Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release

<p>Abstract</p> <p>Background</p> <p>The ALG2-interacting protein X (ALIX)/AIP1 is an adaptor protein with multiple functions in intracellular protein trafficking that plays a central role in the biogenesis of enveloped viruses. The ubiquitin E3-ligase POSH (plenty of SH3) augments HIV-1 egress by facilitating the transport of Gag to the cell membrane. Recently, it was reported, that POSH interacts with ALIX and thereby enhances ALIX mediated phenotypes in <it>Drosophila</it>.</p> <p>Results</p> <p>In this study we identified ALIX as a POSH ubiquitination substrate in human cells: POSH induces the ubiquitination of ALIX that is modified on several lysine residues <it>in vivo </it>and <it>in vitro</it>. This ubiquitination does not destabilize ALIX, suggesting a regulatory function. As it is well established that ALIX rescues virus release of L-domain mutant HIV-1, HIV-1Δ_PTAP, we demonstrated that wild type POSH, but not an ubiquitination inactive RING finger mutant (POSH^V14A), substantially enhances ALIX-mediated release of infectious virions derived from HIV-1Δ_PTAPL-domain mutant (YPX_nL-dependent HIV-1). In further agreement with the idea of a cooperative function of POSH and ALIX, mutating the YPX_nL-ALIX binding site in Gag completely abrogated augmentation of virus release by overexpression of POSH. However, the effect of the POSH-mediated ubiquitination appears to be auxiliary, but not necessary, as silencing of POSH by RNAi does not disturb ALIX-augmentation of virus release.</p> <p>Conclusion</p> <p>Thus, the cumulative results identified ALIX as an ubiquitination substrate of POSH and indicate that POSH and ALIX cooperate to facilitate efficient virus release. However, while ALIX is obligatory for the release of YPX_nL-dependent HIV-1, POSH, albeit rate-limiting, may be functionally interchangeable.</p

Geospatial Information Research: State of the Art, Case Studies and Future Perspectives

Geospatial information science (GI science) is concerned with the development and application of geodetic and information science methods for modeling, acquiring, sharing, managing, exploring, analyzing, synthesizing, visualizing, and evaluating data on spatio-temporal phenomena related to the Earth. As an interdisciplinary scientific discipline, it focuses on developing and adapting information technologies to understand processes on the Earth and human-place interactions, to detect and predict trends and patterns in the observed data, and to support decision making. The authors – members of DGK, the Geoinformatics division, as part of the Committee on Geodesy of the Bavarian Academy of Sciences and Humanities, representing geodetic research and university teaching in Germany – have prepared this paper as a means to point out future research questions and directions in geospatial information science. For the different facets of geospatial information science, the state of art is presented and underlined with mostly own case studies. The paper thus illustrates which contributions the German GI community makes and which research perspectives arise in geospatial information science. The paper further demonstrates that GI science, with its expertise in data acquisition and interpretation, information modeling and management, integration, decision support, visualization, and dissemination, can help solve many of the grand challenges facing society today and in the future

Nighttime wind and scalar variability within and above an Amazonian canopy

Nocturnal turbulent kinetic energy (TKE) and fluxes of energy, CO2 and O3 between the Amazon forest and the atmosphere are evaluated for a 20-day campaign at the Amazon Tall Tower Observatory (ATTO) site. The distinction of these quantities between fully turbulent (weakly stable) and intermittent (very stable) nights is discussed. Spectral analysis indicates that low-frequency, nonturbulent fluctuations are responsible for a large portion of the variability observed on intermittent nights. In these conditions, the lowfrequency exchange may dominate over the turbulent transfer. In particular, we show that within the canopy most of the exchange of CO2 and H2O happens on temporal scales longer than 100 s. At 80 m, on the other hand, the turbulent fluxes are almost absent in such very stable conditions, suggesting a boundary layer shallower than 80 m. The relationship between TKE and mean winds shows that the stable boundary layer switches from the very stable to the weakly stable regime during intermittent bursts of turbulence. In general, fluxes estimated with long temporal windows that account for low-frequency effects are more dependent on the stability over a deeper layer above the forest than they are on the stability between the top of the canopy and its interior, suggesting that low-frequency processes are controlled over a deeper layer above the forest. © Author(s) 2018

