Fig. 4.

Us3 is sufficient to control autophagy in uninfected cells. (A) NHDFs were mock infected or infected with HSV-1 ΔUs3, K220A Us3, or Us3-Repair virus (MOI = 5). At 8 hpi, the cells were treated with DMSO or PP242. Total protein was harvested at 12 hpi, fractionated by SDS/PAGE, and analyzed by immunoblotting using the indicated antisera. (B) HeLa cells were transfected with plasmids expressing Flag-Us3 (WT), Flag-K220A Us3 or an empty vector (control). After 24 h, AAs were maintained (+) or removed (−) from the media for 4 h in the presence or absence of PP242. Total protein was subsequently harvested and analyzed by immunoblotting with the indicated antibodies. Migration of unmodified LC3B-I and lipidated LC3B-II are indicated on the Right of the panel. A representative image is shown. (C) LC3B-I and LC3B-II abundance was quantified using Licor Image Studio software and the LC3BII/LC3BI ratio from 5 (n = 5) separate experiments shown in B was plotted. Bars represent mean ± SEM. *P < 0.05; **P ≤ 0.01 by paired t test using the Bonferroni correction for multiple comparisons.