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. The study describes the implementation of an RNA tag that enables visualization of Zika and chikungunya virus genomes in live cells and identifies critical host proteins that interact with the genomes for these viruses to replicate efficiently.