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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 blocks infection after entry and but before 2-LTR circle formation.Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of nef. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents the data for replicate experiments (n = 3).
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ppat.1005581.g007: SFN blocks infection after entry and but before 2-LTR circle formation.Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of nef. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents the data for replicate experiments (n = 3).

Mentions: SFN blocks single-round HIV-1 and HIV-2 infections (Fig 4). This limits the Nrf2-mediated block to steps that extend from attachment of the virus to the cell through transcription from the provirus. We found that SFN counters infection whether viral entry is mediated through HIV Env or VSV-G glycoproteins (Figs 1B versus 5E). This suggests a post-entry block as these glycoproteins engage different entry mechanisms. To further test whether SFN blocks after entry, we used real time PCR to quantify reverse transcription products in primary macrophage cultures infected in the presence or absence of SFN. We infected cells treated with vehicle, AZT or SFN with VSV-G-pseudotyped HIV-1 and quantified late reverse transcription products. Cultures exposed to the same quantity of heat-inactivated virus served to control for contamination of the viral stocks with the plasmid carrying the proviral clone used to generate the virus. The vehicle- and SFN-treated samples showed similar levels of late reverse transcription product, indicating that SFN, unlike AZT, did not hinder replication steps from viral entry through reverse transcription (Fig 7A). When we tested for 2-LTR circles, non-productive infection by-products that correlate with the transport of viral preintegration complexes into the cell nucleus, we found that these were clearly decreased in the AZT and SFN samples relative to the vehicle controls (Fig 7B). Alu-PCR, testing for integrated proviral sequences, also showed that these were similarly decreased in AZT- and SFN-treated cultures to levels that were significantly lower than in the vehicle-treated controls (Fig 7C).


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 blocks infection after entry and but before 2-LTR circle formation.Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of nef. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents 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.g007: SFN blocks infection after entry and but before 2-LTR circle formation.Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of nef. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents the data for replicate experiments (n = 3).
Mentions: SFN blocks single-round HIV-1 and HIV-2 infections (Fig 4). This limits the Nrf2-mediated block to steps that extend from attachment of the virus to the cell through transcription from the provirus. We found that SFN counters infection whether viral entry is mediated through HIV Env or VSV-G glycoproteins (Figs 1B versus 5E). This suggests a post-entry block as these glycoproteins engage different entry mechanisms. To further test whether SFN blocks after entry, we used real time PCR to quantify reverse transcription products in primary macrophage cultures infected in the presence or absence of SFN. We infected cells treated with vehicle, AZT or SFN with VSV-G-pseudotyped HIV-1 and quantified late reverse transcription products. Cultures exposed to the same quantity of heat-inactivated virus served to control for contamination of the viral stocks with the plasmid carrying the proviral clone used to generate the virus. The vehicle- and SFN-treated samples showed similar levels of late reverse transcription product, indicating that SFN, unlike AZT, did not hinder replication steps from viral entry through reverse transcription (Fig 7A). When we tested for 2-LTR circles, non-productive infection by-products that correlate with the transport of viral preintegration complexes into the cell nucleus, we found that these were clearly decreased in the AZT and SFN samples relative to the vehicle controls (Fig 7B). Alu-PCR, testing for integrated proviral sequences, also showed that these were similarly decreased in AZT- and SFN-treated cultures to levels that were significantly lower than in the vehicle-treated controls (Fig 7C).

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