Fig 1: Tetherin recycles mainly via the retromer-dependent trafficking pathway. HeLa cells on glass coverslips were transfected with plasmids expressing Tetherin–eGFPc along with plasmids expressing dsRed–Vps35, dsRed–Rab4, dsRed–Rab5, dsRed–Arf6, or mCherry–Rab11. After being treated with cycloheximide, cells were fixed and processed for fluorescence microscopy. Shown are confocal images. Boxed regions in merged images were enlarged. Arrows in Insets indicate structures where Tetherin–eGFPc and a particular endosome marker protein were co-localized. Scale bars: 10 µm. Plots beneath are quantitative data of co-localization between Tetherin–GFPc and corresponding endosomal markers. Analysis was conducted with the JACoP plugin of NIH ImageJ/Fiji. Each symbol represents a cell.
Fig 2: VPS35 contributes to the maintenance of genome stability. (A) Representative confocal images of HeLa cells labeled with anti-?H2AX antibody. (B) Numbers of ?H2AX foci per cell were calculated for at least 200 cells of each group and presented as the mean ± S.D. **, p < 0.01 vs. VPS35WT. (C) The nuclear protein extracts were isolated, and the level of ?H2AX was examined by western blotting. (D) A micronucleus assay was performed to assess genomic instability. At least 500 cells were examined, and the frequency of cells with micronuclei was calculated. **, p < 0.01 vs. VPS35WT.
Fig 3: The D620N mutation in the vacuolar protein sorting 35 ortholog (Vps35) imparts a partial loss of function on Herpes simplex virus type 1 (HSV-1) propagation. (A) Outline of experimental schedules to detect effects of Vps35 with or without D620N on HSV-1 entry (B) into cells upon exposure for 2 h, replication (C) inside cells, and release (D,E) into culture media within 36 h after viral exposure. Relative abundance of UL30 in genomic DNAs prepared from infected cells was determined by semi-qPCR (quantitative polymerase chain reaction) with GAPDH as an internal control and used as a measure of HSV-1 virion titers inside cells. Titers of virions released into culture media (conditioned media) were determined by measuring the absolute copy numbers of UL30 in conditioned media (D) and relative abundance of UL30 inside new cells exposed to conditioned media for 2 h (E). The latter was also utilized as a measure of infectivity of virions in conditioned media. Initial viral infection was conducted with a multiplicity of infection (MOI) of 3. Experiments were repeated three times. Data are Mean ± SD. One-way ANOVA followed by Turkey’s analysis for (C, F(2, 20) = 628.4, p < 0.0001) and (E, F(2, 24) = 1252, p < 0.0001) and by Tamhane post hoc multiple comparison for (D, F(2, 25) = 179.3, p < 0.0001) were conducted to determine the statistical significance. ** p < 0.01; # p < 0.0001.
Fig 4: Tetherin mediates the inhibitory effect of Vps35 on HSV-1 propagation. (A) Western blot analysis showed that HEK293T did not express Tetherin at steady state and expressed comparable levels of endogenous Vps35 as well as exogenous Tetherin, dsRed–Vps35, and dsRed–Vps35D620N. (B) Densitometry quantification for (A) from 3 independent experiments. Ectopic expression of Tetherin in HEK293T cells suffocated HSV-1 virion replication (C) and release (D and E). Co-expression of Vps35 or Vps35D620N augmented the restricting activity of Tetherin on HSV-1 (D,E). Experiments were repeated three times. Data are Mean ± SD. Statistical significance in (C–E) was determined by one-way ANOVA followed by Turkey’s analysis. F(3, 20) = 573.3, p < 0.0001 for (C); F(3, 28) = 215.5, p < 0.0001 for (D); F(3, 20) = 84.66, p < 0.0001 for (E); ** p < 0.01; *** p < 0.001; # p < 0.0001.
Fig 5: Tetherin–eGFPc traffics together with Vps35, and the D620N mutation in Vps35 perturbs the dynamics of retromer endosomes. HeLa cells were transfected with plasmids expressing Tetherin-eGFPc along with plasmids expressing dsRed–Vps35 or dsRed–Vps35D620N and imaged in real-time after being treated with cycloheximide. A series of 9 consecutive frames were chosen from movies of cells expressing dsRed-Vps35 or dsRed-Vps35D620N to highlight co-trafficking of Tetherin–eGFPc with dsRed–Vps35 or dsRed–Vps35D620N and the dynamics of Vps35 and Vps35D620N endosomes. The size of structures in each frame were measured and graphed after being manually grouped. Boxed regions in merged frames were enlarged and shown as insets. Arrows in insets trace motile structures containing both Tetherin and Vps35, whereas arrowheads indicate stationary structures. Dashed circles indicate appearances or disappearances of motile structures containing both Tetherin and Vps35, whereas dashed contours indicate morphology changes of large tubulovesicular structures where fission and fusion events were observed. Green highlights in line graphs show frequent changes in size of Tetherin-containing structures in a cell co-expressing dsRed–Vps35, whereas yellow highlights show little or no change in size of structures in a cell co-expressing dsRed–Vps35D620N. Scale bars: 10 µm.
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