Live imaging and interactome analysis of Zika and chikungunya viral RNAs via dual-action aptamer tag.

Arthropod-borne, positive-sense RNA viruses encompass many human pathogens posing significant health threats worldwide. Understanding intracellular dynamics and host-factor interactions of the viral genome is essential for devising effective antiviral strategies. Here, we engineer dual-labeled Zika and chikungunya viruses incorporating a Mango-II aptamer for RNA pulldown and real-time imaging, achieving high-resolution imaging of flavivirus RNA in live cells. Tandem imaging with viral replication proteins revealed intracellular dynamics of replication complexes and RNA molecules of these viruses. We identify >200 high-confidence host interactors of viral RNA and corroborated 355 hits from existing data sets. Spliceosome factor SFPQ is as a common interactor decreasing RNA production in both viruses when depleted. Intracellular colocalization of SFPQ-ZIKV and SFPQ-CHIKV RNA is confirmed through confocal microscopy imaging of infected cells. Thus, we demonstrate Mango-II aptamer tagging as an innovative strategy for identifying spatiotemporal dynamics and virus-host interactions of viral genomes in positive-strand RNA viruses.