The Amazon Tall Tower Observatory (ATTO): Overview of pilot measurements on ecosystem ecology, meteorology, trace gases, and aerosols

The Amazon Basin plays key roles in the carbon and water cycles, climate change, atmospheric chemistry, and biodiversity. It has already been changed significantly by human activities, and more pervasive change is expected to occur in the coming decades. It is therefore essential to establish long-term measurement sites that provide a baseline record of present-day climatic, biogeochemical, and atmospheric conditions and that will be operated over coming decades to monitor change in the Amazon region, as human perturbations increase in the future. The Amazon Tall Tower Observatory (ATTO) has been set up in a pristine rain forest region in the central Amazon Basin, about 150 km northeast of the city of Manaus. Two 80 m towers have been operated at the site since 2012, and a 325 m tower is nearing completion in mid-2015. An ecological survey including a biodiversity assessment has been conducted in the forest region surrounding the site. Measurements of micrometeorological and atmospheric chemical variables were initiated in 2012, and their range has continued to broaden over the last few years. The meteorological and micrometeorological measurements include temperature and wind profiles, precipitation, water and energy fluxes, turbulence components, soil temperature profiles and soil heat fluxes, radiation fluxes, and visibility. A tree has been instrumented to measure stem profiles of temperature, light intensity, and water content in cryptogamic covers. The trace gas measurements comprise continuous monitoring of carbon dioxide, carbon monoxide, methane, and ozone at five to eight different heights, complemented by a variety of additional species measured during intensive campaigns (e.g., VOC, NO, NO2, and OH reactivity). Aerosol optical, microphysical, and chemical measurements are being made above the canopy as well as in the canopy space. They include aerosol light scattering and absorption, fluorescence, number and volume size distributions, chemical composition, cloud condensation nuclei (CCN) concentrations, and hygroscopicity. In this paper, we discuss the scientific context of the ATTO observatory and present an overview of results from ecological, meteorological, and chemical pilot studies at the ATTO site. © Author(s) 2015

Functional and molecular characterization of the HIV-1 accessory protein Vpr with focus on virus - host interaction

