Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization.     

Towards a biogeographic regionalization of the European biota

Aim To determine if it is possible to generate analytically derived regionalizations for multiple groups of European plants and animals and to explore potential influences on the regions for each taxonomic group. Location Europe. Methods We subjected range maps of trees, butterflies, reptiles, amphibians, birds and mammals to k‐means clustering followed by v‐fold cross‐validation to determine the pattern and number of regions (clusters). We then used the mean range sizes of species in each group as a correlate of the number of regions obtained for each taxon, and climate and species richness gradients as correlates of the spatial arrangement of the group‐specific regions. We also included the pattern of tree clusters as a predictor of animal clusters in order to test the ‘habitat templet’ concept as an explanation of animal distribution patterns. Results Spatially coherent clusters were found for all groups. The number of regions ranged from three to eight and was strongly associated with the mean range sizes of the species in each taxon. The cluster patterns of all groups were associated with various combinations of climate, underlying species richness gradients and, in the case of animals, the arrangement of tree clusters, although the rankings of the correlates differed among groups. In four of five groups the tree pattern was the strongest single predictor of the animal cluster patterns. Main conclusions Despite a long history of human disturbance and habitat modification, the European biota retains a discernable biogeographic structure. The primary driver appears to be aspects of climate related to water–energy balance, which also influence richness gradients. For many animals, the underlying habitat structure, as measured by tree distributions, appears to have a strong influence on their biogeographic structure, highlighting the need to preserve natural forest formations if we want to preserve the historical signal found in geographic distributions.     

Synthesis of Bacterial Flagella I. Requirement for Protein and Ribonucleic Acid Synthesis During Flagellar Regeneration in Bacillus subtilis

A relatively simple immunochemical procedure for estimating flagellar protein was developed. This procedure involved measuring the binding of purified, radioactively labeled, antiflagellar antibodies to bacteria. The assay was used to determine the requirements for ribonucleic acid (RNA) and protein synthesis during flagellar regeneration in Bacillus subtilis. Immediate inhibition of flagella development was observed when chloramphenical or puromycin was added to cells. This inhibition indicated the absence of a large pool of flagella precursors that could be assembled in the absence of protein synthesis. When the cells were starved for uracil or treated with actinomycin D to inhibit RNA synthesis, the ability of the cells to regenerate flagella decayed with a half-life of 5.5 min. When B. subtilis auxotrophs were starved for tryptophan, they continued to synthesize flagella, although this process was also inhibited by actinomycin D. On the basis of these results, we concluded that (i) the system involved in flagellar regeneration does not have unusual metabolic stability, (ii) regeneration requires both concomitant protein and RNA syntheses, and (iii) B. subtilis continues to synthesize messenger RNA during tryptophan starvation.     

Intestinal inflammation caused by magnesium deficiency alters basal and oxidative stress-induced intestinal function

The aim of this study was to determine the effect of magnesium deficiency on small intestinal morphology and function. Rats were assigned to 4 groups and placed on magnesium sufficient or deficient diet for 1 or 3 weeks. Infiltration of neutrophils and mucosal injury were assessed in stained sections of small intestine. Magnesium deficiency alone induced a significant increase in neutrophil infiltration and increased vascular ICAM-1 expression, in the absence of changes in mucosal injury or expression of proinflammatory mediators. Magnesium deficiency was associated with hyposecretory epithelial cell responses and vascular macromolecular leak in the small intestine and lung, which was attributed partly to reduced expression of NOS-3. To determine the effect of hypomagnesmia on the intestinal responses to a known oxidative stress, groups of rats were randomized to either sham operation or superior mesenteric artery occlusion for 10 (non-injurious) or 30 (injurious) minutes followed by a 1- or 4-hour reperfusion period. In response to mesenteric ischemia/reperfusion, deficient rats showed exaggerated PMN influx, but similar mucosal injury. Intestinal ischemia in sufficient animals induced vascular macromolecular leak in the small intestine and lung at 4 hours of reperfusion, with levels similar to those observed in untreated deficient rats. Acute magnesium repletion of deficient rats 24 h before surgery attenuated the exaggerated inflammation in deficient rats. These data show that magnesium deficiency induced a subclinical inflammation in the small intestine in the absence of mucosal injury, but with significant functional changes in local and remote organs and increased sensitivity to oxidative stress. The opinions contained herein are those of the authors and are not to be construed as official policy or reflecting the views of the Department of Defense     

The interaction between propagule pressure,habitat suitability and density‐dependent reproduction in species invasion

