Fig 3.

The PP1α but not the beclin-1-binding domain of γ34.5 is required for NEDA. (A) HSV-1 γ34.5 mutants. The γ34.5 protein contains a beclin-1-binding domain (amino acids [aa] 68 to 87), a PP1α binding domain (aa 175 to 188), and an eIF2α binding domain (aa 233 to 248). Aa 68 to 87 are deleted in the HSV-1 Δbeclin-1 virus, and in the HSV-1 ΔPP1α virus, two point mutations inhibit PP1α binding. (B to E) BMA cells were infected with the HSV-1 wt, Δγ34.5, Δbeclin-1, or ΔPP1α virus for 8 h at an MOI of 5. (B) Immunoblot analysis with antibodies against total eIF2α and phosphorylated eIF2α (eIF2α-P). (C) Immunofluorescence with an antibody against cleaved LC3a. (D) Quantification of the number of cells in which LC3a colocalized with the nuclear envelope in immunofluorescence. About 150 cells per condition in three experiments. Error bars, SD; ***, P < 0.001 in a two-sided Student t test. (E) Flow cytometry analysis of LC3a labeling intensity in cells infected with the four viruses.