Nonstructural protein NS17 of grass carp reovirus Honghu strain promotes virus infection by mediating cell-cell fusion and apoptosis

Figures

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  Fig. 1. GCRV-HH196 infection (MOI = 2) leads to cell-cell fusion. GCO cells were infected with GCRV-HH196 for 12 h, fixed, stained with DAPI (blue), and observed by confocal microscopy. Uninfected GCO cells served as controls. Red arrows indicate syncytia. Scar bar = 50 μm.

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  Fig. 2. Phylogenetic tree and sequence analyzes of NS17. (A) Phylogenetic tree constructed using MEGA 11.0 software with the neighbor-joining algorithm. NS17 of GCRV-HH196 is marked with red circle. FAST protein sequences are shown in Table 2. (B) Transmembrane region analyzed by TMHMM Server 2.0. (C) FAST protein structures were further refined based on structural and functional motifs. NS17 of GCRV-HH196 is colored red. NBV, Nelson Bay reovirus; ARV, avian reovirus; RRV, reptilian reovirus; BRV, baboon reovirus; GCRV-873, grass carp reovirus strain 873; GCRV-HH196, grass carp reovirus Honghu strain; AtSRV, Atlantic salmon reovirus; myr, myristate moiety; FP, fusion peptide; TM, transmembrane; PB, polybasic cluster; AH, amphipathic helix; HP, hydrophobic patch; PP, polyproline motif; RPH, motif enriched in arginine, proline and histidine; C, cysteine.

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  Fig. 3. Subcellular localisation of NS17 in GCO cells. (A) Following transfection with pmCherry-N1-NS17 or pmCherry-N1 for 48 h, GCO cells were fixed and stained with DAPI (blue) and inspected by confocal microscopy. Blue fluorescence shows the nucleus, and red fluorescence shows the distribution mCherry-tagged protein. Scar bar = 20 μm. (B) Following transfection with pcDNA3.1-flag-NS17, pmCherry-N1-NS17 or empty vectors for 48 h, GCO cells were lysed and analyzed by western blotting.

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  Fig. 4. NS17 overexpression promotes cell-cell fusion following GCRV-HH196 infection. After transfected with pmCherry-N1 or pmCherry-N1-NS17 for 48 h, GCO cells were infected with GCRV-HH196 (MOI = 0.4) for 48 h, stained with DAPI (blue), and inspected by confocal microscopy. Non-transfected cells served as controls. Scar bar = 100 μm.

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  Fig. 5. NS17 overexpression promotes viral replication. After transfected with pcDNA3.1-flag-NS17 or pcDNA3.1-flag, GCO cells were infected with GCRV-HH196 (MOI = 0.4) and analyzed by qRT-PCR. Non-transfected cells served as controls. Data were acquired from three independently replicated experiments (** p < 0.01).

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  Fig. 6. Flow cytometry analysis of apoptotic cells in the NS17 overexpression group. (A) After transfected with pcDNA3.1-flag-NS17 or pcDNA3.1-flag, GCO cells were collected at the indicated time, stained with Annexin V-FITC and PI, and examined by flow cytometry. Non-transfected cells served as controls. (B) Statistical analysis of the proportion of apoptotic cells. Data were obtained from three independently replicated experiments (* p < 0.05).

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  Fig. 7. Overexpression of NS17 causes DNA fragmentation in GCO cells. (A) Following transfected with pmCherry-N1-NS17 or pmCherry-N1 for 48 and 72 h, GCO cells were incubated with TUNEL reaction mixture (green), nuclei were stained with DAPI (blue) and cells were observed by confocal microscopy. Non-transfected cells served as controls. Scar bar = 20 μm. (B) Proportion of TUNEL-positive cells calculated by from the average of positive signals in four random images. Data were obtained from three independently replicated experiments (** p < 0.01).

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  Fig. 8. ROS levels in GCO cells following overexpression of NS17. After transfected with pmCherry-N1-NS17 or pmCherry-N1, GCO cells were collected at specified times, incubated with ROS probe and analyzed by plate reader. Non-transfected cells served as controls. Data were obtained from three independently replicated experiments (** p < 0.01).