Intercellular calcium waves are a conserved feature of enteric virus infection and drive Tulane virus spread

Joseph Hyser, Baylor College of Medicine

Francesca J. Scribano1,2, Alexandra L. Chang-Graham1, Jacob L. Perry1,3, John T. Gebert1,4,5, Kristen A. Engevik1, Joseph M. Hyser1. 1.Department of Molecular Virology and Microbiology, Baylor College of Medicine, U.S.A 2.Graduate Program in Immunology & Microbiology, Baylor College of Medicine, U.S.A 3.Graduate Program in Molecular Virology and Microbiology, Baylor College of Medicine, U.S.A 4.Medical Scientist (M.D./Ph.D.) Training Program, Baylor College of Medicine, U.S.A 5.Graduate Program in Development, Disease Models, & Therapeutics, Baylor College of Medicine, U.S.A

Entry and Replication

Enteric viruses such as human norovirus (HuNoV) and rotavirus (RV) remain the leading causes of viral gastroenteritis, and calcium signaling is a potential key regulator of virus replication and pathogenesis. HuNoV NS1/2 and RV NSP4 have been identified as viroporins and are used by these viruses to hijack calcium signaling during infection. We found that RV NSP4 produces two distinct calcium signals: “calcium flares” that are NSP4-mediated ER calcium release events, and “intercellular calcium waves” (ICWs) that are ADP and P2Y1 receptor mediated paracrine signals from infected to uninfected cells. We hypothesize that NSP4 and NS1/2 serve analogous functions during infections, that therefore may trigger similar calcium signals. Utilizing live calcium imaging, we demonstrate that Tulane virus (TuV) and murine norovirus, as well as viroporin-expressing enteroviruses (e.g., poliovirus and coxsackievirus) trigger P2Y1-dependent ICWs. Expression of the NS1/2 from TuV and HuNoV alone is sufficient to induce ICWs similar to infection, which correlated with their ability to generate calcium flares similar to those of NSP4. In contrast, infection with TuV NS1/2 viroporin mutants exhibited decreased calcium signaling and severely impaired replication that was transcomplemented by NSP4 expression. Finally, expressing P2Y1 in LLC-MK2 cells, which lack endogenous expression, reconstituted ICWs and this increased the kinetics of TuV spread. Together, these data provide the first evidence that calcium flares and ICWs are conserved between virus families and identify common cellular but divergent viral consequences of viroporin activity. These findings will critically inform the development of broad-spectrum host-directed therapies.