Fangchinoline reduces functional Env expression by interfering with gp160 processing.

<p>(A) HIV-1 NL4-3-infected MT-4 cells were pretreated with 1 µM IDV alone (left panel) or 1 µM IDV plus 2.5 µM fangchinoline (right panel) for 24 hours and then cocultured with C8166 cells. After 24 hours of cocultivation, syncytium formation was examined using light microscopy. (B) 293T cells were transfected with pNL4-3 and treated with the indicated concentration of fangchinoline. The Env protein expression in transfected cells was analyzed by Western blot using a polyclonal antibody directed against HIV-1 gp120 48 hours post-transfection. (C) 293T cells were transfected with pNL4-3. At 3 hours post-transfection, the cell supernatant was removed and fresh medium with or without 10 µM fangchinoline was added. After 24 hours, the cells were labeled for 1 h and chased for indicated times in the presence or absence of fangchinoline. After biotinylated, the newly synthesized protein was collected with streptavidin-coupled magnetic beads. Processing of newly synthesized gp160 was examined by Western blot analysis.</p

Fangchinoline inhibits HIV-1 NL4-3 replication in MT-4 cells.

<p>(A and B) MT-4 cells were infected with HIV-1 NL4-3 at an MOI of 0.02 or mock infected in the presence of serially diluted fangchinoline. On day 4 post-infection, compound cytotoxicity was determined in parallel in mock-infected cells (A), and the level of viral replication in infected cells was determined by p24 antigen capture ELISA (B). The results were presented as the mean values with standard deviations. (C and D) MT-4 cells were infected with HIV-1 NL4-3 at an MOI of 0.02 in the presence of test compounds (AZT, 0.05 µM; fangchinoline, 0.3–2.5 µM), and genomic DNA and mRNA from infected cells were isolated 3 days post-infection. Total viral DNA (C, upper panel), integrated proviral DNA (C, middle panel) were examined by semi-quantitative PCR analysis. Viral mRNA levels were examined by semi-quantitative reverse transcription PCR analysis (D, upper panel). GAPDH was used as both the DNA and RNA input control.</p

Screen of compounds derived from Chinese herbal remedies led to the identification of fangchinoline as a novel anti-HIV-1 agent.

<p>(A) MT-4 cells were infected with HIV-1 LAI at an MOI of 0.1 or mock infected in the presence of serially diluted test compounds. Cell viability was measured 5 days post-infection. The anti-HIV activity of each compound was presented as the maximum inhibition of CPE at various concentrations. (B) The chemical structure of fangchinoline.</p

Anti-HIV activities of test compounds in MT-4 CPE assays and TZM-b1 assays.

<p>Antiviral assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039225#s2" target="_blank">Materials and Methods</a>. Values represent average of at least three independent experiments.</p>a<p>Compound concentration required to reduce the virus-induced CPE by 50%.</p>b<p>Compound concentration required to reduce the luciferase expression in virus infected TZM-b1 cells by 50%.</p

Fangchinoline specifically reduces the incorporation of HIV-1 Env into nascent virus particles.

<p>(A) 293T cells were co-transfected with pSG3^ΔEnv and an envelope expressing plasmid. At 3 hours post-transfection, the cell supernatant was removed, and fresh medium with test compounds (IDV, 1 µM; fangchinoline, 1.3–10 µM) was added. At 48 hours after transfection, the infectivity of the virions produced by the co-transfected cells was determined in the TZM-b1 assay. (B) 293T cells were transfected with pNL4-3. At 3 hours post-transfection, the cell supernatant was removed, and fresh medium with indicated concentration of fangchinoline was added. At 48 hours after transfection, the content of Env in the HIV-1 particles produced by the pNL4-3 transfected 293T cells was analyzed by Western blot. (C) 293T cells were cotransfected with pSG3^Δenv and pVpack-VSV-G. At 48 hours after transfection, the content of VSV-G in the pseudotyped HIV-1 particles produced in the presence or absence of fangchinoline was analyzed by Western blot.</p

Fangchinoline inhibits the production of infectious HIV-1 particles.

<p>(A and B) 293T cells were transfected with the HIV-1 infectious clone pNL4-3. At 3 hours post-transfection, the cell supernatant was removed, and fresh medium with test compounds (AZT, 0.05 µM; IDV, 1 µM; fangchinoline, 0.6–10 µM) was added. At 48 hours after transfection, viral release, viral infectivity and the cell viability of 293T were examined, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039225#s2" target="_blank">Materials and Methods</a>. (C)The Gag protein products derived from virions produced from pNL4-3 transfected 293T cells in the presence of test compounds (AZT, 0.05 µM; IDV, 0.25 µM; fangchinoline, 1.3–10 µM) were examined by Western blot analysis.</p

<i>In vitro</i> antiviral activities of fangchinoline against different HIV-1 laboratory strains.

<p>Antiviral assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039225#s2" target="_blank">Materials and Methods</a>. Values represent average of at least three independent experiments.</p>a<p>Compound concentration required to reduce the production of p24 antigen by 50%.</p>b<p>Compound concentration required to reduce mock-infected cell viability by 50%.</p>c<p>Selective index, ratio of CC₅₀/EC₅₀.</p