To further confirm the role of p17 in the regulation of FAST fusion activity, we examined FAST-mediated cellCcell fusion activity in the presence of either wild-type or mutant p17. in random microscopic fields (100total magnification). Results are expressed as the mean of the results of all samples. ND, not detected. Error bars indicate standard deviations (n = 5).(TIF) Mouse monoclonal to PR ppat.1010553.s002.tif (56K) GUID:?2C8D7288-ED3A-4CC0-918D-333BB0C25210 S3 Fig: CellCcell fusion activity of FAST was enhanced by p17 in YubFKT1 cells. The number of cellCcell fusion events in YubFKT1 cells transfected with NBV p17 and/or FAST expression plasmids at 24 h post-transfection. The number of fusion cells was counted in random microscopic fields (100total magnification). Results are expressed as the mean of the results of all samples. Error bars indicate standard deviations (n = 8). Significant differences compared to the vector control are indicated by asterisks. *p 0.05 (Dunnetts test).(TIF) ppat.1010553.s003.tif (19K) GUID:?718DF948-6055-4E9F-8F35-F0BF8C952042 S4 Fig: Induction of cell-cell fusion by FAST is mediated by FLAG-tagged p17 proteins. (A) The number of cellCcell fusions in DemKT1 cells expressing NBV PD-1-IN-1 FAST and FLAG-tagged wild-type or mutant p17s. The number of fusion cells was counted in random PD-1-IN-1 microscopic fields (100total magnification). Results are expressed as the mean of the results of all samples. ND, not detected. Error bars indicate standard deviations (n = 5). (B) Expression levels of p17 and FAST in transfected DemKT1 cells. FLAG-tagged NBV p17 expression plasmid (0.5 g) was transfected with the NBV FAST expression plasmid (0.5 g) into DemKT1 cells (4 105 cells). At 18 h post-transfection, the samples were collected. Protein expression was detected by immunoblotting.(TIF) ppat.1010553.s004.tif (88K) GUID:?7BA286A2-A71D-4A86-B1A7-4D4C8256312E S1 Table: Amino acid mutations of FAST, p17, and C in recombinant viruses. (TIF) ppat.1010553.s005.tif (256K) GUID:?DD811BB3-F92F-4B6C-97CE-AB78AD886F93 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract Nelson Bay orthoreovirus (NBV), a member of the family belonging to the family are divided into fusogenic and nonfusogenic subgroups, based on their ability to induce cellCcell fusion [28]. The orthoreovirus genome consists of ten segments of double-stranded RNA (dsRNA) (L1CL3, M1CM3, and S1CS4 gene segments) and some fusogenic reovirus genomes, including avian orthoreovirus (ARV), baboon orthoreovirus (BRV), Broome orthoreovirus (BroV), and NBV, encode two unique nonstructural proteins, the fusion-associated small transmembrane (FAST) protein and p17, on the S1 or S4 gene segment [29C32]. FAST proteins are small fusogenic proteins of approximately 10C22 kDa that can induce cellCcell fusion between infected-cells and neighboring cells [32,33]. FAST proteins are composed of three functional domains associated with fusion activity: an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain [34,35]. Unlike the structural proteins of enveloped viruses, nonstructural FAST proteins are dispensable for viral entry [36,37]. While the biological function of FAST proteins is poorly understood, it has been demonstrated that FAST is required for efficient viral propagation and plays a crucial role in pathogenesis [37]. Nonstructural NBV p17, ARV p17, BRV p16, and BroV p16 proteins are encoded by the second open reading frame of the fusogenic reovirus S1 or S4 gene segment [30,31,38,39]. Previous studies have revealed that ARV PD-1-IN-1 p17, also known as CRM1-independent nucleocytoplasmic shuttling protein, possesses a nuclear localization signal with critical C-terminal basic amino acids at residues K122 and R123 conserved among ARV and NBV strains [40]. In addition, it has been shown that ARV p17 activates the p53 signaling pathway and downregulates the PI3K/AKT/mTOR and ERK pathways [41]. The modulation of these fundamental signaling pathways results in the disruption of cellular translation, cell cycle arrest, and the formation of autophagosomes, contributing to efficient viral replication [41C44]. However, PD-1-IN-1 the biological functions of NBV p17 in the viral life cycle, and its involvement in viral pathogenesis, remain unclear. In this study, we PD-1-IN-1 examined the roles of NBV p17 in viral replication in cell lines and viral pathogenesis in a mouse model by using p17-mutant and -deficient viruses. We found that replication of p17-deficient virus was severely impaired in cell lines derived from the fruit bat rescued rsMB/p17-null viral growth and the expression of the viral proteins NS, C, A, and NS (Fig 6A and 6B). By contrast, all p17 constructs encoding uKR and dKR motif mutations failed to restore rsMB/p17-null virus replication (Fig 6A and 6B). We also observed that replication of the p17-null virus was restored by transient transfection with FLAG-tagged wild type p17 but not its mutants (K131A-FLAG, R132A-FLAG, uKR-AA-FLAG, dKR-AA-FLAG,.