Seedling recruitment limitations create a demographic bottleneck that largely determines the viability and structure of plant populations and communities, and pose a core restriction on the colonization of novel habitat. We use a shade‐tolerant, invasive grass, Microstegium vimineum, to examine the interplay between seed and establishment limitations – phenomena that together determine recruitment success but usually are investigated individually. We add increasing amounts of seed to microhabitats containing variable levels of leaf litter thickness – with reduced leaf litter simulating disturbance – to investigate whether reduced seed limitation overcomes the establishment limitation posed by litter cover. We do this across gradients in understory light, moisture and temperature, and quantify germination, survival, and then per capita adult biomass and reproduction in order to understand the implications for invasion across the landscape. We find that the combined effects of seed and establishment limitation influence recruitment; however, propagule pressure overwhelms the inhibitory effects of leaf litter thickness. Leaf litter reduces germination by 22–57% and seedling survival by 13–15% from that observed on bare soil. However, density‐dependent reproduction compensates as 1–3 plants can produce far more seeds (approx. 525) than are required for persistence. As such, just a few plants may establish in understory forest habitat and subsequently overwhelm establishment barriers with copious propagule production. These results, for a widespread, invasive plant, are consistent with the emerging perspective for native plants that seed and establishment limitation jointly influence recruitment. The ability for an exotic plant species to compensate for low population densities with high per capita seed production, that then overrides establishment limitations, makes its invasive potential daunting. Further work is required to test if this is a common mechanism underlying plant invasions.     

Pneumococci in the African Meningitis Belt: Meningitis Incidence and Carriage Prevalence in Children and Adults

Background

The development of optimal vaccination strategies for pneumococcal conjugate vaccines requires serotype-specific data on disease incidence and carriage prevalence. This information is lacking for the African meningitis belt.

Methods

We conducted hospital-based surveillance of acute bacterial meningitis in an urban and rural population of Burkina Faso during 2007–09. Cerebrospinal fluid was evaluated by polymerase chain reaction for species and serotype. In 2008, nasopharyngeal swabs were obtained from a representative population sample (1 month to 39 years; N = 519) and additional oropharyngeal swabs from 145 participants. Swabs were evaluated by culture.

Results

Annual pneumococcal meningitis incidence rates were highest among <6-month-old (58/100,000) and 15- to 19-year-old persons (15/100,000). Annual serotype 1 incidence was around 5/100,000 in all age groups. Pneumococcal carriage prevalence in nasopharyngeal swabs was 63% among <5-year-old children and 22% among ≥5-year-old persons, but adding oropharyngeal to nasopharyngeal swabs increased the estimated carriage prevalence by 60%. Serotype 1 showed high propensity for invasive disease, particularly among persons aged ≥5 years.

Conclusions

Serotype 1 causes the majority of cases with a relatively constant age-specific incidence. Pneumococcal carriage is common in all age groups including adults. Vaccination programs in this region may need to include older target age groups for optimal impact on disease burden.     

Glycogen concentrations and endurance capacity of rats with chronic heart failure

The endurance capacities of rats with myocardial infarctions (MI) and of rats having undergone sham operations (SHAM) were tested during a submaximal exercise regimen that consisted of swimming to exhaustion. During this test, a decrement in the endurance capacity of the MI rat was demonstrated as the SHAM rat swam 25% longer than the MI rat (65 +/- 4 vs. 52 +/- 4 min). Glycogen concentrations were measured in the liver and the white gastrocnemius, plantaris, and soleus muscles of SHAM and MI rats that were randomly divided into four subgroups, which consisted of resting control, swim to exhaustion, swim to exhaustion + 24 h recovery, and swim to exhaustion + 24 h recovery + a second swim to exhaustion. The results demonstrated that the glycogen concentrations found in the liver, white gastrocnemius, plantaris, and soleus muscles of the SHAM and MI rats belonging to the resting control groups were similar. After swimming to exhaustion the glycogen concentrations in these tissues were significantly reduced compared with those found in the resting control groups of rats, and after 24 h of recovery the glycogen concentrations in these tissues were again similar to those found in the resting control groups of rats. Since the magnitude of the glycogen depletion in the liver and the white gastrocnemius, plantaris, and soleus muscles was similar in the SHAM and MI rats and because the SHAM rats consistently swam for longer periods of time in each of the experimental groups, it would be logical to assume that the rates of glycogen utilization for the various tissues may have been greater in the MI rat during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Transmitter Release from Nerve Terminals Isolated from Basal Ganglia and Substantia Nigra

Abstract: The K⁺-induced release of amino acids and dopamine from synaptosomes of basal ganglia and substantia nigra of sheep was studied. K⁺ (56 mM) caused an increase in the release of GABA from caudate, putamen, globus pallidus, and substantia nigra, the increased release being 227, 171, 198, and 366%, respectively, compared with samples incubated without stimulation. The release of glutamate was also increased by 56 mM-K⁺ (136–183%) from all regions except the globus pallidus, and a significant release of aspartate was only seen in response to K⁺ stimulation of synaptosomes from putamen (50%). Veratrine (75 μM) also stimulated a similar pattern of amino acid release from these regions. Regional correlation was shown between the presence of an uptake system for an amino acid and its evoked release. [¹⁴C]Dopamine formed from L-[U-¹⁴C]tyrosine was released only from caudate and putamen synaptosomes by K⁺ stimulation, the increases being 105% and 74%, respectively. Synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine occurred only in synaptosomes prepared from these two regions and was not detected in synaptosomes from substantia nigra or globus pallidus although whole-tissue homogenates of substantia nigra were able to synthesise dopamine.