Joseph Hyser, Baylor College of Medicine

J. Thomas Gebert, Francesca J. Scribano, Ethan M. Huleatt, Michael R. Eledge, and Joseph M. Hyser Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, 77030, USA

Virus Replication: Entry, Exit and Everything in Between

Rotavirus nonstructural protein 4 disrupts host calcium signaling during infection by forming an ion channel within the endoplasmic reticulum (ER). By employing live calcium imaging techniques, we have observed that during RV infection NSP4 triggers an array of dynamic calcium signals, including the release of ER calcium to generate a hypermorphic signal we term a “calcium flare” and a propagating “intercellular calcium wave” (ICWs) mediated by the release of purines from infected cells. Expression of NSP4 alone recapitulates both calcium flares and ICWs but attempts to generate replication competent recombinant rotaviruses with attenuated NSP4 viroporin activity have been largely unsuccessful, underscoring that viroporin function is essential for viral replication and spread. Thus, we mechanistic insights into how calcium flares induce ICWs to better defining the cellular reprogramming caused by NSP4 calcium dysregulation. To overcome these challenges, we developed two model systems: (1) an adeno-associated virus (AAV)-based system for transient NSP4 expression and (2) lentiviral vectors to generate stable NSP4-expressing cell lines. Both models successfully recapitulate NSP4-mediated calcium signaling and show that mutation of the viroporin domain attenuates both calcium flares and ICWs Transcriptomic analyses of both systems will be used to identify key pathways and genes induced by NSP4 calcium signals. These analyses provide insight into how NSP4-driven calcium signaling remodels host transcriptional networks and highlight functional differences between transient and stable expression models. Understanding the mechanistic basis of NSP4-mediated signaling advances our knowledge of rotavirus pathogenesis and may reveal new therapeutic targets to disrupt viral replication.