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Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.

CBF-β is shown to regulate the ability of HIV-1 to evade host restriction mediated by the deaminase APOBEC3. Vif–CBF-β interaction an anti-HIV-1 target The transcription cofactor CBF-β (core binding factor β) regulates the DNA binding activity of RUNX family proteins. Two independent studies now show that CBF-β also regulates the ability of HIV-1 to evade host restriction mediated by the cDNA deaminase APOBEC3G, a host factor that blocks viral replication. They show that it associates with the HIV protein Vif, and is essential for the assembly of the Vif-Cul5 E3 ubiquitin ligase complex, which mediates the ubiquitination and destruction of APOBEC3. Both groups suggest that disrupting the Vif–CBF-β interaction could provide a new therapeutic target against HIV-1 infection. The human APOBEC3 cytidine deaminases are potent inhibitors of diverse retroviruses, including human immunodeficiency virus-1 (HIV-1) 1 , 2 , 3 , 4 , 5 , 6 . HIV-1 Vif forms an E3 ubiquitin ligase complex with cullin 5 (CUL5), elongin B and elongin C 7 , 8 , 9 , which promotes the polyubiquitination and degradation of APOBEC3 substrates 7 , 10 , 11 , 12 , 13 , 14 . Here we demonstrate in human T cells that core binding factor β (CBF-β) is a key regulator of the evasion of HIV-1 from the host defence mediated by APOBEC3. CBF-β, the non-DNA-binding subunit of a heterodimeric transcription factor, regulates the folding and DNA-binding activity of partner RUNX family proteins, which have important roles in the development and differentiation of diverse cell types, including T lymphocytes 15 , 16 . In our study, knockdown of endogenous CBF-β blocked Vif-induced APOBEC3G polyubiquitination and degradation. CBF-β was not required for the interaction between Vif and APOBEC3G, yet was essential for the assembly of the Vif–CUL5 E3-ubiquitin-ligase complex. CBF-β proved to be a unique regulator of primate lentiviral Vif and not a general component of the CUL5 E3 ubiquitin ligase. We show that Vif and CBF-β physically interact, and that the amino-terminal region of Vif is required for this interaction. Furthermore, interactions with Vif required regions in CBF-β that are not involved in RUNX protein binding 17 , 18 , 19 . Considering the importance of the interaction between Vif and CBF-β, disrupting this interaction represents an attractive pharmacological intervention against HIV-1.

Background: The human immune response to mercury is not well characterized despite the body of evidence that suggests that Hg can modulate immune responses, including the induction of autoimmune disease in some mouse models. Dysregulation of cytokine signaling appears to play an important role in the etiology of Hg-induced autoimmunity in animal models. Objectives: In this study, we systematically investigated the human immune response to Hg in vitro in terms of cytokine release. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from 20 volunteers who donated blood six separate times. PBMCs were cultured with lipopolysaccharide and concentrations of mercuric chloride (HgCl₂) up to 200 nM. Seven cytokines representing important pathways in physiologic and pathologic immune responses were measured in supernatants. We used multilevel models to account for the intrinsic clustering in the cytokine data due to experimental design. Results: We found a consistent increase in the release of the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α, and concurrent decrease in release of the antiinflammatory cytokines interleukin 1-receptor antagonist (IL-1Ra) and IL-10 in human PBMCs treated with subcytotoxic concentrations of HgCl₂. IL-4, IL-17, and interferon-γ increased in a concentration-response manner. These results were replicated in a second, independently recruited population of 20 different volunteers. Conclusions: Low concentrations of HgCl₂ affect immune function in human cells by dysregulation of cytokine signaling pathways, with the potential to influence diverse health outcomes such as susceptibility to infectious disease or risk of autoimmunity.

Background HIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-β). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C–terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide. Results Here, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25–30 ( 25 VXHXMY 30 ) reduced the ability for Vif to bind Cul5, but not CBF-β or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-β-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding. Conclusions Taken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.

Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBF[beta]), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBF[beta] interaction. Furthermore, association of Vif with CBF[beta], alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBF[beta]-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBF[beta]-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates.

