Figure 1.

Alternariol 5-O-methyl ether (AME) potentially inhibits early stage of human immunodeficiency virus (HIV)-1 replication. (A) The structure of AME was solved through spectroscopic analysis; (B) The structure of alternariol (AOH); (C) SupT1 cells were infected with HIV-1 NL4-3luc.R-E- pseudoviruses in the presence of different concentrations of AME. 48 h post-infection, cells were lysed and luciferase activity was measured. A dose-response curve was generated using the Origin 8.0 software; (D) SupT1 cells were infected with HIV-1 NL4-3luc.R-E- pseudoviruses before or after treatment with either AME or T-20 at 2 fold IC50. Luciferase activity was measured before (B) or after (A) viral infection. Relative luciferase activity was a ratio of the luciferase activity in cells infected before AME (or T20) treatment over that in cells infected after drug treatment (set as 1); (E) A time of addition (TOA) experiment was carried out to determine the step of HIV-1 infection that AME inhibits. Briefly, after infection of SupT1 cells with HIV-1 NL4-3luc.R-E- pseudoviruses, inhibitors of the indicated concentrations were added at different time points ranging from 1 to 24 h post-infection. The relative inhibition rate was calculated by dividing the inhibition rate at 0 h for each compound. White diamond: AME; black diamond: efavirenz (EFV); triangle: 3TC; rectangular: Raltegravir (RAL); cross: AOH. Viral infection was determined by measuring luciferase activity at 48 h post-infection. As controls, reverse transcriptase (RT) inhibitors EFV and 3TC, integrase strand transfer inhibitors (INSTIs) RAL, as well as inactive congener AOH were also tested; (F) HEK293T cells were transfected with pNL4-3luc.R-E-. Forty-eight h post-transfection, cells were lysed, and Gag expression was determined by Western blotting. Data represent the mean ± SD of three independent experiments.