A dual drug regimen synergistically blocks human parainfluenza virus infection.

International audienceHuman parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 μM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs

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)

Repurposing of approved drugs from the human pharmacopoeia to target Wolbachia endosymbionts of onchocerciasis and lymphatic filariasis

AbstractLymphatic filariasis and onchocerciasis are debilitating diseases caused by parasitic filarial nematodes infecting around 150 million people throughout the tropics with more than 1.5 billion at risk. As with other neglected tropical diseases, classical drug-discovery and development is lacking and a 50year programme of macrofilaricidal discovery failed to deliver a drug which can be used as a public health tool. Recently, antibiotic targeting of filarial Wolbachia, an essential bacterial symbiont, has provided a novel drug treatment for filariasis with macrofilaricidal activity, although the current gold-standard, doxycycline, is unsuitable for use in mass drug administration (MDA). The anti-Wolbachia (A·WOL) Consortium aims to identify novel anti-Wolbachia drugs, compounds or combinations that are suitable for use in MDA. Development of a Wolbachia cell-based assay has enabled the screening of the approved human drug-pharmacopoeia (∼2600 drugs) for a potential repurposing. This screening strategy has revealed that approved drugs from various classes show significant bacterial load reduction equal to or superior to the gold-standard doxycycline, with 69 orally available hits from different drug categories being identified. Based on our defined hit criteria, 15 compounds were then selectively screened in a Litomosoides sigmodontis mouse model, 4 of which were active. These came from the tetracycline, fluoroquinolone and rifamycin classes. This strategy of repurposing approved drugs is a promising development in the goal of finding a novel treatment against filariasis and could also be a strategy applicable for other neglected tropical diseases

A Dominant EV71-Specific CD4+ T Cell Epitope is Highly Conserved Among Human Enteroviruses

CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD), has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine

Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening

Chemical combinations elucidate pathway interactions and regulation relevant to Hepatitis C replication

SREBP-2, oxidosqualene cyclase (OSC) or lanosterol demethylase were identified as novel sterol pathway-associated targets that, when probed with chemical agents, can inhibit hepatitis C virus (HCV) replication.Using a combination chemical genetics approach, combinations of chemicals targeting sterol pathway enzymes downstream of and including OSC or protein geranylgeranyl transferase I (PGGT) produce robust and selective synergistic inhibition of HCV replication. Inhibition of enzymes upstream of OSC elicit proviral responses that are dominant to the effects of inhibiting all downstream targets.Inhibition of the sterol pathway without inhibition of regulatory feedback mechanisms ultimately results in an increase in HCV replication because of a compensatory upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression. Increases in HMGCR expression without inhibition of HMGCR enzymatic activity ultimately stimulate HCV replication through increasing the cellular pool of geranylgeranyl pyrophosphate (GGPP).Chemical inhibitors that ultimately prevent SREBP-2 activation, inhibit PGGT or encourage the production of polar sterols have great potential as HCV therapeutics if associated toxicities can be reduced

Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage

© 2012 Millet 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: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

Background: Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. Methodology/Principal Findings: Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. Conclusions/Significance: These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV. © 2009 Kam et al.published_or_final_versio

Rôles des lectines de type C L-SIGN et DC-SIGN dans l'infection par le virus de l'hépatite C

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

DC-SIGN: a multi-task viral receptor

International audienceThe identification of DC‐SIGN, a lectin expressed at the surface of dendritic cells and macrophages, provides new insight into how viruses interact with their hosts. DC‐SIGN was initially described as a ligand for HIV envelope glycoproteins. This lectin does not play the usual role of a HIV receptor, since it does not allow viral infection in absence of CD4 and co‐receptor(s). However, DC‐SIGN promotes viral capture and transmission to permissive cells. From an immunological point of view, captured virions are processed and presented by MHC molecules, inducing the activation of immune responses. Besides HIV, several viruses (CMV, HCV, Dengue, Sindbis and Ebola viruses) and non viral pathogens also bind DC‐SIGN. This review focuses on our current knowledge of the virological and immunological roles of DC‐SIGN