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Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2.

Furuya AK, Sharifi HJ, Jellinger RM, Cristofano P, Shi B, de Noronha CM - PLoS Pathog. (2016)

Bottom Line: We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles.Interestingly however, neither SAMHD1 nor MX2 were upregulated.This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America.

ABSTRACT
Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

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SFN action blocks spreading infections that rely on the HIV envelope for viral entry.hMDMs were pretreated with vehicle (DMSO), 5 μM AZT, or 10 μM SFN. All cultures were subsequently maintained in their respective treatments for the duration of the experiment. Twenty four hours after initial treatment, the cultures were infected with the HIV-1 clinical isolate 89.6. Culture supernatants were collected 3, 6, 9, and 14 days after infection. (A), Western blots and (B), p24 antigen ELISA assays of viral supernatants were performed. (C), Fourteen days after infection, cells were imaged using phase contrast microscopy. (D), Uninfected replicate cultures were maintained in the presence of vehicle (DMSO) or in 10 μM SFN. After 14 days of treatment, the viability of each cell type was assessed under each condition by measuring water-soluble tetrazolium salt (WST-8) formazan reagent cleavage by cellular dehydrogenases. Continuous treatment of cells with 10 μg/ml of the eukaryotic toxin blasticidin served as a positive control to demonstrate loss of viability. (E), hMDMs were infected with 89.6-Env-pseudotyped HIV-1 encoding firefly luciferase in place of nef. The bar graphs represent the data for replicate experiments (n = 3).
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ppat.1005581.g005: SFN action blocks spreading infections that rely on the HIV envelope for viral entry.hMDMs were pretreated with vehicle (DMSO), 5 μM AZT, or 10 μM SFN. All cultures were subsequently maintained in their respective treatments for the duration of the experiment. Twenty four hours after initial treatment, the cultures were infected with the HIV-1 clinical isolate 89.6. Culture supernatants were collected 3, 6, 9, and 14 days after infection. (A), Western blots and (B), p24 antigen ELISA assays of viral supernatants were performed. (C), Fourteen days after infection, cells were imaged using phase contrast microscopy. (D), Uninfected replicate cultures were maintained in the presence of vehicle (DMSO) or in 10 μM SFN. After 14 days of treatment, the viability of each cell type was assessed under each condition by measuring water-soluble tetrazolium salt (WST-8) formazan reagent cleavage by cellular dehydrogenases. Continuous treatment of cells with 10 μg/ml of the eukaryotic toxin blasticidin served as a positive control to demonstrate loss of viability. (E), hMDMs were infected with 89.6-Env-pseudotyped HIV-1 encoding firefly luciferase in place of nef. The bar graphs represent the data for replicate experiments (n = 3).

Mentions: We exploited the characteristics of VSV-G-pseudotyped, env(‒) reporter viruses to determine that SFN pretreatment blocks infection at or before reporter production from integrated proviruses. Here we tested the impact of continuous SFN treatment on infections with the replication competent, reporter-free, dual-tropic HIV-1 strain HIV-1 89.6 [43]. Primary hMDMs were cultured in media containing DMSO, the vehicle for SFN, 5 μM AZT or 10 μM SFN. Culture supernatant samples were collected 3, 6, 9 and 14 days after infection and tested to determine the concentration of viral capsid protein, p24. Both western blotting and antigen-capture ELISA were employed. The concentration of viral capsid in the cell supernatant increased steadily in the vehicle control, however in the AZT- and 10 μM SFN-treated samples it decreased from residual input levels to background levels (Fig 5A and 5B). The HIV-1 89.6 Env glycoprotein can mediate fusion between adjacent macrophages, making inhibition of virus replication by SFN and AZT readily apparent in the cell cultures (Fig 5C).


Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2.

Furuya AK, Sharifi HJ, Jellinger RM, Cristofano P, Shi B, de Noronha CM - PLoS Pathog. (2016)

SFN action blocks spreading infections that rely on the HIV envelope for viral entry.hMDMs were pretreated with vehicle (DMSO), 5 μM AZT, or 10 μM SFN. All cultures were subsequently maintained in their respective treatments for the duration of the experiment. Twenty four hours after initial treatment, the cultures were infected with the HIV-1 clinical isolate 89.6. Culture supernatants were collected 3, 6, 9, and 14 days after infection. (A), Western blots and (B), p24 antigen ELISA assays of viral supernatants were performed. (C), Fourteen days after infection, cells were imaged using phase contrast microscopy. (D), Uninfected replicate cultures were maintained in the presence of vehicle (DMSO) or in 10 μM SFN. After 14 days of treatment, the viability of each cell type was assessed under each condition by measuring water-soluble tetrazolium salt (WST-8) formazan reagent cleavage by cellular dehydrogenases. Continuous treatment of cells with 10 μg/ml of the eukaryotic toxin blasticidin served as a positive control to demonstrate loss of viability. (E), hMDMs were infected with 89.6-Env-pseudotyped HIV-1 encoding firefly luciferase in place of nef. The bar graphs represent the data for replicate experiments (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836681&req=5

ppat.1005581.g005: SFN action blocks spreading infections that rely on the HIV envelope for viral entry.hMDMs were pretreated with vehicle (DMSO), 5 μM AZT, or 10 μM SFN. All cultures were subsequently maintained in their respective treatments for the duration of the experiment. Twenty four hours after initial treatment, the cultures were infected with the HIV-1 clinical isolate 89.6. Culture supernatants were collected 3, 6, 9, and 14 days after infection. (A), Western blots and (B), p24 antigen ELISA assays of viral supernatants were performed. (C), Fourteen days after infection, cells were imaged using phase contrast microscopy. (D), Uninfected replicate cultures were maintained in the presence of vehicle (DMSO) or in 10 μM SFN. After 14 days of treatment, the viability of each cell type was assessed under each condition by measuring water-soluble tetrazolium salt (WST-8) formazan reagent cleavage by cellular dehydrogenases. Continuous treatment of cells with 10 μg/ml of the eukaryotic toxin blasticidin served as a positive control to demonstrate loss of viability. (E), hMDMs were infected with 89.6-Env-pseudotyped HIV-1 encoding firefly luciferase in place of nef. The bar graphs represent the data for replicate experiments (n = 3).
Mentions: We exploited the characteristics of VSV-G-pseudotyped, env(‒) reporter viruses to determine that SFN pretreatment blocks infection at or before reporter production from integrated proviruses. Here we tested the impact of continuous SFN treatment on infections with the replication competent, reporter-free, dual-tropic HIV-1 strain HIV-1 89.6 [43]. Primary hMDMs were cultured in media containing DMSO, the vehicle for SFN, 5 μM AZT or 10 μM SFN. Culture supernatant samples were collected 3, 6, 9 and 14 days after infection and tested to determine the concentration of viral capsid protein, p24. Both western blotting and antigen-capture ELISA were employed. The concentration of viral capsid in the cell supernatant increased steadily in the vehicle control, however in the AZT- and 10 μM SFN-treated samples it decreased from residual input levels to background levels (Fig 5A and 5B). The HIV-1 89.6 Env glycoprotein can mediate fusion between adjacent macrophages, making inhibition of virus replication by SFN and AZT readily apparent in the cell cultures (Fig 5C).

Bottom Line: We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles.Interestingly however, neither SAMHD1 nor MX2 were upregulated.This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America.

ABSTRACT
Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

Show MeSH
Related in: MedlinePlus