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 enve1ope-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.

Hyperascyrones A–H, polyprenylated spirocyclic acylphloroglucinol derivatives from Hypericum ascyron Linn.

“…Anti-HIV-1 activities of compounds 1-8 were evaluated using a p24 assay (Wan et al, 2012). In the p24 assay, MT-4 cells were infected with HIV-1 LAI at a MOI of 0.01.…”

Validation of Antiviral Potential of Herbal Ethnomedicine

“…Soon after infection, reporter gene expression is induced by a viral transactivator protein Tat. Luciferase activity can be quantified by luminescence and is directly proportional to the number of infectious virus particles present in the initial inoculum [85]. This high-throughput assay requires a 96-well plate, and clonal cells provide enhanced precision and uniformity.…”

“…This high-throughput assay requires a 96-well plate, and clonal cells provide enhanced precision and uniformity. This has been validated for a single-round infection with either uncloned viruses grown in human lymphocytes or molecularly cloned Env-pseudotyped viruses produced by transfection in 293T/17 cells [85].…”

“…The cells then need to be washed twice with PBS, lysed with 1X lysis buffer, and the supernatant added with the substrate can be analyzed for luciferase activity in an optiplate using a fluorimeter. The results are expressed as percentage inhibition as luminescence in the experimental group (test drug or AZT)/luminescence of infected cells without the drug x 100; and percent inhibition can be calculated by subtracting the above value from 100 [44,85].…”

“…The results can be expressed as percentage inhibition: GFP fluorescence in the experimental group/fluorescence in infected cells without the test drug X 100. Percent inhibition should be calculated by subtracting the above value from 100 [85].…”