Using Forward and Reverse Genetics to Understand Calcium Dysregulation in Enteric Viral Virulence

Abstract

Acute gastroenteritis (AGE) remains the second leading cause of death among children under the age of 5 worldwide. AGE becomes fatal when pathogen‐associated upregulation of secretory activity across the intestinal epithelium causes severe volume depletion, hypovolemic shock, and multi‐system failure. While enteric viruses remain the most common cause of fatal AGE in kids, the drivers of their virulence (i.e. how they increase secretory activity) remain poorly understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in kids, release adenosine diphosphate (ADP) to induce coordinated signals known as “intercellular calcium waves,” which spread through uninfected neighboring cells. This dysregulates calcium signaling pathways, which enhances fluid secretion from uninfected cells and thereby contributes to volume depletion. Pharmacological blockade of calcium waves in infected mouse pups decreases disease severity, suggesting they are integral to rotavirus virulence. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. Here, we report data showing that recombinant expression of a single rotavirus protein, non‐structural protein 4 (NSP4), is sufficient to activate intercellular calcium waves. Using the rotavirus reverse genetics system, we show that the diminished calcium wave phenotype associated with an attenuated strain of porcine rotavirus segregates with the NSP4 gene upon reassortment. Furthermore, we show that this attenuation is attributable to a single amino acid polymorphism at the c‐terminal end of NSP4. This implicates the c‐terminal end and a putative PDZ‐binding motif therein as key upstream mediators of intercellular calcium waves. These findings deepen our understanding of enteric virus pathogenesis, characterize a new role for rotavirus non‐structural protein 4, offer novel targets for anti‐secretory therapeutics, and expand foundational knowledge to support the development of improved live‐attenuated vaccines.