hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling

The increased cap-independent translation of anti-apoptotic proteins is involved in the development of drug resistance in lung cancer but signalling events regulating this are poorly understood. Fibroblast growth factor 2 (FGF-2) signalling-induced S6 kinase 2 (S6K2) activation is necessary, but the downstream mediator(s) coupling this kinase to the translational response is unknown. Here, we show that S6K2 binds and phosphorylates hnRNPA1 on novel Ser4/6 sites, increasing its association with BCL-XL and XIAP mRNAs to promote their nuclear export. In the cytoplasm, phosphoS4/6-hnRNPA1 dissociates from these mRNAs de-repressing their IRES-mediated translation. This correlates with the phosphorylation-dependent association of hnRNPA1 with 14-3-3 leading to hnRNPA1 sumoylation on K183 and its re-import into the nucleus. A non-phosphorylatible, S4/6A mutant prevented these processes, hindering the pro-survival activity of FGF-2/S6K2 signalling. Interestingly, immunohistochemical staining of lung and breast cancer tissue samples demonstrated that increased S6K2 expression correlates with decreased cytoplasmic hnRNPA1 and increased BCL-XL expression. In short, phosphorylation on novel N-term sites of hnRNPA1 promotes translation of anti-apoptotic proteins and is indispensable for the pro-survival effects of FGF-2

Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling

TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear. We show that hexagonal assembly triggers N-terminal polyubiquitination of TRIM5 that collectively drives antiviral responses. In uninfected cells, N-terminal monoubiquitination triggers non-productive TRIM5 turnover. Upon TRIM5 assembly on virus, a trivalent RING arrangement allows elongation of N-terminally anchored K63-linked ubiquitin chains (N-K63-Ub). N-K63-Ub drives TRIM5 innate immune stimulation and proteasomal degradation. Inducing ubiquitination before TRIM5 assembly triggers premature degradation and ablates antiviral restriction. Conversely, driving N-K63 ubiquitination after TRIM5 assembly enhances innate immune signaling. Thus, the hexagonal geometry of TRIM5's antiviral lattice converts a capsid-binding protein into a multifunctional antiviral platform

A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles

© 2012 Tang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. Methodology/Findings: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. Conclusions: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 virusesThis work was supported by grants from the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070972), the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, and the RESPARI project of the Institut Pasteur International Network

Extensive Anti-CoA Immunostaining in Alzheimer’s Disease and Covalent Modification of Tau by a Key Cellular Metabolite Coenzyme A

Alzheimer’s disease (AD) is a neurodegenerative disorder, accounting for at least two-thirds of dementia cases. A combination of genetic, epigenetic and environmental triggers is widely accepted to be responsible for the onset and development of AD. Accumulating evidence shows that oxidative stress and dysregulation of energy metabolism play an important role in AD pathogenesis, leading to neuronal dysfunction and death. Redox-induced protein modifications have been reported in the brain of AD patients, indicating excessive oxidative damage. Coenzyme A (CoA) is essential for diverse metabolic pathways, regulation of gene expression and biosynthesis of neurotransmitters. Dysregulation of CoA biosynthesis in animal models and inborn mutations in human genes involved in the CoA biosynthetic pathway have been associated with neurodegeneration. Recent studies have uncovered the antioxidant function of CoA, involving covalent protein modification by this cofactor (CoAlation) in cellular response to oxidative or metabolic stress. Protein CoAlation has been shown to both modulate the activity of modified proteins and protect cysteine residues from irreversible overoxidation. In this study, immunohistochemistry analysis with highly specific anti-CoA monoclonal antibody was used to reveal protein CoAlation across numerous neurodegenerative diseases, which appeared particularly frequent in AD. Furthermore, protein CoAlation consistently co-localized with tau-positive neurofibrillary tangles, underpinning one of the key pathological hallmarks of AD. Double immunihistochemical staining with tau and CoA antibodies in AD brain tissue revealed co-localization of the two immunoreactive signals. Further, recombinant 2N3R and 2N4R tau isoforms were found to be CoAlated in vitro and the site of CoAlation mapped by mass spectrometry to conserved cysteine 322, located in the microtubule binding region. We also report the reversible H_{2}O_{2}-induced dimerization of recombinant 2N3R, which is inhibited by CoAlation. Moreover, CoAlation of transiently expressed 2N4R tau was observed in diamide-treated HEK293/Pank1β cells. Taken together, this study demonstrates for the first time extensive anti-CoA immunoreactivity in AD brain samples, which occurs in structures resembling neurofibrillary tangles and neuropil threads. Covalent modification of recombinant tau at cysteine 322 suggests that CoAlation may play an important role in protecting redox-sensitive tau cysteine from irreversible overoxidation and may modulate its acetyltransferase activity and functional interactions

