Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing

Abstract

The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline). Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.

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

(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.

Figure 2. Fangchinoline inhibits HIV-1 NL4-3 replication in MT-4 cells.

(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.

Figure 3. Fangchinoline inhibits the production of infectious HIV-1 particles.

(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 Materials and Methods. (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.

Figure 4. Fangchinoline specifically reduces the incorporation of HIV-1 Env into nascent virus particles.

(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.

Figure 5. Fangchinoline reduces functional Env expression by interfering with gp160 processing.

(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.