Ziel dieser Arbeit war es am Beispiel von HIV-1 die Abhängigkeit der Virusreplikation von zellulären Faktoren zu untersuchen. Die Interaktion viraler Komponenten mit genetisch stabilen Faktoren der Wirtszelle stellt einen bedeutenden Ansatzpunkt für die antiretrovirale Therapie dar. Durch Identifikation und Inhibition zellulärer Schnittstellen können Resistenzbildungen, wie sie in der Behandlung von HIV-1 Infektionen auftreten, vermieden werden. Das akzessorische Protein Vpr stellt in vielen Abschnitten des HIV-1 Replikationszyklus einen Interaktionspunkt zwischen dem Virus und verschiedenen zellulären Komponenten dar und ist daher ein entscheidender Faktor in der Infektion von Zellen der Monozyten/Makrophagen Linie. Diese stellen einen Teil des Virusreservoirs von HIV-1 im Patienten dar, das es zu eliminieren gilt. Der erste Teil dieser Arbeit baut auf vorangegangenen Studien zur Interaktion von HIV-1 CA mit der zellulären peptidyl-prolyl cis/trans Isomerase Cyclophilin A auf. Hier wurde die Replikation von HIV-1 in Zusammenhang mit der Inkorporation von Cyclophilin A in Virionen gebracht. In der vorliegenden Arbeit konnte gezeigt werden, dass die Replikation einer Cyclophilin A insensitiven HIV-1 Mutante durch die fehlende Interaktion von Cyclophilin A mit Vpr nicht weiter verringert wird. Die Replikation einer Cyclophilin A sensitiven Mutante konnte im verwendeten Zellsystem nicht auf den Einfluss der Vpr-Cyclophilin A Interaktion getestet werden. Der zweite Teil dieser Arbeit beschäftigt sich mit der Lokalisation von Vpr an der nukleären Membran der Wirtszelle und deren Einfluss auf die Induktion des für die Replikation von HIV-1 notwendigen Zellzyklusarrests in der G2 Phase. Konfokale Mikroskopiestudien belegten, dass die Lokalisation von Vpr an der Kernmembran spezifisch durch die Aktivität der zellulären Caspase 3 gesteuert wird. Eine Inhibition der Caspase 3 hatte die Akkumulation von Vpr an der Kernmembran zur Folge. Die Anreicherung von Vpr an der nukleären Membran in wt HIV-1 infizierten T-Zellen nach Caspase 3 Inhibition führte selektiv zu einer gesteigerten Induktion des Vpr abhängigen G2 Zellzyklusarrests. Die Sensitivität von Vpr gegenüber der Caspase 3 Aktivität steht dabei in Abhängigkeit zu der Integrität der N-terminalen Helix α-1. Eine verringerte Induktion der Apoptose durch Vpr nach Caspase 3 Inhibition bei gleichzeitiger Erhöhung des G2 Zellzyklusarrests propagiert die funktionelle Trennung der beiden Funktionen des akzessorischen Proteins. Der selektive Einfluss der Caspase 3 Inhibition auf die Lokalisation von Vpr und die Apoptoseinduktion wurde durch Versuche zur Stabilität und Virion-Inkorporation von Vpr bestätigt. Beide Funktionen zeigen sich insensitiv gegenüber Inhibitoren von Caspase 3. Eine Auswirkung der Caspase 3 Aktivität auf die HIV-1 Virusreplikation konnte im Zuge dieser Arbeit sowohl in PBMCs als auch in Makrophagen nicht gezeigt werden. Der letzte Teil dieser Arbeit untersucht die Freisetzung neu synthetisierter HIV-1 Partikel aus der Wirtszelle in Abhängigkeit der L-Domänen Aktivität von HIV-1 p6. Durch Überexpression des zellulären Faktors ALIX und dessen Bindung an die sekundäre YPXnL-Domäne, welche mit dem Vpr-Bindemotiv in p6 überlappt, konnte der Phänotyp einer Deletion der primären PTAP-Domäne in HIV 1 revidiert und somit dieser Mechanismus bestätigt werden. Die Virusfreisetzung und Infektiosität der Mutante waren nach ALIX Überexpression annähernd vergleichbar mit der Wildtyp Variante. Die in den Vorversuchen generierten und getesteten Mutanten konnten anschließend in infektiöse und nicht infektiöse Vektorsysteme kloniert werden. In weiterführenden Versuchen wurde die Funktion von ALIX in der HIV-1 Freisetzung in Abhängigkeit zu der zellulären RING Finger E3 Ubiquitinligase POSH gebracht. Durch Ubiquitinylierung von ALIX durch POSH wurde der Effekt von ALIX auf die Virusfreisetzung einer HIV-1ΔPTAP Mutante deutlich verstärkt.The aim of this thesis was to analyze the dependence of the HIV-1 replication on cellular factors. Here, the interaction of viral factors with genetically stable components of the host cell provides considerable targets for antiretroviral therapy. By identification and inhibition of host cellular interaction interfaces it might be possible to circumvent the occurrence of resistant virus variants, as observed in conventional HIV-1 treatment. The accessory HIV-1 protein Vpr interacts with a number of host cell proteins during various stages of virus replication. Furthermore, Vpr is an important factor in the infection of terminally differentiated cells of the monocyte/macrophage lineage. These cell types, among others, form the virus reservoir in patients. For the eradication of the virus from the patient, this reservoir has to be eliminated. The first part of this thesis is based on earlier studies regarding the interaction of HIV-1 CA and the host cell cis/trans peptidyl-prolyl isomerase Cyclophilin A. The HIV-1 replication was examined in the context of the incorporation of Cyclophilin A in budding virions. Here it is shown, that inhibition of the Cyclophilin A - Vpr interaction has no influence on the replication of a Cyclophilin A insensitive HIV-1 mutant. However, a Cyclophilin A sensitive mutant could not be analyzed for its replication in the used cell system. In the second part of this thesis the binding of Vpr to the nuclear envelope was analyzed for its implications regarding the Vpr-induced G2 cell cycle arrest that was postulated to be important for HIV-1 replication. In confocal microscopy studies it was shown that the localization of Vpr at the nuclear envelope is dependent on the activity of host cell caspase 3. The inhibition of caspase 3 lead to the accumulation of Vpr at the nuclear envelope. The increased amount of Vpr at the nuclear envelope selectively augmented the Vpr mediated G2 arrest in T-cells infected with wt HIV-1. Additionally, the sensitivity of Vpr for caspase 3 activity is regulated by the integrity of the N-terminal helix α-1 in Vpr. A reduced apoptosis induction that paralleled with an enhanced G2 arrest function of Vpr upon caspase 3 inhibition is a hint for two distinct functions of the accessory protein. Studies on virion incorporation and the stability of Vpr that were both not altered by inhibitor treatment indicated, that the effect of caspase 3 inhibition is specific for localization of Vpr and apoptosis induction. An influence of caspase 3 inhibition on HIV-1 replication could not be observed, neither in human PBMCs nor in macrophages. In the last part of this thesis the release of HIV-1 from host cells in the context of the L-domain activity regulated by the viral p6 protein was analyzed. First studies confirmed the known effect of rescuing the virus release of a PTAP-L-domain mutant by overexpression of the cellular ALIX protein. This rescue is due to the binding of ALIX to the secondary YPXnL-L-domain in HIV-1 p6 that overlaps with the Vpr binding site. ALIX overexpression recovered virus release and the infectivity of released virions. The mutants generated for the preliminary studies were afterwards successfully cloned in infectious and non-infectious vector systems for ongoing studies. In further studies the function of ALIX in HIV-1 release was analyzed in the context of the host cell E3 ubiquitin ligase POSH. The ubiquitination of ALIX by POSH further increased the effect of ALIX in the HIV-1 release

Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2-arrest

AbstractThe HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1NL4–3-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest

Modeling the Autoinhibition of Clarithromycin Metabolism during Repeated Oral Administration▿

Clarithromycin decreases CYP3A4 activity and thus gradually inhibits its own metabolism as well as that of coadministered drugs. The aim of this study was to obtain an understanding of the time course of these changes. The plasma concentration-time profiles of clarithromycin and its active metabolite, 14(R)-hydroxy-clarithromycin, in 12 young healthy volunteers after oral administration of a clarithromycin suspension (500 mg twice a day [b.i.d.] for seven doses) were modeled by population pharmacokinetic analysis in the NONMEM program. The nonlinearity of clarithromycin metabolism was considered during model development, and the metabolite disposition kinetics were assumed to be linear. The absorption kinetics of clarithromycin were best described by a Weibull function model. The pharmacokinetics of clarithromycin and its 14(R)-hydroxyl metabolite were adequately described by a one-compartment model each for clarithromycin and its metabolite as well as an inhibition compartment that reflects the autoinhibition of clarithromycin metabolism. Up to 90% of the apparent total clarithromycin clearance (60 liters/h) was susceptible to reversible autoinhibition, depending on the concentration in the inhibition compartment. The proposed semimechanistic population pharmacokinetic model successfully described the autoinhibition of clarithromycin metabolism and may be used to adjust the doses of other drugs that are metabolized by CYP3A4 and that are coadministered with clarithromycin. Simulations showed that for the standard dose of 500 mg b.i.d., no further increase in the level of exposure occurs after approximately 48 h of treatment. For a 1,000-mg b.i.d. dose, the achievement of steady state is expected to take several days and to achieve a 3.6-fold higher level of clarithromycin exposure than the 500-mg b.i.d. dose. This evaluation provides a rationale for safer and more effective therapy with clarithromycin

An Osteotomy Tool That Preserves Bone Viability: Evaluation in Preclinical and Clinical Settings

The main objectives of this work were to assess the efficiency, ease-of-use, and general performance of a novel osseoshaping tool based on first-user clinical experiences and to compare these observations with preclinical data generated in rodents using a miniaturized version of the instrument. All patients selected for the surgery presented challenging clinical conditions in terms of the quality and/or quantity of the available bone. The presented data were collected during the implant placement of 15 implants in 7 patients, and included implant recipient site (bone quality and quantity) and ridge evaluation, intra-operative handling of the novel instrument, and the evaluation of subsequent implant insertion. The instrument was easy to handle and was applied without any complications during the surgical procedure. Its use obviated the need for multiple drills and enabled adequate insertion torque in all cases. This biologically driven innovation in implant site preparation shows improvements in preserving vital anatomical and cellular structures as well as simplifying the surgical protocol with excellent ease-of-use and handling properties