Kenichi Imai

Involvement of Histone H3 Lysine 9 (H3K9) Methyltransferase G9a in the Maintenance of HIV-1 Latency and Its Reactivation by BIX01294

Elucidating the mechanism of human immunodeficiency virus, type 1 (HIV-1) provirus transcriptional silencing in latently infected cells is crucial for understanding the pathophysiological process of HIV-1 infection. It is well established that hypoacetylation of histone proteins by histone deacetylases is involved in the maintenance of HIV-1 latency by repressing viral transcription. Although histone methylation is involved in the organization of chromatin domains and plays a central epigenetic role in gene expression, the role of histone methylation in the maintenance of HIV-1 latency has not been clarified. Here we present evidence that histone H3 Lys9 (H3K9) methyltransferase G9a is responsible for transcriptional repression of HIV-1 by promoting repressive dimethylation at H3K9 and for the maintenance of viral latency. We observed that G9a significantly inhibited basal, as well as, the induced HIV-1 gene expression by tumor necrosis factor-α or Tat. Mutant G9a, however, lacking the SET domain responsible for the catalytic activity of histone methyltransferase, did not show such an effect. When G9a expression was knocked down by small interfering RNA, HIV-1 replication was augmented from cells transiently transfected with a full-length HIV-1 clone. Moreover, a specific inhibitor of G9a, BIX01294, could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1. Furthermore, chromatin immunoprecipitation assays revealed the presence of G9a and H3K9 dimethylation on nucleosome histones in the vicinity of the HIV-1 long terminal repeat promoter. These results suggest that G9a is responsible for the transcriptional quiescence of latent HIV-1 provirus and provide a molecular basis for understanding the mechanism by which HIV-1 latency is maintained.

Transcriptional Repression of Human Immunodeficiency Virus Type 1 by AP-4

Elucidation of the mechanism of transcriptional silencing of human immunodeficiency virus type 1 (HIV-1) provirus in latently infected cells is crucial to understand the pathophysiology of HIV-1 infection and to develop novel therapies. Here we demonstrate that AP-4 is responsible for the transcriptional repression of HIV-1. We found that AP-4 site within the viral long terminal repeat (LTR) is well conserved in the majority of HIV-1 subtypes and that AP-4 represses HIV-1 gene expression by recruiting histone deacetylase (HDAC) 1 as well as by masking TATA-binding protein to TATA box. AP-4-mediated transcriptional repression was inhibited by an HDAC inhibitor, tricostatin A, and could be exerted even at distant locations from the TATA box. In addition, AP-4 interacted with HDAC1 both in vivo and in vitro. Moreover, chromatin immunoprecipitation assays have revealed that AP-4 and HDAC1 are present in the HIV-1 LTR promoter in latently infected ACH2 and U1 cells, and they are dissociated from the promoter concomitantly with the association of acetylated histone H3, TBP, and RNA polymerase II upon TNF-α stimulation of HIV-1 replication. Furthermore, when AP-4 is knocked down by siRNA, HIV-1 production was greatly augmented in cells transfected with a full-length HIV-1 clone. These results suggest that AP-4 may be responsible for transcriptional quiescence of latent HIV-1 provirus and give a molecular basis to the reported efficacy of combination therapy of conventional anti-HIV drugs with an HDAC inhibitor in accelerating the clearance of HIV-1 from individuals infected with the virus.

Reactivation of Latent HIV-1 Infection by the Periodontopathic Bacterium Porphyromonas gingivalis Involves Histone Modification

Latently infected cells harbor the HIV-1 proviral DNA genome primarily integrated into heterochromatin, allowing the persistence of transcriptionally silent proviruses. Hypoacetylation of histone proteins by histone deacetylases (HDAC) is involved in the maintenance of HIV-1 latency by repressing viral transcription. In addition, periodontal diseases, caused by polymicrobial subgingival bacteria including Porphyromonas gingivalis, are among the most prevalent infections of mankind. Here we demonstrate the effects of P. gingivalis on HIV-1 replication. This activity could be ascribable to the bacterial culture supernatant but not to other bacterial components such as fimbriae or LPS. We found that this HIV-1-inducing activity was recovered in the lower molecular mass (<3 kDa) fraction of the culture supernatant. We also demonstrated that P. gingivalis produces high concentrations of butyric acid, acting as a potent inhibitor of HDACs and causing histone acetylation. Chromatin immunoprecipitation assays revealed that the corepressor complex containing HDAC1 and AP-4 was dissociated from the HIV-1 long terminal repeat promoter upon stimulation with bacterial culture supernatant concomitantly with the association of acetylated histone and RNA polymerase II. We thus found that P. gingivalis could induce HIV-1 reactivation via chromatin modification and that butyric acid, one of the bacterial metabolites, is responsible for this effect. These results suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to the systemic dissemination of the virus.

Transforming growth factor-beta inhibits lipopolysaccharide-stimulated expression of inflammatory cytokines in mouse macrophages through downregulation of activation protein 1 and CD14 receptor expression.

ABSTRACT The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-β1 (TGF-β1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-β1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor. TGF-β1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages. LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fosand c-jun oligonucleotides. Since TGF-β1 pretreatment inhibited LPS-stimulated expression of c-fos and c-jun genes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-β1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1β and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-β1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-β1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-β1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-β1 inhibition of LPS-induced septic shock.

Reactivation of latent HIV-1 by a wide variety of butyric acid-producing bacteria

Latently infected cells harbor human immunodeficiency virus type 1 (HIV-1) proviral DNA copies integrated in heterochromatin, allowing persistence of transcriptionally silent proviruses. It is widely accepted that hypoacetylation of histone proteins by histone deacetylases (HDACs) is involved in maintaining the HIV-1 latency by repressing viral transcription. HIV-1 replication can be induced from latently infected cells by environmental factors, such as inflammation and co-infection with other microbes. It is known that a bacterial metabolite butyric acid inhibits catalytic action of HDAC and induces transcription of silenced genes including HIV-1 provirus. There are a number of such bacteria in gut, vaginal, and oral cavities that produce butyric acid during their anaerobic glycolysis. Since these organs are known to be the major site of HIV-1 transmission and its replication, we explored a possibility that explosive viral replication in these organs could be ascribable to butyric acid produced from anaerobic resident bacteria. In this study, we demonstrate that the culture supernatant of various bacteria producing butyric acid could greatly reactivate the latently-infected HIV-1. These bacteria include Fusobacterium nucleatum (commonly present in oral cavity, and gut), Clostridium cochlearium, Eubacterium multiforme (gut), and Anaerococcus tetradius (vagina). We also clarified that butyric acid in these culture supernatants could induce histone acetylation and HIV-1 replication by inhibiting HDAC. Our observations indicate that butyric acid-producing bacteria could be involved in AIDS progression by reactivating the latent HIV provirus and, subsequently, by eliminating such bacterial infection may contribute to the prevention of the AIDS development and transmission.

Higher Prevalence of Epstein–Barr Virus DNA in Deeper Periodontal Pockets of Chronic Periodontitis in Japanese Patients

Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein–Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤3 mm (shallow) or ≥5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients.

53BP2 induces apoptosis through the mitochondrial death pathway.

The p53 binding protein 2 (53BP2) has been identified as the interacting protein to p53, Bcl‐2, and p65 subunit of nuclear factor κB (NF‐κB). The TP53BP2 gene encodes two splicing variants, 53BP2S and 53BP2L, previously known as apoptosis stimulating protein 2 of p53 (ASPP2). We found that these 53BP2 proteins are located predominantly in the cytoplasm and induce apoptosis as demonstrated by cleavage of poly ADP ribose polymerase (PARP) and annexin V staining. Furthermore, we demonstrate that 53BP2 is located in the mitochondria and induces apoptosis associated with depression of the mitochondrial trans‐membrane potential (ΔΨm) and activation of caspase‐9. From these findings we conclude that 53BP2 induces apoptosis through the mitochondrial death pathway.

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Latently Infected Cells by a Novel IκB Kinase Inhibitor

ABSTRACT In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-κB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-κB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-α) stimulates IκB kinase (IKK) complex, a critical regulator in the NF-κB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-α and IKK-β. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-α-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 μM, whereas its 50% cytotoxic concentration was about 15 μM. Western blot analysis revealed inhibition of IκBα phosphorylation, IκBα degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-κB activation. Furthermore, ACHP inhibited TNF-α-induced NF-κB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-α-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.

Novel histone deacetylase inhibitor NCH-51 activates latent HIV-1 gene expression.

Pharmacological manipulations to purge human immunodeficiency virus (HIV) from latent reservoirs have been considered as an adjuvant therapeutic approach to highly‐active antiretroviral therapy for the eradication of HIV. Our novel histone deacetylase inhibitor NCH‐51 induced expression of latent HIV‐1 with minimal cytotoxicity. Using chromatin immunoprecipitation assays, we observed a reduction of HDAC1 occupancy, histone hyperacetylation and the recruitment of positive transcription factors at the HIV‐1 promoter in latently infected‐cells under the treatment with NCH‐51. Mutation studies of the long terminal repeat (LTR) revealed NCH‐51 mediated gene expression through the Sp1 sites. When Sp1 expression was knocked‐down by small interfering RNA, the NCH‐51‐mediated activation of a stably integrated HIV‐1 LTR was attenuated. Moreover, the Sp1 inhibitor mithramycin A abolished the effects of NCH‐51.

Calpain is involved in the HIV replication from the latently infected OM10.1 cells.

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.