1.A.94 The Rotavirus Non-structural Glycoprotein 4 Viroporin (NSP4) Family 

The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmicmonovalent cations and Ca²⁺ through a phospholipase C-independent pathway (Hyser and Estes 2015; ), which is required for virus replication and morphogenesis. Hyser et al. 2010 identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins that facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca²⁺, but NSP4 viroporin mutants maintained low levels of [Ca²⁺, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca²⁺]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca²⁺]cyto, a critical event that regulates rotavirus replication and virion assembly (Hyser et al. 2010).

Filoviruses, such as Ebola and Marburg viruses, cause severe outbreaks of human infection, including the extensive epidemic of Ebola virus disease (EVD) in West Africa in 2014. The full length glycoprotein (GP), is encoded by the GP gene via RNA editing. Half of the amino acids comprising the 'delta peptide', a 40 amino acid carboxy-terminal fragment of GP, were identical between otherwise widely divergent species. A lysine-rich amphipathic peptide motif was noted at the carboxyl terminus of delta peptide with high structural relatedness to the cytolytic peptide of the non-structural protein 4 (NSP4) of rotavirus. EBOV delta peptide is a candidate viroporin, a cationic pore-forming peptide, and may contribute to EBOV pathogenesis (Gallaher and Garry 2015). Rotavirus maturation depends on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication (Crawford et al. 2019).

The secreted form of NSP4 acts as an enterotoxin that causes phospholipase C-dependent elevation of the intracellular calcium concentration in host intestinal mucosa cells (Bugarcic and Taylor 2006). Increased concentration of intracellular calcium disrupts the cytoskeleton and the tight junctions, raising the paracellular permeability. This potentiates chloride ion secretion through a calcium ion-dependent signaling pathway, inducing age-dependent diarrhea. To perform this enterotoxigenic role in vivo, NSP4 is released from infected enterocytes in a soluble form capable of diffusing within the intestinal lumen and interacting with host plasma membrane integrin receptors on neighboring epithelial cells(Seo et al. 2008).