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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 acts through Nrf2 to block HIV infection in macrophages.PMA-differentiated THP1 cells were pretreated for twenty-four hours with the Nrf2 activators: (A), SFN, (B), DMF, or (C), EGCG at the indicated concentrations. Pretreatment of cultures with 5 μM of the reverse transcription inhibitor zidovudine (AZT) served as a positive control for viral inhibition. Twenty-four hours after treatment, cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the data for replicate experiments (n = 3). (D), Cultures of hMDMs were transfected with either a non-targeting siRNA (control) or siRNA specific for Nrf2 mRNA. siRNA transfected hMDMs were either treated with vehicle (DMSO) or with 10 μM SFN. Twenty-four hours after treatment, the cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the quantified data for replicate experiments (n = 3). (E) and (F), Representative samples from (D) were lysed and proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. Densitometric analysis was performed on the Nrf2 and NQO1 (an indicator of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the Nrf2 and NQO1 bands were then graphed. The data shown is representative of n = 3.
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ppat.1005581.g006: SFN acts through Nrf2 to block HIV infection in macrophages.PMA-differentiated THP1 cells were pretreated for twenty-four hours with the Nrf2 activators: (A), SFN, (B), DMF, or (C), EGCG at the indicated concentrations. Pretreatment of cultures with 5 μM of the reverse transcription inhibitor zidovudine (AZT) served as a positive control for viral inhibition. Twenty-four hours after treatment, cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the data for replicate experiments (n = 3). (D), Cultures of hMDMs were transfected with either a non-targeting siRNA (control) or siRNA specific for Nrf2 mRNA. siRNA transfected hMDMs were either treated with vehicle (DMSO) or with 10 μM SFN. Twenty-four hours after treatment, the cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the quantified data for replicate experiments (n = 3). (E) and (F), Representative samples from (D) were lysed and proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. Densitometric analysis was performed on the Nrf2 and NQO1 (an indicator of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the Nrf2 and NQO1 bands were then graphed. The data shown is representative of n = 3.

Mentions: We found that like SFN, DMF and EGCG hinder reporter virus expression in single-round HIV-1 infections of PMA-differentiated THP1 cells and exhibit minimal toxicity (Fig 6A–6C and S2A–S2C Fig). This extends the previous work with DMF [40] by showing that the block is at a replication step at or before transcription from the provirus. Our observation that EGCG blocked VSV-G-pseudotyped virus eliminates the possibility that EGCG is thwarting a specific gp120/CD4 interaction in our system [48]. While we did not see SFN-mediated infection inhibition in HeLa cells (Fig 2A) we tested whether reporter expression from the provirus is inhibited by EGCG or the other compounds, as this could reflect inhibition of Tat function or transcription from the viral promoter. PMA-differentiated THP1 cells were pre-treated with AZT, DMSO, SFN, DMF or EGCG and then infected with VSV-G-pseudotyped luciferase reporter-encoding HIV-1 (S3A Fig). Replicate cultures were infected with the same reporter virus and, 5 days later, treated for twenty-four hours with DMSO, AZT, SFN, DMF or EGCG. While pretreatment with AZT, SFN, DMF or EGCG significantly inhibited luciferase activity, treatment after infection had much less or no impact. This suggests that each compound is likely acting before transcription in THP-1 infections, although we cannot rule out some impact after integration, especially for SFN. Neither DMF nor EGCG suppressed reporter activity to the same degree as SFN in pretreated cultures, perhaps because SFN is one of the most potent naturally occurring Nrf2 activators [49–52] or because SFN may also have an additional, albeit smaller, impact after proviral integration. These observations prompted us to test directly whether SFN blocks HIV-1 through Nrf2.


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 acts through Nrf2 to block HIV infection in macrophages.PMA-differentiated THP1 cells were pretreated for twenty-four hours with the Nrf2 activators: (A), SFN, (B), DMF, or (C), EGCG at the indicated concentrations. Pretreatment of cultures with 5 μM of the reverse transcription inhibitor zidovudine (AZT) served as a positive control for viral inhibition. Twenty-four hours after treatment, cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the data for replicate experiments (n = 3). (D), Cultures of hMDMs were transfected with either a non-targeting siRNA (control) or siRNA specific for Nrf2 mRNA. siRNA transfected hMDMs were either treated with vehicle (DMSO) or with 10 μM SFN. Twenty-four hours after treatment, the cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the quantified data for replicate experiments (n = 3). (E) and (F), Representative samples from (D) were lysed and proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. Densitometric analysis was performed on the Nrf2 and NQO1 (an indicator of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the Nrf2 and NQO1 bands were then graphed. The data shown is representative of n = 3.
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ppat.1005581.g006: SFN acts through Nrf2 to block HIV infection in macrophages.PMA-differentiated THP1 cells were pretreated for twenty-four hours with the Nrf2 activators: (A), SFN, (B), DMF, or (C), EGCG at the indicated concentrations. Pretreatment of cultures with 5 μM of the reverse transcription inhibitor zidovudine (AZT) served as a positive control for viral inhibition. Twenty-four hours after treatment, cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the data for replicate experiments (n = 3). (D), Cultures of hMDMs were transfected with either a non-targeting siRNA (control) or siRNA specific for Nrf2 mRNA. siRNA transfected hMDMs were either treated with vehicle (DMSO) or with 10 μM SFN. Twenty-four hours after treatment, the cells were either mock infected or infected with VSV-G-pseudotyped HIV-1 encoding firefly luciferase in place of nef. Twenty-four hours after infection, the cells were harvested and luciferase activity was measured by photon emission. The bar graphs represent the quantified data for replicate experiments (n = 3). (E) and (F), Representative samples from (D) were lysed and proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. Densitometric analysis was performed on the Nrf2 and NQO1 (an indicator of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the Nrf2 and NQO1 bands were then graphed. The data shown is representative of n = 3.
Mentions: We found that like SFN, DMF and EGCG hinder reporter virus expression in single-round HIV-1 infections of PMA-differentiated THP1 cells and exhibit minimal toxicity (Fig 6A–6C and S2A–S2C Fig). This extends the previous work with DMF [40] by showing that the block is at a replication step at or before transcription from the provirus. Our observation that EGCG blocked VSV-G-pseudotyped virus eliminates the possibility that EGCG is thwarting a specific gp120/CD4 interaction in our system [48]. While we did not see SFN-mediated infection inhibition in HeLa cells (Fig 2A) we tested whether reporter expression from the provirus is inhibited by EGCG or the other compounds, as this could reflect inhibition of Tat function or transcription from the viral promoter. PMA-differentiated THP1 cells were pre-treated with AZT, DMSO, SFN, DMF or EGCG and then infected with VSV-G-pseudotyped luciferase reporter-encoding HIV-1 (S3A Fig). Replicate cultures were infected with the same reporter virus and, 5 days later, treated for twenty-four hours with DMSO, AZT, SFN, DMF or EGCG. While pretreatment with AZT, SFN, DMF or EGCG significantly inhibited luciferase activity, treatment after infection had much less or no impact. This suggests that each compound is likely acting before transcription in THP-1 infections, although we cannot rule out some impact after integration, especially for SFN. Neither DMF nor EGCG suppressed reporter activity to the same degree as SFN in pretreated cultures, perhaps because SFN is one of the most potent naturally occurring Nrf2 activators [49–52] or because SFN may also have an additional, albeit smaller, impact after proviral integration. These observations prompted us to test directly whether SFN blocks HIV-1 through Nrf2.

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