HTLV-1 bZIP factor impairs DNA mismatch repair system.

Adult T-cell leukemia (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Microsatellite instability (MSI) has been observed in ATL cells. Although MSI results from impaired mismatch repair (MMR) pathway, no null mutations in the genes encoding MMR factors are detectable in ATL cells. Thus, it is unclear whether or not impairment of MMR causes the MSI in ATL cells. HTLV-1 bZIP factor (HBZ) protein interacts with numerous host transcription factors and significantly contributes to disease pathogenesis and progression. Here we investigated the effect of HBZ on MMR in normal cells. The ectopic expression of HBZ in MMR-proficient cells induced MSI, and also suppressed the expression of several MMR factors. We then hypothesized that the HBZ compromises MMR by interfering with a transcription factor, nuclear respiratory factor 1 (NRF-1), and identified the consensus NRF-1 binding site at the promoter of the gene encoding MutS homologue 2 (MSH2), an essential MMR factor. The luciferase reporter assay revealed that NRF-1 overexpression enhanced MSH2 promoter activity, while co-expression of HBZ reversed this enhancement. These results supported the idea that HBZ suppresses the transcription of MSH2 by inhibiting NRF-1. Our data demonstrate that HBZ causes impaired MMR, and may imply a novel oncogenesis driven by HTLV-1.

Critical role of PP2A-B56 family protein degradation in HIV-1 Vif mediated G2 cell cycle arrest.

HIV-1 Vif forms an E3 ubiquitin ligase complex with host proteins to counteract host restrictive APOBEC3, and is also known to accumulate infected cells at the G2 phase to promote viral replication. However, the underlying mechanism of how Vif induces G2 arrest is not fully understood, and more specifically, direct target molecules of G2 arrest have not been identified. Here we show that degradation of B56 family proteins (PP2A-B56), one of the regulatory subunits of protein phosphatase 2A, is critical for the Vif-induced G2 arrest. NL4-3 Vif caused degradation of PP2A-B56, and complementation of PP2A-B56 overcome the Vif-induced arrest. Supportively, knockdown of PPP2R5D, one of PP2A-B56, by siRNA itself induced cell cycle arrest of non-infected cells. We also identified Vif residues I31 and R or K33 are determinants for inducing G2 arrest, and Vif variants that did not cause G2 arrest did not induce PPP2R5D degradation, although it maintain the ability to induce APOBEC3G degradation, showing strong correlation between Vif-induced arrest and PP2A-B56 degradation. In a sequence database of HIV-1 isolates, Vif strains harboring residues that presumably induce cell cycle arrest are approximately 43%, suggesting Vif-induced G2 arrest contributes to HIV-1 infection in vivo and spread. Our data help understand the mechanism of Vif-mediated arrest, and gain insights into general cell cycle regulation.

A screening for DNA damage response molecules that affect HIV-1 infection.

Host DNA damage response molecules affect retroviral infection, as DNA intermediates of the viruses play essential roles in the viral life cycles. Although several such molecules have been reported, interactions between HIV-1 and host DNA damage response molecules have not been fully elucidated. To screen DNA damage response molecules that might affect HIV-1 infection, a set of 32 DNA-repair-deficient DT40 isogenic mutant cells were tested for HIV-1 infectivity. Seven out of the 32 clones showed less than 50% infectivity compared to parental DT40 cells, implying that DNA repair molecules deficient in these cells might support HIV-1 infection. Of these, EXO1 -/-, TP53BP1 -/- and WRN -/- cells showed more than twofold accumulation of two long terminal repeat circles and less than 50% integrated proviral DNA in quantitative-PCR analyses, indicating that the integration step is impaired. RAD18 -/- cells showed twofold higher HIV-1 infectivity and increased reverse transcription products at earlier time points, suggesting that RAD18 suppresses reverse transcription. The HIV-1 suppressive effects of RAD18 were confirmed by over-expression and knockdown experiments in human cells. L274P, a DNA-binding-impaired mutant of RAD18, showed impaired HIV-1 suppression and DNA binding, suggesting that binding HIV-1 DNA intermediates is critical for RAD18 to suppress reverse transcription and HIV-1 infection. Our data help understand interactions between host DNA damage response molecules and viral DNA.

The findings of musculoskeletal ultrasonography on primary Sjögren's syndrome patients in childhood with articular manifestations and the impact of anti-cyclic citrullinated peptide antibody.

