Figure 5. Enhanced WSSV entry mediated by rCq-GABARAP was dependent on CME and positively correlated with autophagy.
(A) WSSV entry promoted by rCq-GABARAP in a CME-dependent manner. (a) Hpt cells were pretreated with CPZ (2.5 μM), MβCD (500 μM), rottlerin (1 μM) or solvent controls for 30 min followed by infection accordingly with WSSV pre-incubated with rCq-GABARAP. The cells were then harvested for immunoblotting against the viral envelope protein VP28 at 1 hpi (lower panel). The band intensities of three independent experiments were calculated using the Quantity One program (upper panel). (b) Hpt cells were pretreated with dsRNA of Cq-CLC, Cq-AP50 or GFP followed by infection with WSSV pre-incubated with rCq-GABARAP accordingly and then harvested for immunoblotting against VP28 at 1 hpi (lower panel). The band intensities of three independent experiments were calculated using the Quantity One program (upper panel). (B) Promotion of rCq-GABARAP-mediated WSSV entry by gene knockdown of Cq-Rac1. The RNAi efficiency of Cq-Rac1 was examined by qRT-PCR (upper panel). WSSV entry was examined by immunoblotting against the viral envelope protein VP28 (lower panel). (C) rCq-GABARAP-mediated WSSV entry positively correlated with cellular autophagy activity. The cellular autophagy activity was changed by pretreating Hpt cell with an autophagy inducer (rapamycin, 50 nM) or inhibitor (L-Asn, 30 mM). WSSV entry was assessed by immunoblotting against the viral envelope protein VP28. The data are presented as the mean ± SEM from at least three independent experiments and were analyzed using Student’s t test (*P < 0.05).