The NS16 protein of aquareovirus-C is a fusion-associated small transmembrane (FAST) protein, and its activity can be enhanced by the nonstructural protein NS26.

Orthoreoviruses and aquareoviruses represent two different genera in the family Reoviridae, but they share many common characteristics in structural organization and pathogenesis. Similar to fusogenic orthoreoviruses, aquareoviruses can induce cell-cell fusion and multinucleated syncytium formation. Sequence analysis indicated that the nonstructural protein NS16 might be a fusion protein responsible for aquareovirus-C (AqRV-C) syncytiogenesis. To understand the basis of AqRV-C in syncytium formation, the properties of NS16 in mediating cell-cell fusion were investigated in this study. Bioinformatics analysis indicated that NS16 shares basic structural motifs with reovirus fusion-associated small transmembrane (FAST) proteins. However, the relative arrangement of these predicted structural motifs is different from the identified FAST proteins, suggesting that NS16 may present a new member of the FAST protein family. Further transfection assays showed that NS16 was able to induce cell-cell fusion. Nevertheless, the fusion activity was less efficient in comparison with that of the viral infection. In addition, NS16 was defined to display an N-terminus-outside/C-terminus-inside orientation, and the N-terminal ectodomain was critical for effective fusion. Moreover, immunofluorescence assays revealed that NS16 colocalized with nonstructural protein NS26 in cotransfected cells. And the enhanced fusion efficiency could be detected when NS16 was coexpressed with NS26, implying that NS26 may participate in cell-cell fusion through cooperation with NS16 in aquareovirus infection. Our study provided a basis for further characterization of cell-cell fusion mediated by AqRV-C.