Figure 1: Production of the tiger frog virus (TFV) was inhibited by dominant-negative (DN) and RNA interference of VPS4A.

(A) HepG2 cells were transfected with 0.1, 0.2, 0.4, or 0.8 μg of pVPS4A-DN-myc. Protein samples were collected and analyzed by Western blot, and GAPDH was included as control. (B) HepG2 cells were infected with TFV for 1 h after transfection with 0.8 μg of pVPS4A-DN-myc or pCMV-myc and then cultured for 66 h. Cells and supernatant were lysed to extract DNA. Samples were then subjected to absolute real-time quantitative PCR (qPCR) using major capsid protein (MCP) primers to quantify TFV genomic concentration. Statistical analysis was performed using Student’s t-test (0.05 < *p ≤ 0.1, 0.01 < **p ≤ 0.05, 0.01 ≤ ***p). (C) 100 nM of siRNA specific for VPS4A and control RNA (siGFP) were transfected into cells. The cells were collected from 24 h to 72 h and subjected to Western blot. (D) 100 nM siVPS4A or 100 nM siGFP were transfected into cells 6 h prior to infection with TFV for 1 h and then cultured for 66 h. Cells and supernatant were lysed to extract DNA. Samples were then subjected to qPCR using MCP primers to quantify TFV genomic concentration. Statistical analysis was performed using Student’s t-test (0.05 < *p ≤ 0.1, 0.01 < **p ≤ 0.05, 0.01 ≤ ***p). (E) EM analysis of HepG2 cells transfected with 100 nM siVPS4A or siGFP prior to infection with TFV for 66 h. Arrows indicated virions tethered to the plasma membrane.