Class II ADP-ribosylation factors are required for efficient secretion of Dengue viruses

This article is available open access through the publisher’s website.Identification and characterization of virus-host interactions are very important steps toward a better understanding of the molecular mechanisms responsible for disease progression and pathogenesis. To date, very few cellular factors involved in the life cycle of flaviviruses, which are important human pathogens, have been described. In this study, we demonstrate a crucial role for class II Arf proteins (Arf4 and Arf5) in the dengue flavivirus life cycle. We show that simultaneous depletion of Arf4 and Arf5 blocks recombinant subviral particle secretion for all four dengue serotypes. Immunostaining analysis suggests that class II Arf proteins are required at an early pre-Golgi step for dengue virus secretion. Using a horseradish peroxidase protein fused to a signal peptide, we show that class II Arfs act specifically on dengue virus secretion without altering the secretion of proteins through the constitutive secretory pathway. Co-immunoprecipitation data demonstrate that the dengue prM glycoprotein interacts with class II Arf proteins but not through its C-terminal VXPX motif. Finally, experiments performed with replication-competent dengue and yellow fever viruses demonstrate that the depletion of class II Arfs inhibits virus secretion, thus confirming their implication in the virus life cycle, although data obtained with West Nile virus pointed out the differences in virus-host interactions among flaviviruses. Our findings shed new light on a molecular mechanism used by dengue viruses during the late stages of the life cycle and demonstrate a novel function for class II Arf proteins.Research Fund for Control of Infectious Diseases of Hong Kong and BNP Paribas Corporate and Investment Banking

Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins.

© 2009 Wang 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: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. Methodology/Principal Findings: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. Conclusions/Significance: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.This work was supported by the 6th European Framework programme DENFRAME and by the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070952)

Investigation of the functional roles of host cell proteins involved in Coronavirus infection using highly specific and scalable RNA interference (RNAi) approach

Since its identification in the 1990s, the RNA interference (RNAi) pathway has proven extremely useful in elucidating the function of proteins in the context of cells and even whole organisms. In particular, this sequence-specific and powerful loss-of-function approach has greatly simplified the study of the role of host cell factors implicated in the life cycle of viruses. Here, we detail the RNAi method we have developed and used to specifically knock down the expression of ezrin, an actin binding protein that was identified by yeast two-hybrid screening to interact with the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) spike (S) protein. This method was used to study the role of ezrin, specifically during the entry stage of SARS-CoV infection

KDEL receptors assist dengue virus exit from the endoplasmic reticulum

Membrane receptors at the surface of target cells are key host factors for virion entry; however, it is unknown whether trafficking and secretion of progeny virus requires host intracellular receptors. In this study, we demonstrate that dengue virus (DENV) interacts with KDEL receptors (KDELR), which cycle between the ER and Golgi apparatus, for vesicular transport from ER to Golgi. Depletion of KDELR by siRNA reduced egress of both DENV progeny and recombinant subviral particles (RSPs). Coimmunoprecipitation of KDELR with dengue structural protein prM required three positively charged residues at the N terminus, whose mutation disrupted protein interaction and inhibited RSP transport from the ER to the Golgi. Finally, siRNA depletion of class II Arfs, which results in KDELR accumulation in the Golgi, phenocopied results obtained with mutagenized prME and KDELR knockdown. Our results have uncovered a function for KDELR as an internal receptor involved in DENV trafficking.published_or_final_versio

DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a 'hydration matrisome' that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface (http://www.sbms.hku.hk/dclab/DIPPER/), will be valuable references in the field of IVD biology and proteomic analytics

Effect of improving aerobics classes at the level of flexibility of female students

The features of the influence of a set of special exercises with gymnastic stick and elements of stretching to develop flexibility while improving aerobics classes. In pedagogical experiment involved 20 girls 17 - 18 years old, first-year student. During the final part of the session used a set of special exercises aimed at increasing the range of motion when the torso and the mobility of the shoulder joints. The dynamics of indicators of flexibility. Determined that the selected set of special exercises in the classroom improving aerobics has a positive effect on the performance increase flexibility. Significant increase in the results can be seen in test 1 tilt body forward, which is characterized by increased mobility of the joints of the spine. It was determined that the transfer of gymnastic stick behind his back indicates an increase in the mobility of the shoulder joints

Effect of improving aerobics classes at the level of flexibility of female students

The features of the influence of a set of special exercises with gymnastic stick and elements of stretching to develop flexibility while improving aerobics classes. In pedagogical experiment involved 20 girls 17 - 18 years old, first-year student. During the final part of the session used a set of special exercises aimed at increasing the range of motion when the torso and the mobility of the shoulder joints. The dynamics of indicators of flexibility. Determined that the selected set of special exercises in the classroom improving aerobics has a positive effect on the performance increase flexibility. Significant increase in the results can be seen in test 1 tilt body forward, which is characterized by increased mobility of the joints of the spine. It was determined that the transfer of gymnastic stick behind his back indicates an increase in the mobility of the shoulder joints

Cellular enhancing and restricting factors of dengue virus egress

This is an open access article available to view at: http://www.hkmj.org/abstracts/v20n4s4/44.htmDengue viruses of four serotypes (DV1-4) are the pathogens of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. An estimated 50 to 100 millions such cases (including 25 000 deaths) occur every year. There is no established treatment for such infections.This study was supported by the Research Fund for the Control of Infectious Diseases, Food and Health Bureau, Hong Kong SAR Government (#08070952)

Composite Membranes Containing Nanoparticles of Inorganic Ion Exchangers for Electrodialytic Desalination of Glycerol

Abstract Composite membranes were obtained by modification of heterogeneous polymer cation and anion-exchange membranes with nanoparticles of zirconium hydrophosphate and hydrated zirconium dioxide, respectively. The ion-exchange materials were investigated with the methods of electron microscopy, potentiometry, voltammetry, and impedance spectroscopy. Single nanoparticles, which were precipitated in aqueous media, form aggregates, when the composites are in a contact with polar organic solvent. Both single nanoparticles (up to 10 nm) and their aggregates (up to 200 nm) were precipitated in ion-exchange polymers in glycerol media. Non-aggregated nanoparticles improve electrical conductivity of the ion-exchange materials, the aggregates are barriers against fouling. The membranes were applied to NaCl removal from highly concentrated glycerine-water mixture containing organic additives (byproduct of biodiesel production). As opposite to pristine materials, the composites demonstrate stability against fouling