FIG 6.

pUL38 antagonizes the function of USP24 and prevents ferritin degradation in lysosomes. (A) MRC5 cells were transduced with lentivirus expressing control shRNA (shc) or USP24-specific shRNA (shUSP24-1) and then mock infected or infected with wild type (wt) or pUL38-deficient (mut) HCMV at an MOI of 3. Cell lysates were collected at the indicated time points after virus infection and analyzed by immunoblotting with the indicated antibodies. (B) MRC5 cells were transduced with empty vector (vector), or with pUL38- or pUL38 TFV/AAA-expressing lentivirus and then mock infected or infected with wild type (wt) or pUL38-deficient (mut) HCMV at an MOI of 3. Cell lysates were collected at 72 hpi and analyzed by immunoblotting with the indicated antibodies. (C) MRC5 cells were transduced with mCherry-FTH1- and shc/shUSP24-1-expressing lentiviruses and then infected with wild-type (wt) or pUL38-deficient (mut) HCMV at an MOI of 0.3. Cells were fixed at 96 hpi and stained with the indicated antibodies. Images were taken using an Olympus FV1200 laser scanning microscope. (D) Colocalization of mCherrry-FTH1 and LAMP1 was assessed using ImageJ. Data shown represent the mean ± SD for nine cells. ***, P < 0.001; ns, not significant. (E) RNAi knockdown of USP24 reduces NCOA4 protein stability. MRC5 cells were transduced with shc/shUSP24-1-expressing lentivirus and then treated with 100 μg/ml cycloheximide (CHX). Cell lysates were collected at the indicated time points and analyzed by immunoblotting. (F) Quantification of NCOA4 protein levels in two independent CHX chase experiments. Data shown represent the mean ± SD from two independent experiments.