FIG 5.

Knockdown of A3B suppresses deaminase activity during HAdV infection. (A) A3B shRNAs target only A3B expression. mRNA from HBEC3-KT cells stably expressing A3B-targeting shRNAs (A3B shRNAs) or a nontargeting control shRNA (NTC shRNA) or wild-type HBEC3-KT cells (no shRNA) was extracted. The histogram reports A3B, A3C, A3F, and A3G mean mRNA levels expressed relatively to the three housekeeping genes (HKGs). (B to D) HBEC3-KT cells stably expressing A3B-targeting shRNAs or a nontargeting control shRNA or wild-type HBEC3-KT cells were infected with HAdV-A12, -B3, -C2, or mock control. Four days postinfection, A3B mRNA level (B), A3B protein abundance (C), and deaminase activity (D) were assessed. (B) The histogram reports A3B mean mRNA levels, quantified by RT-qPCR and expressed relatively to three housekeeping genes (HKGs). (C) A3B protein levels were assessed by Western blotting and quantified by densitometry. HSP90 was used as loading control. (D) Whole-cell protein extracts were subjected to deamination activity assay. HBEC-A3B was used as positive control (last two lanes). Error bars show standard deviations, and Student’s t test was used to assess significance (NS, nonsignificant, *, P < 0.05, **, P < 0.01.). The experiment was replicated at least 3 times and representative images are shown.