FIG 11.
Inhibition of the ISR prevents the coalescence of HRD1 and UL148 into discrete structures during infection. (A) Representative 3D maximum intensity projections of confocal imaging Z-stacks obtained from cells infected at an MOI of 1 for 96 h with HCMV strain TB40/E carrying an HA-tagged UL148, TB_148HA, and maintained in the presence of ISRIB (200 nM), GSK2606414 (1.1 μM), or DMSO carrier alone (0.1% [vol/vol]). In merged images, HA signal is shown in magenta, HRD1 in green, and DAPI counterstaining in blue. (B) The percentages of HRD1 antibody signals involved in discrete structures at 96 hpi were calculated for a minimum of 30 cells per condition using Imaris x64 9.3.0 software. Statistical significance was determined using a one-way ANOVA followed by Tukey’s posttest; ****, P < 0.0001. The arithmetic mean for colocalization analysis results are shown as blue lines with data points for individual cells analyzed plotted as circles, squares, or triangles, as indicated. (C and D) Western blot analyses of fibroblasts infected at an MOI of 1 with TB_148HA and maintained in the presence of ISRIB (200 nM), GSK2606414 (1.1 μM), or DMSO carrier alone (0.01%); hpi, h postinfection. Note that a phospho-specific antibody was used for detection of eIF2α phosphorylated at Ser51 (eIF2α-P).