Figure 3

MG132 suppressed virus production, viral DNA replication and lytic genes expression. (a–e) Suppression of extracellular HSV-1 production by MG132. Vero (a), HepG2 (b), H1299 (c), ME180 (d) and MCF7 (e) were infected with HSV-1 at an MOI of 1 for 30 min. Cells were cultured with 0.75 μM MG132 (Vero and HepG2), 0.3 μM MG132 (H1299) or 0.25 μM MG132 (ME180 and MCF7) and 10 μM ACV for 24 hours, except for Vero cells. Vero cells were cultured with drug for 12 hours. The culture supernatants containing virus were harvested, and virus DNA were purified from culture supernatants and measured by qPCR. (f) The effect of MG132 on intracellular HSV-1 DNA replication. HepG2 cells were infected with HSV-1 at an MOI of 1 for 30 min and subsequently cultured with the drug for 43 hours. Viral DNA were isolated from cells and quantified by qPCR using UL19 specific primers. (g,h) Suppression of lytic genes transcription by MG132. HepG2 were infected with HSV-1 for 30 min and cultured with 0.75 μM MG132 for 20 hours, Total RNA was purified from cells and subjected to RT-qPCR to measure mRNA expression of Us12 (IE/α gene) and UL19 (L/γ gene). The mRNA expression of Us12 and UL19 were normalized by GAPDH mRNA expression. (a–h) Standard deviations were determined by analyzing the data from three experiments. ^**P < 0.01 and ^***P < 0.001 indicate the statistical significance compared with vehicle (DMSO) treated cells. N.S. indicates not statistically significant. (i) Analysis of viral replication cycle by one-step growth curve in the presence of MG132. HepG2 cells were infected with HSV-1 at 1 PFU/cell for 30 min and cultured with vehicle (DMSO), 0.75 μM MG132 or 10 μM ACV. The culture media was harvested at 0, 6, 12, 24 and 48 hours postinfection, and amount of virus in culture media was measured by a plaque reduction assay using Vero cells. The black square, white square and gray triangle indicate DMSO-, MG132- and ACV-treatment, respectively.