EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport

Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.     

Comparative Analysis of Nucleotide Fluorescent Analogs for Registration of DNA Conformational Changes Induced by Interaction with Formamidopyrimidine-DNA Glycosylase Fpg

Russian Journal of Bioorganic Chemistry - DNA-substrates containing fluorescent DNA base analogs are widely used to study protein–nucleic acid interactions. In the case of DNA-recognizing...     

Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species

Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named "hexacoordinate" because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K(H)). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K(H). Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K(H) is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.     

Impact of Stakeholders Influence,Geographic Level and Risk Perception on Strategic Decisions in Simulated Foot and Mouth Disease Epizootics in France

Comparison of control strategies against animal infectious diseases allows determining optimal strategies according to their epidemiological and/or economic impacts. However, in real life, the choice of a control strategy does not always obey a pure economic or epidemiological rationality. The objective of this study was to analyze the choice of a foot and mouth disease (FMD) control strategy as a decision-making process in which the decision-maker is influenced by several stakeholders (government, agro-food industries, public opinion). For each of these, an indicator of epizootic impact was quantified to compare seven control strategies. We then determined how, in France, the optimal control strategy varied according to the relative weights of stakeholders and to the perception of risk by the decision-maker (risk-neutral/risk-averse). When the scope of decision was national, whatever their perception of risk and the stakeholders'' weights, decision-makers chose a strategy based on vaccination. This consensus concealed marked differences between regions, which were connected with the regional breeding characteristics. Vaccination-based strategies were predominant in regions with dense cattle and swine populations, and in regions with a dense population of small ruminants, combined with a medium density of cattle and swine. These differences between regions suggested that control strategies could be usefully adapted to local breeding conditions. We then analyzed the feasibility of adaptive decision-making processes depending on the date and place where the epizootic starts, or on the evolution of the epizootic over time. The initial conditions always explained at least half of the variance of impacts, the remaining variance being attributed to the variability of epizootics evolution. However, the first weeks of this evolution explained a large part of the impacts variability. Although the predictive value of the initial conditions for determining the optimal strategy was weak, adaptive strategies changing dynamically according to the evolution of the epizootic appeared feasible.