Changing Selective Pressure during Antigenic Changes in Human Influenza H3

The rapid evolution of influenza viruses presents difficulties in maintaining the optimal efficiency of vaccines. Amino acid substitutions result in antigenic drift, a process whereby antisera raised in response to one virus have reduced effectiveness against future viruses. Interestingly, while amino acid substitutions occur at a relatively constant rate, the antigenic properties of H3 move in a discontinuous, step-wise manner. It is not clear why this punctuated evolution occurs, whether this represents simply the fact that some substitutions affect these properties more than others, or if this is indicative of a changing relationship between the virus and the host. In addition, the role of changing glycosylation of the haemagglutinin in these shifts in antigenic properties is unknown. We analysed the antigenic drift of HA1 from human influenza H3 using a model of sequence change that allows for variation in selective pressure at different locations in the sequence, as well as at different parts of the phylogenetic tree. We detect significant changes in selective pressure that occur preferentially during major changes in antigenic properties. Despite the large increase in glycosylation during the past 40 years, changes in glycosylation did not correlate either with changes in antigenic properties or with significantly more rapid changes in selective pressure. The locations that undergo changes in selective pressure are largely in places undergoing adaptive evolution, in antigenic locations, and in locations or near locations undergoing substitutions that characterise the change in antigenicity of the virus. Our results suggest that the relationship of the virus to the host changes with time, with the shifts in antigenic properties representing changes in this relationship. This suggests that the virus and host immune system are evolving different methods to counter each other. While we are able to characterise the rapid increase in glycosylation of the haemagglutinin during time in human influenza H3, an increase not present in influenza in birds, this increase seems unrelated to the observed changes in antigenic properties

Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses

<p>Abstract</p> <p>Background</p> <p>Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAα2,6Gal and Maackia amurensis agglutinin (MAA) for SAα2,3Gal in the respiratory tract of normal adults and children.</p> <p>Methods</p> <p>We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.</p> <p>Results</p> <p>We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.</p> <p>Conclusion</p> <p>The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAα2,3 binding viruses, such as influenza or human parainfluenza are to be made.</p

Towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method

Immunodiagnostic microneedles provide a novel way to extract protein biomarkers from the skin in a minimally invasive manner for analysis in vitro. The technology could overcome challenges in biomarker analysis specifically in solid tissue, which currently often involves invasive biopsies. This study describes the development of a multiplex immunodiagnostic device incorporating mechanisms to detect multiple antigens simultaneously, as well as internal assay controls for result validation. A novel detection method is also proposed. It enables signal detection specifically at microneedle tips and therefore may aid the construction of depth profiles of skin biomarkers. The detection method can be coupled with computerised densitometry for signal quantitation. The antigen specificity, sensitivity and functional stability of the device were assessed against a number of model biomarkers. Detection and analysis of endogenous antigens (interleukins 1α and 6) from the skin using the device was demonstrated. The results were verified using conventional enzyme-linked immunosorbent assays. The detection limit of the microneedle device, at ≤10 pg/mL, was at least comparable to conventional plate-based solid-phase enzyme immunoassays

Glycosylation Focuses Sequence Variation in the Influenza A Virus H1 Hemagglutinin Globular Domain

Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the “flow index” (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation

Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to “right next door”: one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors

Two Glycosylation Sites in H5N1 Influenza Virus Hemagglutinin That Affect Binding Preference by Computer-Based Analysis

Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs