Dendritic cell aggresome-like-induced structure formation and delayed antigen presentation coincide in influenza virus-infected dendritic cells

Influenza virus infection induces maturation of murine dendritic cells (DCs), which is most important for the initiation of an immune response. However, in contrast to EL-4 and MC57 cells, DCs present viral CTL epitopes with a delay of up to 10 h. This delay in Ag presentation coincides with the up-regulation of MHC class I molecules as well as costimulatory molecules on the cell surface and the accumulation of newly synthesized ubiquitinated proteins in large cytosolic structures, called DC aggresome-like-induced structures (DALIS). These structures were observed previously after LPS-induced maturation of DCs, and it was speculated that they play a role in the regulation of MHC class I Ag presentation. Our findings provide the first evidence for a connection between DC maturation, MHC class I-restricted Ag presentation, and DALIS formation, which is further supported by the observation that DALIS contain ubiquitinated influenza nucleoprotein.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.     

Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.Retrovirus assembly is driven by the oligomerization of Gag, a multidomain protein, including an N-terminal membrane binding domain (MA), a two-domain structural component (CA), and an RNA binding domain (NC). The Gag proteins of all orthoretroviruses, including the alpha-, beta-, and lentiretroviruses discussed here, share this conserved modular architecture (Fig. ​(Fig.1).1). Despite very weak sequence conservation, the tertiary structures of MA, CA, and NC are conserved among retroviruses. Outside these conserved domains the Gag proteins of different retroviruses exhibit substantial variability. Other domains may be present or absent, and the length and sequence of linker peptides may also vary (12) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Modular architecture of the full-length Gag proteins of HIV, M-PMV, and RSV. White rectangles illustrate Gag polyprotein cleavage products. The extent of the constructs used in the electron microscopic analysis is specified under each protein as a black rectangle. Gray triangles specify cleavage sites. Residue numbers are counted from the beginning of Gag.Oligomerization of Gag in an infected cell leads to the formation of roughly spherical immature virus particles, where Gag is arranged in a radial fashion with the N-terminal MA domain associated with a surrounding lipid bilayer, and the more C-terminal NC pointing toward the center of the particle (15, 44, 46). Subsequent multiple cleavages of Gag by the viral protease lead to a rearrangement of the virus. NC and the RNA condense in the center of the particle, CA assembles into a capsid or shell around the nucleoprotein, and MA remains associated with the viral membrane. This proteolytic maturation is required to generate an infectious virion (2). In contrast to the mature CA lattice, which has been extensively studied (11, 16, 36), the Gag lattice in immature particles is incompletely understood.Gag itself contains all of the necessary determinants for particle assembly. For example, the expression of Gag alone in an insect cell expression system is sufficient to generate viruslike particles (3, 17, 22, 38). Retroviral Gag proteins also can be assembled in vitro in the presence of nucleic acids to form spherical particles (9, 19, 39, 43, 47). The arrangement of Gag within these in vitro-assembled Gag particles is indistinguishable from that found in immature virus particles (6), and the in vitro assembly systems have proved valuable for unraveling the principles of virus assembly (18, 28, 29, 39). Multiple layers of interaction promote the assembly of Gag in vivo, including MA-membrane-MA interactions, CA-CA interactions, and NC-RNA-NC interactions. An extensive body of literature has explored which regions of Gag are required for assembly and which can be replaced or deleted without compromising assembly. MA-membrane-MA interactions contribute but are not essential. NC-RNA-NC interactions appear to function to nonspecifically link Gag molecules together and can be replaced both in vivo and in vitro by other interaction domains such as leucine zippers (4, 13, 20, 32, 48). The C-terminal domain of CA (referred to here as C-CA) and the stretch of amino acids immediately following this domain (termed the spacer peptide [SP] region) are critical for assembly and sensitive to mutation (1, 22, 27, 30).We set out to understand how the substantial sequence variation among Gag proteins in different retroviruses is manifested in structural differences in the immature Gag lattice. To do this, we studied three retroviruses from different genera: the lentivirus human immunodeficiency virus type 1 (HIV-1), the betaretrovirus Mason-Pfizer monkey virus (M-PMV), and the alpharetrovirus Rous sarcoma virus (RSV). These retroviruses are those for which in vitro assembly was first established and has been most extensively studied (6, 19, 24, 28, 29, 35, 43, 47).The domain structures of the three retroviruses differ most substantially upstream of CA. Both M-PMV and RSV have domains located between MA and CA that are absent in HIV (Fig. ​(Fig.1).1). In M-PMV there are 198 residues forming the pp24 and p12 domains; in RSV there are 84 residues forming the p2a, p2b, and p10 domains. The three retroviruses have different requirements for regions upstream of CA during assembly. The C-terminal 25 residues of p10 are essential for proper immature RSV assembly, both in vitro and in vivo, and these residues are inferred to interact directly with N-CA to stabilize the hexamer by forming contacts between adjacent N-CA domains (35). An equivalent assembly domain has not been described for other retroviruses. Within M-PMV p12 is the so-called internal scaffolding domain that is not essential for assembly in vitro (43) but is required for particle assembly when the precursor is expressed under the control of the M-PMV promoter (41). It is a key domain for the membrane-independent assembly of immature capsids (40).In HIV, five residues upstream of CA must be present for assembly of immature virus-like spherical particles in vitro, although larger upstream extensions, including part of MA, are required for efficient assembly of regular particles, both for HIV and RSV. For HIV, if the entire MA domain is included, in vitro assembly requires the presence of inositol penta- or hexakis phosphate (8). If no sequences upstream of CA are present, the in vitro particles in both of these viruses adopt a mature-type tubular morphology (10, 18). It has been hypothesized that cleavage at the N terminus of N-CA during maturation leads to the N-terminal residues of CA folding back into the N-CA structure to form a β-hairpin. The β-hairpin is important for assembly of the mature CA lattice, whereas its absence is important for immature assembly (23, 42). These requirements explain why, in HIV and RSV, immature Gag lattice-like structures are formed only if regions upstream of CA are present (18). In M-PMV, an immature Gag lattice can be produced when the regions upstream of CA are deleted if this is combined with mutations (such as deleting the initial proline of CA), which prevent β-hairpin formation (43).During maturation, HIV and RSV Gag proteins are cleaved twice between CA and NC to release a small peptide called SP1 or SP. In RSV the most N-terminal of these two cleavages can occur at one of two possible positions such that the released peptide is either 9 or 12 amino acids long (33). In M-PMV only one cleavage occurs between CA and NC, and no short peptide is produced. The region between the final helix of CA and the Zn fingers has been proposed to adopt a helical bundle architecture in HIV and RSV based on bioinformatic prediction, on mutational analysis, and on structural studies (1, 22, 27, 45). In all three viruses, C-CA and the residues immediately downstream are critical for assembly and are sensitive to mutation. C-CA contains the major homology region, a group of residues that are highly conserved across the retroviruses.Cryo-electron tomography (cET) studies of immature virus particles (6, 45) have resolved the electron density of the HIV Gag lattice in three dimensions at low resolution. Using these methods, we have also described the three-dimensional architecture of in vitro-assembled HIV Gag particles (6). In immature viruses and in vitro-assembled particles, Gag is seen to adopt an 8 nm hexameric lattice, as was predicted from previous Fourier analysis of two-dimensional images (7, 46). The hexameric lattice is interrupted by irregularly shaped holes and cracks in the lattice (6, 45). A similar observation has been made using AFM of in vitro-assembled particles of M-PMV Gag (26). These holes and cracks allow an otherwise planar hexameric lattice to form the surface of an approximately spherical particle.The radial positions of the MA, CA, and NC domains had been assigned previously from cryo-electron micrographs (44, 46). Based on these assignments and the shape of the density, the position and relative orientations of CA domains can be modeled into the low-resolution structure of the HIV lattice (6, 45). Density ascribed to the N-terminal domain of CA (N-CA) forms rings around large holes at the 6-fold symmetry positions in the lattice. Below this layer, at the expected radius of the C-CA, are 2-fold densities, interpreted as corresponding to dimers of C-CA. These densities are linked by rodlike densities, which descend into the NC-nucleic acid layer.HIV is the only retrovirus for which the arrangement of Gag in the immature particle has been described in three dimensions. Prior to this work, important open questions were therefore: which features of the arrangement of Gag are conserved between genera and therefore reflect general principles of Gag-Gag interactions, and which features are specific to certain genera? We have applied subtomogram averaging of cryo-electron tomograms to generate reconstructions of in vitro-assembled Gag particles from HIV, M-PMV, and RSV. These allow identification of the general and variable features of the arrangement of Gag and the architecture of immature retroviruses.     

