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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).

This family belongs to the: Viroporin-1.

References associated with 1.A.94 family:

Bugarcic, A. and J.A. Taylor. (2006). Rotavirus nonstructural glycoprotein NSP4 is secreted from the apical surfaces of polarized epithelial cells. J. Virol. 80: 12343-12349. 17035333
Crawford, S.E., J.M. Criglar, Z. Liu, J.R. Broughman, and M.K. Estes. (2019). COPII Vesicle Transport Is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms. J. Virol. 94:. 31597778
Gallaher, W.R. and R.F. Garry. (2015). Modeling of the Ebola virus delta peptide reveals a potential lytic sequence motif. Viruses 7: 285-305. 25609303
Hyser, J.M. and M.K. Estes. (2015). Pathophysiological Consequences of Calcium-Conducting Viroporins. Annu Rev Virol 2: 473-496. 26958925
Hyser, J.M., B. Utama, S.E. Crawford, and M.K. Estes. (2012). Genetic divergence of rotavirus nonstructural protein 4 results in distinct serogroup-specific viroporin activity and intracellular punctate structure morphologies. J. Virol. 86: 4921-4934. 22357281
Hyser, J.M., B. Utama, S.E. Crawford, J.R. Broughman, and M.K. Estes. (2013). Activation of the endoplasmic reticulum calcium sensor STIM1 and store-operated calcium entry by rotavirus requires NSP4 viroporin activity. J. Virol. 87: 13579-13588. 24109210
Hyser, J.M., M.R. Collinson-Pautz, B. Utama, and M.K. Estes. (2010). Rotavirus disrupts calcium homeostasis by NSP4 viroporin activity. MBio 1:. 21151776
Pham, T., J.L. Perry, T.L. Dosey, A.H. Delcour, and J.M. Hyser. (2017). The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel. Sci Rep 7: 43487. 28256607
Scott, C. and S. Griffin. (2015). Viroporins: structure, function and potential as antiviral targets. J Gen Virol 96: 2000-2027. 26023149
Seo, N.S., C.Q. Zeng, J.M. Hyser, B. Utama, S.E. Crawford, K.J. Kim, M. Höök, and M.K. Estes. (2008). Integrins alpha1beta1 and alpha2beta1 are receptors for the rotavirus enterotoxin. Proc. Natl. Acad. Sci. USA 105: 8811-8818. 18587047