Journal: The Journal of Cell Biology

Article Title: Septins suppress the release of vaccinia virus from infected cells

doi: 10.1083/jcb.201708091

Figure Lengend Snippet: Loss of septin promotes virus release and spread. (A) Schematic depicting key events at the plasma membrane during vaccinia virus egress. A36, the actin tail nucleator phosphorylated by Src and Abl family kinases, and B5, exposed on the surface of CEV, are integral viral membrane proteins. (B) Immunoblot analysis confirms that SEPT7 siRNA treatment of A549 cells for 72 h leads to loss of SEPT7 as well as reduction in the levels of SEPT2, SEPT9, and SEPT11. (C) Images of plaques formed on A549 cell monolayers treated with control (Allstar) or SEPT7 siRNA for 72 h under liquid overlay after 3 d of infection with WR or the YdF strain of virus, which is deficient in actin tail formation. Plaque comets are seen as a diffuse spray emanating from WR plaques in liquid overlay conditions. The graphs show the quantification of plaque size ( n > 190) with error bars representing the SEM from three independent experiments. (D) Quantification of virus release and total intracellular virus in A549 cells in the presence or absence of SEPT7 at 18 h after infection with WR or the YdF virus. Error bars represent SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Bar, 1 cm.

Article Snippet: The following primary antibodies were used for immunofluorescence and immunoblotting: A36 , AP-2 (2730; Abcam), Clathrin Heavy Chain (21679; Abcam), B5 ( ; the B5 antibody was labeled with the Molecular Probes Alexa Fluor 488 Monoclonal Antibody Labeling kit [Invitrogen] to generate the Alexa Fluor 488-B5 antibody [ ]), dyn II for immunofluorescence ( ; provided by M. McNiven, Mayo Clinic, Rochester, NY) and Western (A303-513A; Bethyl), FHOD1 (A304-825A; Bethyl), GAPDH (ab8245; Abcam), SEPT2 (60075–1-Ig; ProteinTech), SEPT7 (18991; IBL), SEPT9 (10769–1-AP; ProteinTech) for immunofluorescence and SEPT9 (A302-353A; Bethyl) for Western, and SEPT11 (A304-176A; Bethyl).

Techniques: Virus, Clinical Proteomics, Membrane, Western Blot, Control, Infection

Journal: The Journal of Cell Biology

Article Title: Septins suppress the release of vaccinia virus from infected cells

doi: 10.1083/jcb.201708091

Figure Lengend Snippet: Septin suppresses actin tail formation. (A) Immunoblot analysis confirms that SEPT7 siRNA treatment of HeLa cells for 72 h reduces the levels of SEPT7, SEPT2, SEPT9, and SEPT11. (B) Representative immunofluorescence images showing that loss of SEPT7 in uninfected HeLa cells has no obvious impact on the actin cytoskeleton (green). Bar, 50 µm. (C) Immunoblot analysis confirming that infection of HeLa cells with WR vaccinia virus does not alter the level of SEPT2, SEPT7, SEPT9, or SEPT11. (D) Representative images of actin tails (green) induced by WR (red labeled with B5 antibody) in HeLa cells treated with Allstar (control) or SEPT7 siRNA. Bar, 2 µm. The graphs show the quantification of the number of CEVs inducing actin tails and their length. Error bars represent SEM from three independent experiments in which a total of 30 cells were analyzed for actin tail number and 370 tails were measured for length. (E) Quantification of the velocity, directionality, time to form, and duration of actin tails in the absence of SEPT7. Error bars represent SEM from three independent experiments in which a total of >150 actin tails were measured for duration, >50 events for tail initiation from four independent experiments, and >300 actin tails for speed and directionality. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: The following primary antibodies were used for immunofluorescence and immunoblotting: A36 , AP-2 (2730; Abcam), Clathrin Heavy Chain (21679; Abcam), B5 ( ; the B5 antibody was labeled with the Molecular Probes Alexa Fluor 488 Monoclonal Antibody Labeling kit [Invitrogen] to generate the Alexa Fluor 488-B5 antibody [ ]), dyn II for immunofluorescence ( ; provided by M. McNiven, Mayo Clinic, Rochester, NY) and Western (A303-513A; Bethyl), FHOD1 (A304-825A; Bethyl), GAPDH (ab8245; Abcam), SEPT2 (60075–1-Ig; ProteinTech), SEPT7 (18991; IBL), SEPT9 (10769–1-AP; ProteinTech) for immunofluorescence and SEPT9 (A302-353A; Bethyl) for Western, and SEPT11 (A304-176A; Bethyl).

Techniques: Western Blot, Immunofluorescence, Infection, Virus, Labeling, Control