Determinants Flanking the CD4 Binding Loop Modulate Macrophage Tropism of Human Immunodeficiency Virus Type 1 R5 Envelopes

Human immunodeficiency virus type 1 R5 viruses vary extensively in phenotype. Thus, R5 envelopes (env) in the brain tissue of individuals with neurological complications are frequently highly macrophage-tropic. Macrophage tropism correlates with the capacity of the envelope to exploit low CD4 levels for infection. In addition, the presence of an asparagine at residue 283 within the CD4 binding site has been associated with brain-derived envelopes, increased env-CD4 affinity, and enhanced macrophage tropism. Here, we identify additional envelope determinants of R5 macrophage tropism. We compared highly macrophage-tropic (B33) and non-macrophage-tropic (LN40) envelopes from brain and lymph node specimens of one individual. We first examined the role of residue 283 in macrophage tropism. Introduction of N283 into LN40 (T283N) conferred efficient macrophage infectivity. In contrast, substitution of N283 for the more conserved threonine in B33 had little effect on macrophage infection. Thus, B33 carried determinants for macrophage tropism that were independent of N283. We prepared chimeric B33/LN40 envelopes and used site-directed mutagenesis to identify additional determinants. The determinants of macrophage tropism that were identified included residues on the CD4 binding loop flanks that were proximal to CD4 contact residues and residues in the V3 loop. The same residues affected sensitivity to CD4-immunoglobulin G inhibition, consistent with an altered env-CD4 affinity. We predict that these determinants alter exposure of CD4 contact residues. Moreover, the CD4 binding loop flanks are variable and may contribute to a general mechanism for protecting proximal CD4 contact residues from neutralizing antibodies. Our results have relevance for env-based vaccines that will need to expose critical CD4 contact residues to the immune system.Human immunodeficiency virus type 1 (HIV-1) requires interactions between viral envelope glycoproteins and cell surface CD4 and coreceptors to trigger fusion and entry into cells. HIV-1 R5 viruses that specifically use CCR5 as a coreceptor are those predominantly transmitted (3). Yet, our knowledge of R5 virus variation in different biological properties is still limited. In vivo, HIV-1 infection is limited mostly to cells that express CD4 and appropriate coreceptors. Thus, HIV-1 infects CD4⁺ T cells, monocyte/macrophage lineage cells, and dendritic cells. CCR5 is expressed on each of these cell lineages, although on T cells, CCR5 is restricted mainly to RO45⁺ memory cells (1, 16). Early in infection, R5 viruses target and decimate mucosal CD4⁺ memory T cells (2, 18, 26). R5 viruses are also predominant in tissues in which monocyte/macrophage lineage cells are prevalent, and several reports have described the presence of highly macrophage-tropic R5 viruses in brain tissue (11, 12, 20, 22). Previously, we used PCR to amplify HIV-1 envelope genes directly from patient tissues. We found that R5 virus envelopes amplified from brain tissue frequently conferred highly efficient infection of macrophages, while the majority of those from lymph nodes, blood, and semen infected macrophages inefficiently (20, 22). Although those studies examined relatively few infected individuals, they demonstrated over 1,000-fold variation in macrophage-tropic HIV-1 R5 viruses. Such variation is likely to have a significant impact on transmission and pathogenesis.The envelope (env) determinants of R5 macrophage-tropic strains are poorly understood. Several studies have shown that highly macrophage-tropic brain envelopes are able to exploit low levels of CD4 on macrophages for infection, consistent with an enhanced interaction between gp120 and CD4. Dunfee et al. reported that an asparagine residue at position 283 in the C2 part of the CD4 binding site was present in 41% of envelope sequences from brain tissue specimens of patients with HIV-associated dementia and in only 8% of envelopes from non-HIV-associated dementia brain tissue (8). The same study showed that the presence of N283 (rather than the more conserved T283) led to an increased affinity of gp120 for CD4, probably because the side chain of asparagine could more readily form a hydrogen bond with Q40 on CD4. However, our previous data showed that N283 is not the only determinant of macrophage infectivity, since several macrophage-tropic R5 envelopes from brain and semen specimens lacked N283, while non-macrophage-tropic envelopes from lymph node specimens carrying N283 were identified (22). Dunfee et al. also reported that a glycosylation site at residue 386, close to the CD4 binding loop, influenced exposure of the CD4 binding site and had an impact on macrophage tropism and sensitivity to the CD4 binding site antibody b12 (9). We have recently confirmed a role for N386 in resistance to the CD4 binding site monoclonal antibody (MAb) b12. However, resistance was dependent on the presence of a proximal residue, R373, which acted together with N386 to block b12 (7).Here, we have further investigated envelope determinants of macrophage tropism by preparing chimeric envelopes from highly macrophage-tropic and non-macrophage-tropic R5 envelopes from brain and lymph node specimens from the same subject. We show that R5 macrophage tropism is controlled by several determinants in gp120 that are focused on amino acids flanking the CD4 binding loop, with a contribution from residues in the V3 loop.     

