations slowing down the processing of the ns polyprotein. For different strains of Semliki Forest virus, the elevated synthesis of rPAMPs correlates with a previously reported virulent phenotype, while for mutants of chikungunya virus, the situation is reversed. Thus, synthesis of rPAMPs is a universal property of alphavirus replicases; these molecules are used to regulate virus infection, and their functional impact depends on their amount as well as the virus species.IMPORTANCEAlphaviruses are important mosquito-borne emerging pathogens. Their ability to interact with cellular defenses, including type I IFN, is crucial for infection. Here, we found that alphavirus replicases have a universal ability to synthesize type I IFN-inducing RNAs using non-viral templates, and that their synthesis varies greatly among viruses and their strains. Production of these RNAs was increased by mutations slowing down the maturation of the viral replicase. The abundance of non-viral type I IFN-inducing RNAs correlated with neurovirulence of Semliki Forest virus, indicating their role in virus pathogenicity; however, for chikungunya virus, their excess correlated with virus attenuation. These data are important to promote the understanding of mechanisms of alphavirus pathogenesis and virus interactions with the host immune system. As alphaviruses represent promising platforms for development of advanced mRNA vaccines, the data can also be used for rational optimization of alphavirus-based vaccine candidates.