Objective: We researched the findings of musculoskeletal ultrasound sonography (MSUS) on primary Sjogren's syndrome in childhood (pSS-C) with articular manifestations. The correlation of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (ACPA) were investigated to evaluate the usefulness of MSUS on their articular prognosis. Method: The objective patients are pSS-C cases who visited our hospital complaining joint pain and/or joint swelling and for whom MSUS was performed. Result: Eight patients included 6 female and 2 male, 5 RF-positive patients and 3 ACPA- positive patients. The mean age of onset was 11.1 ± 3.0 years (352 physical joint findings and 284 MSUS findings. The number of joints found clinical articular manifestations was 58/352 joints, and arthritis detected by MSUS was 30/284 joints). In multivariate analysis, the odds ratio of clinical articular manifestations was significant high in RF-positivity (2.9, 95%CI 1.5-6.2). The odds ratio of arthritis detected by MSUS in ACPA-positivity was significant high (3.7, 95%CI 1.5-11.6), although odds ratio in RF-positivity had no statistical significance and a similar trend was seen in odds ratios of subclinical arthritis (4.9, 95%CI 1.6-18.0). Conclusion: It was indicated that MSUS is useful for pSS-C. ACPA-positive pSS-C patients have arthritis and subclinical arthritis more frequently than ACPA-negative patients.

Dual inhibition of the mTORC1 and mTORC2 signaling pathways is a promising therapeutic target for adult T-cell leukemia.

Adult T-cell leukemia (ATL) has a poor prognosis as a result of severe immunosuppression and rapid tumor progression with resistance to conventional chemotherapy. Recent integrated-genome analysis has revealed mutations in many genes involved in the T-cell signaling pathway, suggesting that the aberration of this pathway is an important factor in ATL pathogenesis and ATL-cell proliferation. We screened a siRNA library to examine signaling-pathway functionality and found that the PI3K/Akt/mTOR pathway is critical to ATL-cell proliferation. We therefore investigated the effect of mammalian target of rapamycin (mTOR) inhibitors, including the dual inhibitors PP242 and AZD8055 and the mTORC1 inhibitors rapamycin and everolimus, on human T-cell leukemia virus type 1 (HTLV-1)-infected-cell and ATL-cell lines. Both dual inhibitors inhibited the proliferation of all tested cell lines by inducing G1-phase cell-cycle arrest and subsequent cell apoptosis, whereas the effects of the 2 mTORC1 inhibitors were limited, as they did not induce cell apoptosis. In the ATL-cell lines and in the primary ATL samples, both dual inhibitors inhibited phosphorylation of AKT at serine-473, a target of mTORC2, as well as that of S6K, whereas the mTORC1 inhibitors only inhibited mTORC1. Furthermore, AZD8055 more significantly inhibited the in vivo growth of the ATL-cell xenografts than did everolimus. These results indicate that the PI3K/mTOR pathway is critical to ATL-cell proliferation and might thus be a new therapeutic target in ATL.

Core Binding Factor ß Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor ß (CBFß). CBFß is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFß up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFß might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFß preferentially interact with MDM2 and that overexpression of CBFß disrupts the interaction between MDM2 and Vif. Knockdown of CBFß reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFß, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFß is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFß binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFß.

Vif hijacks CBF-ß to degrade APOBEC3G and promote HIV-1 infection.

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-ß to this ubiquitin ligase complex. CBF-ß, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-ß is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-ß to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-ß-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.

Classical NF-κB pathway is responsible for APOBEC3B expression in cancer cells.

APOBEC3B (A3B) is a DNA cytosine deaminase and catalyzes cytosine deamination, resulting in mutations in genomic DNA. A3B is aberrantly expressed in a variety of cancers and considered to be a source of genomic mutations that contribute to cancer progression and metastasis. However, the mechanisms through which A3B expression is dysregulated in cancer cells are not fully elucidated. Here we report that the classical NF-κB pathway plays a crucial role in the transcriptional regulation of A3B in various cancer cells, including lymphoid malignancies. PMA, a strong activator of PKC, induces A3B at both mRNA and protein levels in cancer cell lines, and specific inhibitors of both PKC and IKK downregulate A3B expression. Using luciferase reporter and EMSA assays, we identify 3 NF-κΒ binding sites in the A3B promoter and reveal that NF-κB p65/p50 and p65/c-Rel heterodimers are important for A3B transcription. These results suggest that the classical NF-κB pathway is responsible for activation of A3B mRNA expression and further imply that inhibition of PKC and IKK might augment cancer treatment by reducing cancer progression and metastasis through downregulation of A3B expression.

Small molecules that inhibit Vif-induced degradation of APOBEC3G.

