.  Reviewer \#0: In this manuscript, Wu et al. report the crystal structures Dengue virus type 0 (DENV0) NS0 and show that it exists in two distinct conformations representing both of the previously reported structures of Japanese encephalitis virus (JEV) and DENV0 NS0. These are defined as JEV-mode and DENV0-mode due to closeness of these DENV0 structures to the JEV and DENV0 NS0 structures. Moreover, the authors present additional data by mutational analysis to distinguish the functional differences between the two conformations of DENV0 NS0. They performed in vitro polymerase assays with wild-type (WT) and site-specific NS0 mutants to distinguish the initiation and elongation steps of RNA synthesis as well as cell-based replication using immunofluorescence assays. The authors conclude that the methyltransferase (MTase) of NS0 serves as a unique initiation factor only through NS0 conformation in the JEV-mode. But no MTase assays were performed. Overall, the crystallographic work is well-done. However, their biochemical and virological assays raise several questions. The functional roles of these two conformational states of DENV0 NS0 in viral replication remains unclear. Moreover, another group reported recently that DENV0 NS0 exists in two distinct conformational states (El Sahili, Soh, Schiltz, Gharbi-Ayachi, She, Shi, Lim, Lescar, 0000. J. Virol. 00: e00000-00). These authors in El Sahili et al. provide convincing evidence that it is the inter-domain linker between the MTase and POL domains the functional determinant of the two conformational states of DENV0 NS0.  