Fig 3.

Inhibition of autophagic flux requires expression of viral genes. (A) Representative images of MRC5 cells transfected with mRFP-GFP-LC3 plasmid and infected with HCMV or UV-inactivated HCMV. Red and yellow dots indicate GFP⁻ RFP⁺ and GFP⁺ RFP⁺ puncta, respectively. (B) Autolysosomes were quantified by counting GFP⁻ RFP⁺ puncta per cell. The results are the means of three independent experiments. Twenty cells were analyzed per assay. *, P < 0.05 (t test). Amino acid-starved cells (EBSS) were used as a positive control (C) Immunoblot analysis of p62 levels in cells infected with HCMV or UV-inactivated HCMV for the indicated times. Lysosomal inhibitors E64/pepstatin A were added to all conditions, to block the lysosomal degradation. Actin immunoblotting was used as a loading control. (D) Effect of HCMV infection on long-lived protein degradation. [¹⁴C]valine-radiolabeled MRC5 cells were mock infected or infected with HCMV or UV-inactivated HCMV for 8 or 24 h, and proteolysis was measured as described in Materials and Methods. Cells were treated with 200 nM wortmannin (Plus WN) or not (No WN). The results are the means of three independent experiments. *, P < 0.05.