Silencing of endogenous CBF-b (lanes 4-6) impeded the ability of Vif to reduce the expression of APOBEC3G (lane 6). Because HIV-1 Vif mainly reduces the expression of APOBEC3G by inducing its degradation, these data indicated that CBF-b is required for the HIV-1 Vif-mediated degradation of APOBEC3G (Fig. 1d, compare lanes 2 and 3 to lanes 5 and 6).

Educating the next generation of nanoelectronics researchers, engineers, and scientists presents several challenges due to the fast‐moving and interdisciplinary nature of the field. This chapter outlines some of the considerations involved with designing modern university‐level programs in nanoelectronics. In overviewing traditional programs that confer degrees related to nanoelectronics, specific challenges related to bridging disparate disciplines are addressed. Integrating theory, design, and lab work and enabling new industrial applications are described in detail. A case study involving educating students in the use of nanoelectronics in the health care industry is presented, followed by a discussion of the future of university programs aiming to give students a holistic view of the field. Future educational programs should be based around competencies required to generate solutions that can adequately address industry and societal needs: cross‐discipline subjects; nanoelectronics‐related education and research framework; and soft‐skills elective subjects.

An international group of cancer geneticists review the level of evidence for the association of gene variants with the risk of breast cancer. It is difficult to draw firm conclusions from the data because of ascertainment bias and the lack of data from large populations. Advances in sequencing technology have made multigene testing, or “panel testing,” a practical option when looking for genetic variants that may be associated with a risk of breast cancer. In June 2013, the U.S. Supreme Court 1 invalidated specific claims made by Myriad Genetics with respect to the patenting of the genomic DNA sequence of BRCA1 and BRCA2 . Other companies immediately began to offer panel tests for breast cancer genes that included BRCA1 and BRCA2 . The subsequent flourishing of gene-panel testing services (Table 1, and Table S1 in the Supplementary Appendix, available with the full text of this article at . . .

In combination with existing observations and detailed circumstellar models, the detection of hydrogen deuteride emission from the star TW Hydrae implies a circumstellar disk mass of more than 0.05 solar masses, which is enough to form a planetary system like our own. The search for planetary potential The mass of a protoplanetary disk is a key influence on its likely fate, and a new way of estimating that mass, requiring fewer assumptions than other methods, should aid these studies. For planets to have formed, our Solar System at birth required a minimum disk mass of about 0.01 solar masses within around 100 astronomical units of its centre. This study reports the detection of the fundamental rotational transition of hydrogen deuteride (HD) in the circumstellar disk of the star TW Hydrae. Distribution of this gas mirrors that of molecular hydrogen, and its spectral emission is thought to trace the total mass. Its presence, together with modelling data, implies a disk mass of more than 0.05 solar masses, sufficient to form a planetary system like our own. At 3–10 million years old, TW Hydrae is considered old for a protoplanetary disk, yet it is still young enough to develop into a planetary system. From the masses of the planets orbiting the Sun, and the abundance of elements relative to hydrogen, it is estimated that when the Solar System formed, the circumstellar disk must have had a minimum mass of around 0.01 solar masses within about 100 astronomical units of the star 1 , 2 , 3 , 4 . (One astronomical unit is the Earth–Sun distance.) The main constituent of the disk, gaseous molecular hydrogen, does not efficiently emit radiation from the disk mass reservoir 5 , and so the most common measure of the disk mass is dust thermal emission and lines of gaseous carbon monoxide 6 . Carbon monoxide emission generally indicates properties of the disk surface, and the conversion from dust emission to gas mass requires knowledge of the grain properties and the gas-to-dust mass ratio, which probably differ from their interstellar values 7 , 8 . As a result, mass estimates vary by orders of magnitude, as exemplified by the relatively old (3–10 million years) star TW Hydrae 9 , 10 , for which the range is 0.0005–0.06 solar masses 11 , 12 , 13 , 14 . Here we report the detection of the fundamental rotational transition of hydrogen deuteride from the direction of TW Hydrae. Hydrogen deuteride is a good tracer of disk gas because it follows the distribution of molecular hydrogen and its emission is sensitive to the total mass. The detection of hydrogen deuteride, combined with existing observations and detailed models, implies a disk mass of more than 0.05 solar masses, which is enough to form a planetary system like our own.