Model membrane studies for characterization of different antibiotic activities of lipopeptides from Pseudomonas

Lipopeptides (LPs) are a structurally diverse class of amphipathic natural products that were in the past mainly known for their surfactant properties. However, the recent discovery of their antimicrobial and cytotoxic bioactivities have fueled and renewed the interest in this compound class. Propelled by the antimicrobial potential of this compound class, in this study a range of six underinvestigated LPs from Pseudomonads were examined with respect to their antibiotic activities towards bacteria. The assays revealed that only the glycosylated lipodipeptide SB-253514, produced by Pseudomonas strain SH-C52, showed significant antibacterial activity. Since the bioactivity of LPs is commonly attributed to membrane interactions, we analyzed the molecular interactions between the LPs and bacteria-like lipid model membranes in more detail via complementary biophysical approaches. Application of the quartz crystal microbalance (QCM) showed that all LPs possess a high binding affinity towards the model membranes. Despite their similar membrane affinity, monolayer studies displayed different tendencies of LPs to incorporate into the membrane. The degree of membrane incorporation could be correlated with specific structural features of the investigated LPs, such as distance between the peptidic macrocycle and the fatty acid, but did not fully reflect their respective antibacterial activity. Cyclic voltammetry (CV) experiments further demonstrated that SB-253514 showed no membrane permeabilization effects at inhibitory concentrations. Collectively, these results suggests that the antibacterial activity of SB-253514 cannot be explained by an unspecific detergent-like mechanism generally proposed for amphiphilic molecules but instead appears to occur via a defined structural target.     

Tirucallane triterpenoids from the stem bark of Araliopsis synopsis

Two new tirucallane triterpenoids, 21-methoxy-21,23-epoxy-tirucalla-7,24-dien-3α-ol (1) and 21-methoxy-21,23-epoxy-tirucalla-7,24-diene-1α,3α-diol (2), together with thirteen known compounds were isolated from the CH₂Cl₂ extract of the stem bark of Araliopsis synopsis. The structures of the compounds were determined by comprehensive analyses of their 1D and 2D NMR, mass spectral (EI and ESI) data and comparison with previously known analogs. Compounds 1–10 were tested against bacteria, fungi and plant pathogen oomycetes by the paper disk agar diffusion assay resulting in missing to low activities corresponding with MICs > 1 mg/mL. However, compounds 5–10 exhibited high cytotoxic activity against the human Caucasian prostate adenocarcinoma cell PC-3 line, with IC₅₀ 8.5–12.5 μM compared to the standard Doxorubicin with IC₅₀ = 0.9 μM, while compounds 1, 3 and 4 showed low activity.     

Changes in neuronal DNA content variation in the human brain during aging

The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age-related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide-based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age-related CNS disorders. Alternatively, a potential selection bias of "healthy aging brains" needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer's disease.     

Erythropoietin mimetic compound AGEM400(HES) binds to the same receptor as erythropoietin but displays a different spectrum of activities

EPO mimetic peptides (EMPs) have a completely different structure than erythropoietin (EPO) or new generation recombinant erythropoiesis stimulating agents (ESAs) like Darbepoietin alfa (Aranesp) and continuous erythropoiesis stimulating agent (CERA). This study intended to compare the effects of a novel compound called AGEM400(HES), consisting of a dimeric EMP conjugated to hydroxyethyl starch (HES), to those of recombinant EPO. AGEM400(HES) efficiently stimulated erythropoiesis in vitro and efficiently stimulated survival of EPO-dependent cell line UT7/EPO. It also efficiently induced phosphorylation of signaling proteins in these models. However, AGEM400(HES) was shown to have weak or absent effects on survival of, and signaling in, three different EPO-responsive hematopoietic cell lines. In the latter models, when added in excess to moderate concentrations of EPO, AGEM400(HES) inhibited the activity of EPO in a fashion indicating receptor binding competition between EPO and AGEM400(HES). It was furthermore shown, using stably transfected BA/F3 cells, that the degree of responsiveness of a cell to AGEM400(HES) relative to its responsiveness to EPO, correlated with the level of EPO receptor surface expression. The findings presented raise intriguing possibilities because they imply that not all side-effects said to be associated with EPO must necessarily be elicited by AGEM400(HES) too.     

Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1

HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.