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Title: Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2}
Article:
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.
Introduction:
Highly active anti-retroviral treatment( HAART) is saving countless lives, however its application is accompanied by high financial costs, the emergence of resistant viruses and short- and long-term side-effects. A better understanding of how to activate cellular anti-viral defenses promises new therapeutic alternatives to overcome these limitations and to support prevention and cure strategies. Here we show for the first time that sulforaphane( SFN), a natural product recognized for its health benefits, blocks HIV infection in macrophages. These cells play a critical role in HIV infection and pathogenesis, forming long-lived viral reservoirs[1, 2] and carrying virus into restricted compartments like the brain[3]. Most monocytes, precursors of macrophages, are largely refractory to HIV infection until they differentiate to replenish the macrophage pool[4, 5]. A small but specific subset of these cells however may be readily infectable, even in HAART-treated patients[6–9]. Infected macrophages have been observed in asymptomatic, untreated patients[10] and in HAART-treated individuals[11]. Other work, while not directly probing infection in situ, shows that rectal and vaginal macrophages share markers with HIV-susceptible macrophages[12, 13]. Importantly, macrophages can powerfully increase T cell infection[14] and may thereby exacerbate T cell depletion in infected individuals. SFN is generated in cruciferous vegetables after mechanical damage, like chewing, causes admixing of glucoraphanin with the enzyme myrosinase. It reaches various tissues after consumption where it maintains biological function[15]. In humans SFN is absorbed in the gut and peak plasma levels are achieved within 90 minutes[16]. In mice SFN can reach the prostate after ingestion[17, 18] where biological activity is maintained[19–21]. SFN is a powerful mobilizer of the transcription factor nuclear factor erythroid 2-related factor 2( Nrf2)[22–24]. Nrf2, a master regulator of the antioxidant response[25, 26], is held in check by its constitutive degradation through CUL3 ubiquitin ligase complexes using the protein KEAP1 as a substrate adaptor. SFN and other Nrf2-activators modify KEAP1 to prevent engagement of Nrf2[27]. As Nrf2 accumulates, it translocates into the nucleus, partners with musculoaponeurotic fibrosarcoma protein( Maf) and initiates transcription from antioxidant response elements( ARE). The consensus Nrf2 binding motif, TGACTCAGCA, was derived from known anti-oxidant response genes and Nrf2 binding was reconfirmed in chromatin immunoprecipitation and transcription analyses[28]. Nrf2 activates several types of genes[29] which predominantly encode proteins that counter oxidizing conditions[30]. Three viruses, Marburg virus[31, 32] Kaposi's sarcoma-associated herpesvirus( KSHV)[33] and Dengue virus[34] trigger Nrf2 activation. KSHV and Dengue virus appear to cause Nrf2 mobilization indirectly. Marburg virus however boosts Nrf2 levels directly with a protein designated VP24. This protein assembles with Keap1 to, like SFN, hamper engagement of Nrf2 and thus Nrf2 turnover[31, 32]. All three viruses benefit from increased Nrf2 expression[31–34]. The impact of SFN and Nrf2 on HIV-1 and HIV-2 infection, in primary cells, has not been determined. Here we show that SFN, acting through Nrf2, triggers a strong block against HIV infection in myeloid-derived cells including primary macrophages, but not in primary T cells. SFN blocks infection after reverse transcription but before the formation of 2-LTR circles. Together this work shows that, in contrast to Marburg, Dengue and KSHV, HIV is blocked through Nrf2 and sets the stage for discovering the mechanism underlying this restriction.
Discussion:
Here we have, for the first time, shown that SFN stops HIV infection in primary macrophages by triggering a block to infection that impacts the virus after reverse transcription but before 2-LTR circle formation. The SFN-regulated restriction relies on Nrf2, a transcription regulator of anti-oxidant genes. Importantly, we found that macrophages express sufficient Nrf2 to maintain a partial restriction even in the absence of SFN. This underscores that the anti-viral activity, while significantly enhanced by SFN through its augmentation of Nrf2 levels, does not require SFN to function. Our work showing that( 1) SFN blocks macrophage infection mediated by either VSV-G or HIV-1 Env,( 2) SFN does not block infection in all cell-types and( 3) blocks replication after late reverse transcription products have been formed, all support a model in which the restriction is well after entry into the cell but likely before the viral pre-integration complex is imported into the cell nucleus. The lack of anti-viral SFN action in numerous cell types and the dependence in macrophages solely on Nrf2 also indicates that SFN is neither inhibiting HIV directly by modifying a viral component nor is it modifying and thereby inhibiting a cellular component that the virus needs for successful infection. Similarly it is unlikely that SFN is activating a protein, post-translationally, that blocks HIV directly. Our experiments comparing SFN action on GFP reporter virus with that on luciferase reporter virus further support a model in which the block is before the level of transcription. We used the GFP reporter virus to quantify individual infected cells and the fraction of those infected cells closely paralleled the population-wide reporter activity measured with the luciferase reporter virus. This indicates that the SFN-mediated block reduced the number of infected cells rather than suppressing transcription from similar numbers of infected cells. HIV-1 Tat was reported to increase Nrf2 levels in HeLa-derived MAGI cells which corresponded with decreased Tat-mediated HIV-1 LTR transactivation[47]. While we did not test MAGI cells, SFN did not