BACKGROUND: HIV-1 Vif is essential for virus replication in natural target cells such as T cells and macrophages. Vif recruits a ubiquitin ligase to degrade restrictive APOBEC3 proteins. APOBEC3G is one of the most potent retroviral restriction factors targeted by Vif and, as such, the Vif-APOBEC3G interaction has emerged as a promising HIV-1 therapeutic target. METHODS: 20,000 small molecules were used in live-cell screens for those that preserve EGFP-APOBEC3G fluorescence and luciferase-APOBEC3G luminescence in the presence of HIV-1 Vif. RESULTS: 2 compounds with similar core structures preserved APOBEC3G levels in the presence of Vif. 10 µM of compound restored APOBEC3G to levels sufficient for incorporation into vif-proficient virus particles and restriction of virus infectivity. Vif-dependent APOBEC3G polyubiquitination and general proteasomal activity were unaffected at the same concentration. CONCLUSIONS: The small molecules described here preserve APOBEC3G levels and activity in the presence of Vif. These molecules are starting points for further development as antiretrovirals.

Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G.

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

Quantitative profiling of the full APOBEC3 mRNA repertoire in lymphocytes and tissues: implications for HIV-1 restriction.

The human APOBEC3 proteins are DNA cytidine deaminases that impede the replication of many different transposons and viruses. The genes that encode APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H were generated through relatively recent recombination events. The resulting high degree of inter-relatedness has complicated the development of specific quantitative PCR assays for these genes despite considerable interest in understanding their expression profiles. Here, we describe a set of quantitative PCR assays that specifically measures the mRNA levels of each APOBEC3 gene. The specificity and sensitivity of each assay was validated using a full matrix of APOBEC3 cDNA templates. The assays were used to quantify the APOBEC3 repertoire in multiple human T-cell lines, bulk leukocytes and leukocyte subsets, and 20 different human tissues. The data demonstrate that multiple APOBEC3 genes are expressed constitutively in most types of cells and tissues, and that distinct APOBEC3 genes are induced upon T-cell activation and interferon treatment. These data help define the APOBEC3 repertoire relevant to HIV-1 restriction in T cells, and they suggest a general model in which multiple APOBEC3 proteins function together to provide a constitutive barrier to foreign genetic elements, which can be fortified by transcriptional induction.

A single amino acid in human APOBEC3F alters susceptibility to HIV-1 Vif.

Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu(324) as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu(324) is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins.

Evolution of HIV-1 isolates that use a novel Vif-independent mechanism to resist restriction by human APOBEC3G.

The human APOBEC3G protein restricts the replication of Vif-deficient HIV-1 by deaminating nascent viral cDNA cytosines to uracils, leading to viral genomic strand G-to-A hypermutations. However, the HIV-1 Vif protein triggers APOBEC3G degradation, which helps to explain why this innate defense does not protect patients. The APOBEC3G-Vif interaction is a promising therapeutic target, but the benefit of the enabling of HIV-1 restriction in patients is unlikely to be known until Vif antagonists are developed. As a necessary prelude to such studies, cell-based HIV-1 evolution experiments were done to find out whether APOBEC3G can provide a long-term block to Vif-deficient virus replication and, if so, whether HIV-1 variants that resist restriction would emerge. APOBEC3G-expressing T cells were infected with Vif-deficient HIV-1. Virus infectivity was suppressed in 45/48 cultures for more than five weeks, but replication was eventually detected in three cultures. Virus-growth characteristics and sequencing demonstrated that these isolates were still Vif-deficient and that in fact, these viruses had acquired a promoter mutation and a Vpr null mutation. Resistance occurred by a novel tolerance mechanism in which the resistant viruses packaged less APOBEC3G and accumulated fewer hypermutations. These data support the development of antiretrovirals that antagonize Vif and thereby enable endogenous APOBEC3G to suppress HIV-1 replication.

Alternative splicing of APOBEC3D generates functional diversity and its role as a DNA mutator.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family members have cytidine deaminase activity and can induce cytosine to uracil transition in nucleic acid. The main function of APOBEC3 (A3) proteins is to trigger an innate immune response to viral infections. Recent reports have shown that several APOBEC family proteins such as A3B can induce somatic mutations into genomic DNA and thus promote cancer development. However, the role of A3D on somatic mutations is unclear. Here, we identified the alternative splicing of A3D, and investigated each splice variant's subcellular localization and role in DNA mutagenesis. We identified four A3D variants, which all have one or two cytidine deaminase domains. The full-length form of A3D (variant 1) and truncated forms of A3D (variant 2, 6, 7) showed the ability to induce C/G to T/A transitions in foreign DNA and genomic DNA and retained antiretroviral activity. Furthermore, we demonstrated that A3D and A3B could induce deletions that are possibly repaired by microhomology-mediated end joining (MMEJ). Taken together, our experiments illustrated that alternative splicing generates functional diversity of A3D, and some variants can act as DNA mutators in genomic DNA.

