EGCG targets synthetic PAP248–286-derived amyloid fibrils. (A) Amyloid fibrils were formed by agitation of the fresh PAP248–286 solutions (1 and 5 mg/mL) at 37 °C. Fibrillar aggregates were exposed to various concentrations of EGCG (□, 20 mM; ●, 10 mM; ■, 5 mM; and ▲, 1 mM) and detected by Congo red staining at the indicated time points. Addition of PBS alone (solvent for EGCG) served as negative control (×). (B) Preformed PAP248–286-derived fibrils (SEVI) were treated with increasing concentrations of EGCG (▲, 1 mM; △, 2 mM; □, 4 mM; ○, 8 mM; and ●, 10 mM) for 48 h and analyzed as before. Reactions containing EGCG alone (■, 10 mM) or PBS alone (×) served as controls. (C) HIV-1 particles (X4 strain NL4/3) were preincubated for 20 min with or without the indicated concentrations of SEVI. Subsequently, Jurkat 1G5 luciferase indicator cells were exposed to the respective virus/SEVI mixtures for 5 h. At 24 h after infection, RLU/s was determined. Error bars represent three independent experiments. (D) Cellular metabolic activity was tested in uninfected Jurkat 1G5 cells by alamarBlue assay after 5 h of exposure to the indicated concentrations of SEVI. (E) Viabilities of uninfected Jurkat 1G5 cells were determined after 5 h exposure to the indicated EGCG concentrations as before.

Transmission electron microscopy analysis of EGCG-treated SEVI in a closed system. Corresponding images are arranged side by side. (A) Dissolved EGCG is shown inside the ultrathin sectioned microtubes and at different magnifications. The overview shows the interface between the tube wall (arrow) and the tube's lumen filled with EGCG. (Magnification: 3000×.) EGCG aggregates are organized in different patterns. (Magnifications: 35,000× and 75,000×.) (B) The SEVI solution shows a high density of SEVI-specific amyloid fibrils. (Magnification: 3,000×.) Because of the embedding angle, the fibrils often run out of the section plane. (Magnification: 28,000×.) Nevertheless, their length differs between 30 and 90 nm. (C) Appearance of SEVI after 12 h of incubation in EGCG. The small EGCG aggregates diffused from the outside through the porous wall into the lumen of the tube covering the surface of the fibrils. (Magnification: 6,300×.) (D) Degradation of the majority of SEVI after 60 h of incubation in 10 mM EGCG.

The green tea catechin EGCG inhibits SEVI-mediated HIV infection. (A) Jurkat 1G5 cells were infected with HIV-1 NL4/3 in presence of the indicated concentrations of EGCG. The rate of infection was monitored by measuring the luciferase activity in the respective cell cultures (indicated as RLU/s; bars), and cell viabilities were determined by alamarBlue assay (red plot) 24 h after infection. (B) HIV-1 NL4/3 or the multiple antiretroviral drug-resistant virus isolate BE4 was preincubated for 20 min with 50 μg of SEVI and the indicated concentrations of EGCG. Using this mixture, 1G5 cells were subsequently infected for 5 h. At 24 h after infection, luciferase activity was measured as before. (C) Constant amounts (2.5 mM) of EGCG or GC were incubated together with SEVI (5 mg/mL) for the indicated time periods. The degradation of SEVI in each reaction was monitored by Congo red staining (EGCG: black plot; GC: green plot). Subsequently, equal aliquots of each reaction were used to infect 1G5 cells with HIV-1 NL4/3. The respective cultures were assayed as before (EGCG: black bars; GC: green bars).

EGCG abrogates SE-enhanced virus infection. (A) TZM-bl cells were infected with 50 ng of p24 antigen of replication-competent HIV-1 NL4/3 in the absence or presence of the indicated dilutions of a randomly selected human SE sample (SE#1). Noninfected cells and infected SE-untreated cells served as controls. Infectivity and cell viability (red plot) was determined essentially as before. (B) Six different SE samples (SE#s 1–6), diluted 1:4 in PBS, were analyzed in parallel as before. Experiments were performed in triplicate. (C) HIV-1 NL4/3 was exposed for 20 min to individual SE samples (SE#s 1 and 2) (1:4 dilution) in the presence or absence of the indicated EGCG concentrations. Subsequently, these mixtures were used for infection of TZM-bl cell cultures. Luciferase activity and cell viability (red plot) were determined at 24 h after infection as before. Error bars represent three independent experiments.