detectably impact LTR-directed luciferase reporter expression in HeLa or other non-macrophage-like cells. Our finding that SFN strongly inhibits infection at or before 2-LTR formation could account for the entire SFN-mediated block however, based on data shown in S3 Fig, we cannot fully rule out the possibility that SFN also impacts transcription from virus that evaded the initial block. Our findings also put into perspective previous work that tested the neuroprotective effects of di- and monomethyl fumarate( MMF). This study showed that DMF and MMF can attenuate spreading HIV infections in hMDMs[40]. Our work, employing single-round infections, allowed us to determine more specifically that DMF hinders a process before expression of new viral proteins. DMF and MMF, like SFN, increase levels of Nrf2[55], suggesting that these compounds also block HIV in macrophages through Nrf2. Interestingly, in our experiments SFN blocked infections more effectively at lower concentrations than DMF did[40]. None of the genes known to be modulated by Nrf2 direct production of recognized anti-viral products[28]. It is however possible that Nrf2 initiates a different expression pattern in macrophages than it does in previously tested B lymphocyte-derived cell lines[28]. In considering gene expression changes that could occur in human macrophages after SFN treatment, our attention was drawn to work describing murine macrophages treated with oxidized phospholipids[56]. These cells, designated Mox, adopt an Nrf2-dependent phenotype that is distinct from that of both M1 and M2 macrophages. Like SFN treated B lymphocyte-derived cells[28] or the THP1 cells in our work, and unlike M1 macrophages, Mox cells do not upregulate expression of known anti-viral genes. SFN treatment, on the other hand, may elicit a transcription pattern distinct from that described for Mox cells as we observed increases in both GCLM and NQO1( Fig 8) while only GCLM was upregulated after treatment with oxidized phospholipids. Future experiments, measuring the infectivity of heterokaryons formed by fusing cells that block HIV in response to SFN and those that don’t, will help to define the SFN-mediated block as a lack of a factor required for infection or the induction of a factor that blocks infection. This will help to guide the search for the mechanism underlying the SFN-mediated block. Presently however, the lack of HIV-associated factors among SFN-modulated genes is not surprising since new cellular anti-HIV factors and HIV co-factors continue to be identified[53, 54, 57–67]. We thus have discovered in SFN, a new way to activate an anti-viral state that HIV may not have previously encountered. It is therefore unlikely that the virus has developed an effective countermeasure. SFN, and other Nrf2 stimulators, could be used therapeutically to block HIV infection of macrophages and possibly of other important HIV targets. The capacity to hinder both HIV-1 and HIV-2 provides an advantage over several protease inhibitors as well as the fusion inhibitor enfuvirtide and first generation non-nucleoside reverse transcription inhibitors which only block HIV-1[68]. Further investigation will be required to determine why SFN fails to block infection in T cells, whether the same pathway could be exploited there as well or whether the restriction can be activated directly by other means. Drugs based on their electrophilic potential, like SFN, have been widely used. These include β-lactam antibiotics[69] and drugs that generate electrophilic intermediates through metabolism like the heartburn medicine omeprazole, or clopidogrel which is used against strokes and heart attacks[70–74]. Afatanib, another such drug, was approved by the FDA for the treatment of lung cancer[75]. Beloranib, also an agent with an electrophilic center, is in trials for the treatment of obesity[76]. The isothiocyante group of SFN can be replaced with other groups to further optimize this compound as a therapeutic agent[77]. Work in this regard has shown that substituting the sulfoxide group of SFN with a ketone group resulted in a compound that retained the biological activity of SFN itself[78]. Finally, the discovery that Nrf2 mobilization counters HIV infection in macrophages stands in contrast with recent work showing that Marburg and Dengue virus as well as KSHV initiate and benefit from increased Nrf2 expression[30, 32, 34, 41]. All four viruses share tropism for cells within the myeloid lineage but KSHV, Dengue and Marburg virus, unlike HIV, also share the capacity to infect endothelial cells[79–81]. The consequences of how HIV, KSHV and Marburg virus interface with Nrf2 may be a reflection of this tropism difference, especially if the putative Nrf2-regulated anti-viral defense is not active in endothelial cells.
Nrf2 turns on anti-oxidant genes in response to pharmaceuticals like oltipratz , environmental agents like heavy metals and cigarette smoke , endogenous agents like nitrous oxide and nitro-fatty acids and even plant products like sulforaphane ( SFN ) and epigallocatechin gallate ( EGCG ) . An increasing body of work is showing that some viruses activate and benefit from Nrf2 . In this work we tested the impact of Nrf2 on HIV . We used SFN , abundant in cruciferous vegetables and often used as a dietary supplement , to activate Nrf2 . Here we show , for the first time , that in immune cells isolated from donor blood , SFN halts HIV infection in macrophages , but not in T cells . We further show that upon SFN treatment the virus is blocked after it has transcribed its RNA-encoded genome into DNA , but before this genetic material is inserted into host chromosomes . Importantly this block is indeed dependent on Nrf2 . Interestingly , Nrf2 does not activate recognized anti-viral genes . Thus , unlike viruses recently found to benefit from Nrf2 activation , HIV can be blocked by its activation . This highlights the opportunity to activate a heretofore unrecognized anti-viral function by triggering an antioxidant response with a common dietary component .