A small molecule inhibitor of alpha4 integrin-dependent cell migration

A small molecule inhibitor of alpha4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with gamma-parvin. This molecule should serve as a useful tool to study alpha4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune diseases.     

Integration of the barley genetic and seed proteome maps for chromosome 1H, 2H, 3H, 5H and 7H

Two-dimensional gel electrophoresis was used to screen spring barley cultivars for differences in seed protein profiles. In parallel, 72 microsatellite (simple sequence repeat (SSR)) markers and 11 malting quality parameters were analysed for each cultivar. Over 60 protein spots displayed cultivar variation, including peroxidases, serpins and proteins with unknown functions. Cultivars were clustered based on the spot variation matrix. Cultivars with superior malting quality grouped together, indicating malting quality to be more closely correlated with seed proteomes than with SSR profiles. Mass spectrometry showed that some spot variations were caused by amino acid differences encoded by single nucleotide polymorphisms (SNPs). Coding SNPs were validated by mass spectrometry, expressed sequence tag and 2D gel data. Coding SNPs can alter function of affected proteins and may thus represent a link between cultivar traits, proteome and genome. Proteome analysis of doubled haploid lines derived from a cross between a malting (Scarlett) and a feed cultivar (Meltan) enabled genetic localisation of protein phenotypes represented by 48 spot variations, involving e.g. peroxidases, serpins, α-amylase/trypsin inhibitors, peroxiredoxin and a small heat shock protein, in relation to markers on the chromosome map. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Genome-wide Association Study of Lung Cancer Identifies a Region of Chromosome 5p15 Associated with Risk for Adenocarcinoma

Three genetic loci for lung cancer risk have been identified by genome-wide association studies (GWAS), but inherited susceptibility to specific histologic types of lung cancer is not well established. We conducted a GWAS of lung cancer and its major histologic types, genotyping 515,922 single-nucleotide polymorphisms (SNPs) in 5739 lung cancer cases and 5848 controls from one population-based case-control study and three cohort studies. Results were combined with summary data from ten additional studies, for a total of 13,300 cases and 19,666 controls of European descent. Four studies also provided histology data for replication, resulting in 3333 adenocarcinomas (AD), 2589 squamous cell carcinomas (SQ), and 1418 small cell carcinomas (SC). In analyses by histology, rs2736100 (TERT), on chromosome 5p15.33, was associated with risk of adenocarcinoma (odds ratio [OR] = 1.23, 95% confidence interval [CI] = 1.13–1.33, p = 3.02 × 10⁻⁷), but not with other histologic types (OR = 1.01, p = 0.84 and OR = 1.00, p = 0.93 for SQ and SC, respectively). This finding was confirmed in each replication study and overall meta-analysis (OR = 1.24, 95% CI = 1.17–1.31, p = 3.74 × 10⁻¹⁴ for AD; OR = 0.99, p = 0.69 and OR = 0.97, p = 0.48 for SQ and SC, respectively). Other previously reported association signals on 15q25 and 6p21 were also refined, but no additional loci reached genome-wide significance. In conclusion, a lung cancer GWAS identified a distinct hereditary contribution to adenocarcinoma.     

The adaptive value of cued seed dispersal in desert plants: Seed retention and release in Mammillaria pectinifera (Cactaceae), a small globose cactus

Serotiny, or delayed seed dispersal, is common in fluctuating environments because it hedges the risks of establishment. Mammillaria pectinifera (Cactaceae) facultatively expels fruits in the year they are produced or retains them to disperse the seed over several years. We tested whether M. pectinifera increased fruit expulsion as a response to increased rainfall. While no fruit expulsion was observed in 1997, a dry year, in the wetter 1998 around 20% of all fruits formed were expelled from the maternal plant. A greenhouse experiment showed that high moisture results in the plants expelling all their fruits. Because in 1998 establishment was five times higher than in 1997, this response seems to be highly adaptive: Active fruit expulsion and consequent seed release increases the probability of establishment during pulses of high precipitation.     

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ_(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ_(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.     

