Role of Rotavirus NSP4 Viroporin and Enterotoxin Activity in Aberrant Calcium Signaling and Replication

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ABSTRACT Rotavirus (RV) is the leading cause of life-threatening diarrheal disease in young children, and a hallmark of RV- infection is the elevation of cytosolic calcium ([Ca2+]cyto) in the host cell. RV NSP4 is a multifunctional protein that acts as a viral ion channel (e.g., viroporin) through its viroporin domain (VD) to cause an elevation in ([Ca2+]cyto), whereas the enterotoxin domain (ED) elicits a receptor-mediated transient Ca2+ signal and causes diarrhea in mice. Interestingly, the RV-induced elevation in Ca2+ manifests as a complex dynamic signaling regime that is comprised of at least two distinct Ca2+ signal types: Ca2+ puffs and intercellular waves (ICWs). Both of these are known phenomenon in host Ca2+ signaling but have not been previously associated with virus-induced Ca2+ signaling. We observed that RV-induced Ca2+ puffs occur early during infection as subcellular, perinuclear Ca2+ release events. Later during infection, after the onset of the Ca2+ puffs, RV-infected cells trigger ICWs, which we previously characterized are mediated by the infected cell releasing ADP that in turn diffuses and activates P2Y1 purinergic receptors on surrounding cells. While these aberrant Ca2+ signals are caused by NSP4, the relative contribution of the NSP4 VD and ED to these signals are not known. First, I propose to utilize reverse genetic engineering of RV NSP4 VD and ED to probe the function of this protein through mutagenesis of key amino acid residues in the context of a native viral infection. Furthermore, these disruptions in NSP4 will give key insights into multiple aspects of virus replication and pathogenesis by observing Ca2+ signaling phenotype, growth kinetics, protein production, viroplasm formation, Cl- secretion, and serotonin secretion. Secondly, using live cell Ca2+ imaging technology, I propose that RV-induced Ca2+ puffs are from the RV NSP4 VD and not from host Ca2+ channels. Using the NSP4 VD mutants and live confocal Ca2+ imaging, I will characterize the RV Ca2+ puffs, and determine whether these elementary Ca2+ release events are host or NSP4 generated. The information gained through this proposal will allow us to gain mechanistic insights about how viruses utilize viral Ca2+ channels to commandeer host Ca2+ signaling pathways and reprogram cells to facilitate viral replication. Furthermore, these ideas and experiments will broaden our understanding of how other Ca2+-conducting viroporins function during infections, aiding in the discovery of other viruses that disrupt Ca2+ signaling pathways for virus replication strategies. In conclusion, these new ideas and discoveries from the use of the new RV reverse genetics platform and live cell Ca2+ imaging will usher in a new era of research driving advancements in the field of molecular virology and physiology.