Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, 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, the RESPARI Pasteur Network

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

Parameter optimization by using differential elimination: a general approach for introducing constraints into objective functions

<p>Abstract</p> <p>Background</p> <p>The investigation of network dynamics is a major issue in systems and synthetic biology. One of the essential steps in a dynamics investigation is the parameter estimation in the model that expresses biological phenomena. Indeed, various techniques for parameter optimization have been devised and implemented in both free and commercial software. While the computational time for parameter estimation has been greatly reduced, due to improvements in calculation algorithms and the advent of high performance computers, the accuracy of parameter estimation has not been addressed. </p> <p>Results</p> <p>We propose a new approach for parameter optimization by using differential elimination, to estimate kinetic parameter values with a high degree of accuracy. First, we utilize differential elimination, which is an algebraic approach for rewriting a system of differential equations into another equivalent system, to derive the constraints between kinetic parameters from differential equations. Second, we estimate the kinetic parameters introducing these constraints into an objective function, in addition to the error function of the square difference between the measured and estimated data, in the standard parameter optimization method. To evaluate the ability of our method, we performed a simulation study by using the objective function with and without the newly developed constraints: the parameters in two models of linear and non-linear equations, under the assumption that only one molecule in each model can be measured, were estimated by using a genetic algorithm (GA) and particle swarm optimization (PSO). As a result, the introduction of new constraints was dramatically effective: the GA and PSO with new constraints could successfully estimate the kinetic parameters in the simulated models, with a high degree of accuracy, while the conventional GA and PSO methods without them frequently failed.</p> <p>Conclusions</p> <p>The introduction of new constraints in an objective function by using differential elimination resulted in the drastic improvement of the estimation accuracy in parameter optimization methods. The performance of our approach was illustrated by simulations of the parameter optimization for two models of linear and non-linear equations, which included unmeasured molecules, by two types of optimization techniques. As a result, our method is a promising development in parameter optimization. </p

Genetic characterization of H5N1 influenza viruses isolated from chickens in Indonesia in 2010

Since 2003, highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry in Indonesia every year, producing the highest number of human victims worldwide. However, little is known about the H5N1 influenza viruses that have been circulating there in recent years. We therefore conducted surveillance studies and isolated eight H5N1 viruses from chickens. Phylogenic analysis of their hemagglutinin and neuraminidase genes revealed that all eight viruses belonged to clade 2.1.3. However, on the basis of nucleotide differences, these viruses could be divided into two groups. Other viruses genetically closely related to these two groups of viruses were all Indonesian isolates, suggesting that these new isolates have been evolving within Indonesia. Among these viruses, two distinct viruses circulated in the Kalimantan islands during the same season in 2010. Our data reveal the continued evolution of H5N1 viruses in Indonesia

Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity.

Many genetic loci affect circulating lipid levels, but it remains unknown whether lifestyle factors, such as physical activity, modify these genetic effects. To identify lipid loci interacting with physical activity, we performed genome-wide analyses of circulating HDL cholesterol, LDL cholesterol, and triglyceride levels in up to 120,979 individuals of European, African, Asian, Hispanic, and Brazilian ancestry, with follow-up of suggestive associations in an additional 131,012 individuals. We find four loci, in/near CLASP1, LHX1, SNTA1, and CNTNAP2, that are associated with circulating lipid levels through interaction with physical activity; higher levels of physical activity enhance the HDL cholesterol-increasing effects of the CLASP1, LHX1, and SNTA1 loci and attenuate the LDL cholesterol-increasing effect of the CNTNAP2 locus. The CLASP1, LHX1, and SNTA1 regions harbor genes linked to muscle function and lipid metabolism. Our results elucidate the role of physical activity interactions in the genetic contribution to blood lipid levels

Adaptation of High-Growth Influenza H5N1 Vaccine Virus in Vero Cells: Implications for Pandemic Preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 108 TCID50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes

A Novel PAN/Apple Domain-Containing Protein from Toxoplasma gondii: Characterization and Receptor Identification

Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677) is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG) interaction. Chondroitin sulfate (CS), a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite

A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice

<p>Abstract</p> <p>Background</p> <p>Developing vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.</p> <p>Methods</p> <p>Mice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.</p> <p>Results</p> <p>Challenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.</p> <p>Conclusion</p> <p>A single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.</p

Elucidating the Mechanisms of Influenza Virus Recognition by Ncr1

Natural killer (NK) cells are innate cytotoxic lymphocytes that specialize in the defense against viral infection and oncogenic transformation. Their action is tightly regulated by signals derived from inhibitory and activating receptors; the later include proteins such as the Natural Cytotoxicity Receptors (NCRs: NKp46, NKp44 and NKp30). Among the NCRs, NKp46 is the only receptor that has a mouse orthologue named Ncr1. NKp46/Ncr1 is also a unique marker expressed on NK and on Lymphoid tissue inducer (LTI) cells and it was implicated in the control of various viral infections, cancer and diabetes. We have previously shown that human NKp46 recognizes viral hemagglutinin (HA) in a sialic acid-dependent manner and that the O-glycosylation is essential for the NKp46 binding to viral HA. Here we studied the molecular interactions between Ncr1 and influenza viruses. We show that Ncr1 recognizes influenza virus in a sialic acid dependent manner and that N-glycosylation is important for this binding. Surprisingly we demonstrate that none of the predicted N-glycosilated residues of Ncr1 are essential for its binding to influenza virus and we thus conclude that other, yet unidentified N-glycosilated residues are responsible for its recognition. We have demonstrated that N glycosylation play little role in the recognition of mouse tumor cell lines and also showed the in-vivo importance of Ncr1 in the control of influenza virus infection by infecting C57BL/6 and BALB/c mice knockout for Ncr1 with influenza

In vitro antimicrobial effects of aztreonam, colistin, and the 3-drug combination of aztreonam, ceftazidime and amikacin on metallo-β-lactamase-producing Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>There are limited choice of antimicrobial agents to treat infection with metallo-<it>β</it>-lactamase-producing <it>Pseudomonas aeruginosa</it>. We evaluate the antimicrobial effects of aztreonam alone, colistin alone and the 3-drug combination of aztreonam, ceftazidime and amikacin on 23 strains of metallo-<it>β</it>-lactamase-producing <it>P. aeruginosa </it>by time-killing tests.</p> <p>Methods</p> <p>Strains used were from different hospitals in Japan and had different pulse-field gel electrophoresis patterns by restriction with <it>Spe</it>I. The minimum inhibitory concentrations of 11 antimicrobial agents (piperacillin, piperacillin/tazobactam, imipenem, meropenem, aztreonam, ceftazidime, amikacin, tobramycin, arbekacin, ciprofloxacin and colistin) were determined using the agar dilution test. The effects of aztreonam, colistin and the combination of aztreonam, ceftazidime and amikacin were determined by time-killing studies.</p> <p>Results</p> <p>Bacteriostatic effects after 6 hours of drug exposure were observed in 12 strains (52.2%) of 23 strains of metallo-<it>β</it>-lactamase-producing <it>P. aeruginosa </it>with 48 mg/l aztreonam, in 19 strains (82.6%) with the 3-drug combination of 16 mg/l aztreonam, 16 mg/l ceftazidime, and 4 mg/l amikacin, and in 23 strains (100%) with 2 mg/l colistin. Bactericidal effects after 6 h drug exposure were observed in 1 strain (4.3%) with 48 mg/l aztreonam, in 8 strains (30.4%) with the 3-drug combination and in all 23 strains (100%) with 2 mg/l colistin.</p> <p>Conclusion</p> <p>Evaluation of <it>in vitro </it>antimicrobial effects on metallo-<it>β</it>-lactamase-producing <it>P. aeruginosa </it>revealed relatively good effects of the 3-drug combination of aztreonam, ceftazidime and amikacin and marked effects of colistin.</p