Alternative Translation Initiation Generates Cytoplasmic Sheep Prion Protein

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein (^GFPPrP) in N2a cells, with variable sequence context surrounding the start codon Met¹. ^GFPPrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of ^GFPPrP were detected intracellularly, starting in frame from Met¹⁷. When ^GFPPrP was expressed with a compromised Kozak sequence (^GFPPrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of ^GFPPrP*, whereas the N-terminal fragments starting from Met¹⁷ were still present. Formation of these N-terminal fragments was completely abolished when Met¹⁷ was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met¹⁷ is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.PrP^{C 2} is a cell surface glycoprotein with an essential role in the pathogenesis of transmissible neurodegenerative prion diseases (1, 2). According to the prion hypothesis, a misfolded, pathogenic form of the protein (PrP^Sc) is the sole constituent of transmissible prions (3, 4), but the molecular details and required environs for the misfolding are incompletely understood. As would be expected for a glycosylphosphatidylinositol-anchored protein with N-linked glycans, PrP^C is observed at the outer leaflet of the plasma membrane, the end point of the secretory route. The half-time at the plasma membrane is fairly short, because the protein may undergo shedding or endocytic internalization (5–9). Thus, PrP^C can be encountered throughout the secretory and endocytic routes and is also able to leave cells via exosomes derived from multivesicular endosomes (10). In agreement with this, studies of the subcellular distribution of PrP^C in mammalian brain have identified localization to the outer cell membrane, in the Golgi apparatus, and in endosomal vesicles (11, 12). However, others have found that PrP^C is not solely associated with membranes, but, in some subpopulations of neurons, is localized to the cytoplasm (13, 14). In line with the latter observations, transgenic mice expressing PrP carrying a C-terminal GFP tag demonstrated intense cytoplasmic fluorescence from a limited number (approximately 1%) of the neurons in certain brain areas, such as the hippocampus (15). Immunohistochemical detection of intracellular, possibly cytoplasmic, PrP has also been reported from large mononuclear cells in the gut wall of sheep (16) and from enteric neurons in mice (17). The recent observations of pronounced cytoplasmic aggregation of PrP in pancreatic β-cells of rats prone to development of diabetes mellitus provide a perplexing example of nonstandard PrP localization in non-neuronal cells (18).The flexibility observed in the subcellular localization of PrP^C has been suggested to be a requirement for normal functions of the protein (14, 19, 20), but how cytoplasmic and nuclear variants arise has not been established. Cytoplasmic PrP could be a result of retro-translocation from the endoplasmic reticulum (ER), as part of an unfolded protein response (21–23) or from attenuated ER import of PrP under conditions of lumenal stress in the ER (24, 25). The finding of intact ER-targeting signal sequences on cytoplasmic PrPs (25, 26) favors the latter mechanism, namely a reduced ER import of PrP, possibly caused by saturation of the ER translocation machinery or an overload of unfolded proteins within the ER. However, no signs of stress or pathology could be detected in neurons of wild-type mice expressing cytoplasmic PrP (14), which led to the suggestion that the cytoplasmic appearance of PrP could constitute a physiologically relevant, but minor, pathway for the protein.Forced cytoplasmic expression of PrP in transgenic mice (22) and in the nematode Caenorhabditis elegans (27) resulted in neurodegenerative disease, suggesting that toxic mislocalization of PrP could be part of the pathogenic mechanism in prion diseases (28). However, transgenic mice expressing cytoplasmic PrP, on a PrP-null background, developed cerebellar atrophy but were resistant to experimental prion infection (29), suggesting that cytoplasmic PrP is unlikely to serve as substrate for prion replication. Furthermore, data obtained from transgenic mice expressing an anchorless secretory PrP show that, although these mice accumulate PrP-containing amyloid plaques upon challenge with PrP^Sc, they fail to develop clinical prion disease (30). Thus, membrane-attached PrP appears to be a prerequisite for development of prion-derived neurodegeneration.In eukaryotes, ribosomes bind specifically to linear mRNAs carrying a 7-methylguanosine 5′-end cap and slide along the mRNA in the 5′ → 3′ direction until they encounter the first start codon (AUG), from which the protein translation starts exclusively. Therefore, eukaryotic mRNAs are generally monocistronic. However, deviations from this standard principle have been reported, in which protein translation is initiated at alternative start codons either up or downstream from the primary AUG. The best characterized mechanism is known as context-dependent leaky ribosomal scanning (LRS) (31). This cap-dependent mechanism is particularly operative when the optimal (5′-GCCRCCaugG-3′) sequence context surrounding the first AUG codon is compromised, most notably at positions R⁻³ (where R= purine, A or G, but optimally G) and G⁺⁴ (32, 33).In this work, we report that in a cell culture system, sheep PrP mRNA displays a tendency to allow alternative translation initiation through LRS. Met¹⁷ serves as an internal in-frame alternative start codon giving rise to PrP with a severely shortened ER-targeting peptide.Although the LRS mechanism is active in sheep PrP, it appears to occur much less in mouse PrP (34). The molecular explanation and possible pathophysiological relevance of these observations in relation to PrP function await further studies. Interestingly, during the review process of this paper, observations of cytoplasmic PrP similar to some of those described herein were reported for human and hamster PrP (35).     

A multi-component LC–MS/MS method for detection of ten plant-derived psychoactive substances in urine

A sensitive and specific LC–MS/MS method for simultaneous detection of 10 plant-derived psychoactive substances (atropine, N,N-dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine and yohimbine) in urine was developed. Direct injection of urine diluted with 3 deuterated internal standards allowed for a readily accessible method suitable for application in clinical intoxication cases. Separation was achieved using reversed phase chromatography and gradient elution with a total analysis time of 14 min. Electrospray ionization was used and ions were monitored in the positive selected reaction monitoring mode. The calibration curves were linear (r² > 0.999) and the total imprecision at high (1000 μg/L) and low (50 μg/L) substance concentrations were 4.9–13.8% and 8.3–26%, respectively. Infusing the analytes post column and injecting matrix samples showed limited influence by ion suppression. The multi-component method proved to be useful for investigation of authentic cases of intoxication with plant-derived psychoactive drugs and was indicated to cover the clinically relevant concentration ranges.