Genetic divergence of rotavirus nonstructural protein 4 results in distinct serogroup-specific viroporin activity and intracellular punctate structure morphologies.

Nonstructural protein 4 (NSP4) viroporin activity is critical for the replication and assembly of serogroup A rotavirus (RVA); however, the dramatic primary sequence divergence of NSP4s across serogroups raises the possibility that viroporin activity is not a common feature among RVs. We tested for NSP4 viroporin activity from divergent strains, including RVA (EC and Ty-1), RVB (IDIR), and RVC (Cowden). Canonical viroporin motifs were identified in RVA, RVB, and RVC NSP4s, but the arrangement of basic residues and the amphipathic α-helices was substantially different between serogroups. Using Escherichia coli and mammalian cell expression, we showed that each NSP4 tested had viroporin activity, but serogroup-specific viroporin phenotypes were identified. Only mammalian RVA and RVC NSP4s induced BL21-pLysS E. coli cell lysis, a classical viroporin activity assay. In contrast, RVA, RVB, and RVC NSP4 expression was universally cytotoxic to E. coli and disrupted reduction-oxidation activities, as measured by a new redox dye assay. In mammalian cells, RVB and RVC NSP4s were initially localized in the endoplasmic reticulum (ER) and trafficked into punctate structures that were mutually exclusive with RVA NSP4. The punctate structures partially localized to the ER-Golgi intermediate compartment (ERGIC) but primarily colocalized with punctate LC3, a marker for autophagosomes. Similar to RVA NSP4, expression of RVB and RVC NSP4s significantly elevated cytosolic calcium levels, demonstrating that despite strong primary sequence divergence, RV NSP4 has maintained viroporin activity across serogroups A to C. These data suggest that elevated cytosolic calcium is a common critical process for all rotavirus strains.