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Aquareoviruses AqRVs have a close relationship with orthoreoviruses. However, they contain an additional genome segment, S11, which encodes nonstructural protein NS26. We previously showed that NS26 can enhance the fusogenic activity of the fusion-associated small transmembrane FAST protein NS16 from AqRV. In this study, a TLPK motif in NS26 was identified as being important for the enhancement. When the TLPK motif was deleted from NS26, the enhanced efficiency of the NS16-mediated cellcell fusion was significantly impaired. Further mutational analysis showed that the lysine K residue in the TLPK motif was critical for the enhancement. Additionally, deletion of the TLPK motif prevented NS26 from interacting with lysosomes. These findings suggested that the TLPK motif is important for NS26 to enhance the fusogenic activity of NS16, and NS26 may utilize the lysosome to benefit the fusion process.
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