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

Endogenous APOBEC3B Overexpression Constitutively Generates DNA Substitutions and Deletions in Myeloma Cells.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminases have emerged as potential genomic mutators in various cancers. Multiple myeloma accumulates APOBEC signature mutations as it progresses; however, the mechanisms underlying APOBEC signature acquisition and its consequences remain elusive. In this study, we examined the significance and clinical impact of APOBEC3B (A3B) activity in multiple myeloma. Among APOBECs, only highly expressed A3B was associated with poor prognosis in myeloma patients, independent of other known poor prognostic factors. Quantitative PCR revealed that CD138-positive primary myeloma cells and myeloma cell lines exhibited remarkably high A3B expression levels. Interestingly, lentiviral A3B knockdown prevented the generation of deletion and loss-of-function mutations in exogenous DNA, whereas in control cells, these mutations accumulated with time. A3B knockdown also decreased the basal levels of γ-H2AX foci, suggesting that A3B promotes constitutive DNA double-strand breaks in myeloma cells. Importantly, among control shRNA-transduced cells, we observed the generation of clones that harboured diverse mutations in exogenous genes and several endogenous genes frequently mutated in myeloma, including TP53. Taken together, the results suggest that A3B constitutively mutates the tumour genome beyond the protection of the DNA repair system, which may lead to clonal evolution and genomic instability in myeloma.

Protein kinase A inhibits tumor mutator APOBEC3B through phosphorylation.

APOBEC3B cytidine deaminase (A3B) catalyzes cytosine into uracil in single-strand DNA and induces C-to-T mutations in genomic DNA of various types of tumors. Accumulation of APOBEC signature mutations is correlated with a worse prognosis for patients with breast cancer or multiple myeloma, suggesting that A3B activity might be a cause of the unfavorable DNA mutations and clonal evolution in these tumors. Phosphorylation of conserved threonine residues of other cytidine deaminases, activation induced deaminase (AID) and APOBEC3G, inhibits their activity. Here we show that protein kinase A (PKA) physically binds to A3B and phosphorylates Thr214. In vitro deaminase assays and foreign DNA editing assays in cells confirm that phosphomimetic A3B mutants, T214D and T214E, completely lose deaminase activity. Molecular dynamics simulation of A3B phosphorylation reveals that Thr214 phosphorylation disrupts binding between the phospho-A3B catalytic core and ssDNA. These mutants still inhibit retroviral infectivity at least partially, and also retain full anti-retrotransposition activity. These results imply that PKA-mediated phosphorylation inhibits A3B mutagenic activity without destructing its innate immune functions. Therefore, PKA activation could reduce further accumulation of mutations in A3B overexpressing tumors.

Extensive mutagenesis experiments corroborate a structural model for the DNA deaminase domain of APOBEC3G.

APOBEC3G is a single-strand DNA cytosine deaminase capable of blocking retrovirus and retrotransposon replication. APOBEC3G has two conserved zinc-coordinating motifs but only one is required for catalysis. Here, deletion analyses revealed that the minimal catalytic domain consists of residues 198-384. Size exclusion assays indicated that this protein is monomeric. Many (31/69) alanine substitution derivatives of APOBEC3G198-384 retained significant to full levels of activity. These data corroborated an APOBEC2-based structural model for the catalytic domain of APOBEC3G indicating that most non-essential residues are solvent accessible and most essential residues cluster within the protein core.

HTLV-1 bZIP factor suppresses TDP1 expression through inhibition of NRF-1 in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). We recently reported that abacavir, an anti-HIV-1 drug, potently and selectively kills ATL cells. This effect was attributed to the reduced expression of tyrosyl-DNA-phosphodiesterase 1 (TDP1), a DNA repair enzyme, in ATL cells. However, the molecular mechanism underlying the downregulation of TDP1 in ATL cells remains elusive. Here we identified the core promoter of the TDP1 gene, which contains a conserved nuclear respiratory factor 1 (NRF-1) binding site. Overexpression of NRF-1 increased TDP1-promoter activity, whereas the introduction of dominant-negative NRF-1 repressed such activity. Overexpression of NRF-1 also upregulated endogenous TDP-1 expression, while introduction of shNRF-1 suppressed TDP1 in Jurkat T cells, making them susceptible to abacavir. These results indicate that NRF-1 is a positive transcriptional regulator of TDP1-gene expression. Importantly, we revealed that HTLV-1 bZIP factor (HBZ) protein which is expressed in all ATL cases physically interacts with NRF-1 and inhibits the DNA-binding ability of NRF-1. Taken together, HBZ suppresses TDP1 expression by inhibiting NRF-1 function in ATL cells. The HBZ/NRF-1/TDP1 axis provides new therapeutic targets against ATL and might explain genomic instability leading to the pathogenesis of ATL.