Transmission electron microscopic analysis of the fibrillar shape of PAP248-286.

<p>2 mg/ml PAP248-286 was incubated in the presence or absence of 30 µg/ml polyanions. The amyloid fibril shapes were visualized by negative-stain EM at different time points (12 h, 36 h, 60 h). Images are shown relative to a scale bar of 500 nm (magnification: 18,500×).</p

Chemical structures of polyanion-based microbicides and GAGs.

<p>Chemical structures of the molecules include cellulose sulfate (A), λ-carrageenan (B), CAP (C), polystyrene sulfonate (D), PRO 2000 (E), heparin sulfate (F) and methyl cellulose (G).</p

Interaction between PAP248-286 and polyanionic candidate microbicides.

<p>300 µg/ml PAP248-286 was incubated with various polyanions at graded concentration and then the remaining peptides in the supernatants were electrophoresed on 10% native polyacrylamide continuous gels. Gels were either stained with Coomassie Blue (A) or subjected to immunoblotting with anti-PAP248-286 polyclonal antibody (B). The concentrations of polyanions were 0, 300, 150, 75, 37.5, 18.75, 9.375, 4.7 µg/ml (from lane 1 to lane 8), respectively.</p

Schematic presentation of drug-protein interaction that result in the poor efficacy of polyanionic candidate microbicides in clinical trials.

<p>Prior to the sexual intercourse, candidate microbicide was intravaginally applied and these anionic macromolecules remained on the vaginal surface rather than distributed in the inner tissue. When the viruses arrived, polyanions neutralized the viruses and stopped HIV at the gate (I). However, during sexual intercourse, viruses were delivered in semen, in which cationic peptides/proteins competitively interacted with polyanions so that there might not be sufficient polyanions to interfere with HIV-1 infection (II). Even worse, the interaction between polyanions and PAP derived fragments led to a facilitated process of amyloid fibril formation. The accelerated amyloid fibril formation increased the risk of HIV sexual transmission (III). Green particles, polyanionic molecules, purple particles, PAP derived fragments, orange particles, non-PAP derived cationic peptides/proteins.</p

A brief schematic of the assays for testing the effect of polyanions on HIV-1 infection in the presence of PAP248-286 or SE-F.

<p>Polyanions at graded concentration was mixed with an indicated concentration of PAP248-286 or SE-F at an indicated interval with agitation as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059777#s2" target="_blank">Materials and Methods</a>”. After centrifugation, the supernatants were collected for testing the inhibitory activity of the unbounded polyanions on HIV-1 infection. The pellets containing the PAP248-286 (SEVI) - or SE-F-derived amyloid fibrils were re-suspended in 100 µl medium for testing the enhancing effect on HIV-1 infection.</p

Enhancement of HIV-1 infection of SEVI in the presence of polyanions.

<p>Fibril formation of PAP248-286 was promoted by agitation in the presence of polyanions (30 µg/ml) or PBS for 60 h. PAP248-286 at different concentration were mixed with R5 tropic (A) and X4 tropic viruses (B), respectively. The mixtures were then added to TZM-b1 cells. After culture at 37°C for 3 h, the medium was replaced. Luciferase activity was detected 72 h later. Experiments were repeated once and similar results were obtained. Shown are average values (± standard deviations) of triplicate measurements of a representative experiment.</p

Time courses of PAP248-286 aggregation in the absence or presence of polyanionic candidate microbicides.

<p>2 mg/ml PAP248-286 was agitated at 37°C and 1200 rpm in the presence or absence of various polyanions (30 µg/ml). The status of peptide aggregation is monitored by Thioflavin T staining (A) or Congo red staining (B). The facilitation of the fibrillogenesis of PAP248-286 mediated by cellulose sulfate is dose-dependant as revealed by Thioflavin T staining (C) and Congo red staining (D). The data presented were the median values obtained from one experiment performed in triplicate. Experiments were repeated once that yielded similar trends.</p

Enhancement of HIV-1 infection of SE-F in the presence of polyanions.

<p>SE-F or PBS (as a control) was agitated with various polyanions at graded concentration for 5 h. The pellet portion of the mixture was mixed with R5 tropic (A) and X4 tropic viruses (B), respectively. The mixtures were added to TZM-b1 cells. After culture at 37°C for 3 h, the medium was replaced. Luciferase activity was detected 72 h later. The experiment was repeated once and similar results were obtained. Shown are average values (± standard deviations) of triplicate measurements of a representative experiment. *vs. HIV-1 in SE, <i>P</i><0.05 by <i>t</i>-test.</p

No significant cytotoxicity of SE-F and polyanions at the concentrations used in the present study.

<p>(<b>A</b>) Cytotoxicity of SE-F to different target cells. Seminal fluids (1∶500) and seminal pellet (1∶200) were incubated with TZM-bl cells or MT-2 cells respectively. After 48-h culture, cytotoxicity was determined by the XTT cell viability assay. The results were expressed as the mean of three different experiments. (<b>B</b>) Cytotoxicity of polyanions to different target cells. Various polyanions (50 µg/ml) were incubated with TZM-bl cells or MT-2 cells respectively. After 48-h culture, cytotoxicity was determined by the XTT cell viability assay. The results were expressed as the mean of three different experiments.</p

Effect of PAP248-286 on the antiviral activity of polyanionic candidate microbicides against laboratory-adapted HIV-1 strains^a.

a<p>IC₅₀s are means ± standard deviations (n = 3).</p