Mari Shimura

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.

HIV-1 Vpr induces TLR4/MyD88-mediated IL-6 production and reactivates viral production from latency

Vpr, a HIV‐1 accessory protein, was believed to be present in the plasma of HIV‐1‐positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients’ viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL‐6 was identified as a responsible factor. The induction of IL‐6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr‐induced IL‐6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr‐induced formation of OxPC, the phosphorylation of C/EBP‐β, a substrate of MAPK, and IL‐6 production. As rIL‐6 reactivated viral replication in latently infected cells, our data indicate that rVpr‐induced oxidative stress triggers cell‐based innate immune responses and reactivates viral production in latently infected cells via IL‐6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti‐AIDS therapeutics targeting Vpr.

Chromatin Compaction Protects Genomic DNA from Radiation Damage

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.

HIV-1 Vpr induces DNA double-strand breaks.

Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1-induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1-infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome-related tumor development.

Element Array by Scanning X-ray Fluorescence Microscopy after Cis-Diamminedichloro-Platinum(II) Treatment

Minerals are important for cellular functions, such as transcription and enzyme activity, and are also involved in the metabolism of anticancer chemotherapeutic compounds. Profiling of intracellular elements in individual cells could help in understanding the mechanism of drug resistance in tumors and possibly provide a new strategy of anticancer chemotherapy. Using a recently developed technique of scanning X-ray fluorescence microscopy (SXFM), we analyzed intracellular elements after treatment with cis-diamminedichloro-platinum(II) (CDDP), a platinum-based anticancer agent. The images obtained by SXFM (element array) revealed that the average Pt content of CDDP-resistant cells was 2.6 times less than that of sensitive cells, and the zinc content was inversely correlated with the intracellular Pt content. Data suggested that Zn-related detoxification is responsible for resistance to CDDP. Of Zn-related excretion factors, glutathione was highly correlated with the amount of Zn. The combined treatment of CDDP and a Zn(II) chelator resulted in the incorporation of thrice more Pt with the concomitant down-regulation of glutathione. We propose that the generation of an element array by SXFM opens up new avenues in cancer biology and treatment.

Micronuclei formation and aneuploidy induced by Vpr, an accessory gene of human immunodeficiency virus type 1

Vpr, an accessory gene of HIV‐1, induces cell cycle abnormality with accumulation at G2/M phase and increased ploidy. Since abnormality of mitotic checkpoint control provides a molecular basis of genomic instability, we studied the effects of Vpr on genetic integrity using a stable clone, named MIT‐23, in which Vpr expression is controlled by the tetracycline‐responsive promoter. Treatment of MIT‐23 cells with doxycycline (DOX) induced Vpr expression with a giant multinuclear cell formation. Increased micronuclei (MIN) formation was also detected in these cells. Abolishment of Vpr expression by DOX removal induced numerous asynchronous cytokinesis in the multinuclear cells with leaving MIN in cytoplasm, suggesting that the transient Vpr expression could cause genetic unbalance. Consistent with this expectation, MIT‐23 cells, originally pseudodiploid cells, became aneuploid after repeated expression of Vpr. Experiments using deletion mutants of Vpr revealed that the domain inducing MIN formation as well as multinucleation was located in the carboxy‐terminal region of Vpr protein. These results suggest that Vpr induces genomic instability, implicating the possible role in the development of AIDS‐related malignancies.—Shimura, M., Tanaka, Y., Nakamura, S., Minemoto, Y., Yamashita, K., Hatake, K., Takaku, F., Ishizaka, Y. Micronuclei formation and aneuploidyinduced by Vpr, an accessory gene of human immunodeficiency virus type 1. FASEB J. 13, 621–637 (1999)

Binding of 14-3-3β but not 14-3-3σ controls the cytoplasmic localization of CDC25B: Binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3β, 14-3-3ϵ and 14-3-3σ, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3β bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3σ bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3β drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3σ and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3β, even when other putative 14-3-3 binding sites were mutated. 14-3-3ϵ resembled 14-3-3β with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3σ has effects on CDC25B other than the control of its subcellular localization.

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination.

An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR–Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of γ-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-SceI, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.

Differential Anti-APOBEC3G Activity of HIV-1 Vif Proteins Derived from Different Subtypes

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.

Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation

The HIV-1 protein Vpr disrupts higher-order chromatin structure by altering histone modification and displacing important heterochromatin proteins, resulting in chromatid cohesion defects.