Figure 4.

IRS1 and TRS1 are involved in the inhibition of autophagy after HCMV infection. (A) MRC5 cells were mock-infected or infected with HCMV, ΔIRS1-ΔTRS1, ΔIRS1 and ΔTRS1 mutant viruses at an MOI of 1, transfected with GFP-LC3 and then fixed at 24 h pi. Cells were fixed and immunostained for pp65 or IEA viral proteins as indicated (scale bar: 10 µm). (B) GFP-LC3 dots were quantified in pp65- or IEA-positive cells. The results are the mean of 6 independent experiments. Twenty-five cells were counted per assay. **, P<0.01; ***, P<0.001 (One-way ANOVA). (C and D) Immunoblot analysis of SQSTM1 and LC3 proteins in mock-infected MRC5 cells or infected with WT HCMV or the indicated mutant viruses for 24 h. (E) Immunoblot analysis of LC3 protein in cells transfected with the indicated constructs for 24 h and grown in starvation medium for 4 h before cell lysis. (D and E) Chloroquine was added 4 h before lysis to monitor autophagic flux.