g(m3)-ganglioside and Influenza--Human

The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor β (PDGFRβ), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRβ-containing endosomal compartment. PDGFRβ/GM3-dependent virus internalization involved PDGFRβ phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRβ by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRβ signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.

Topics: Animals; Cell Line; CHO Cells; Cricetulus; Dogs; G(M3) Ganglioside; HEK293 Cells; Humans; Influenza, Human; Madin Darby Canine Kidney Cells; Orthomyxoviridae; Orthomyxoviridae Infections; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Quinolines; Receptor Protein-Tyrosine Kinases; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Virus Internalization

Following the mass vaccinations against pandemic influenza A/H1N1 virus in 2009, a sudden increase in juvenile onset narcolepsy with cataplexy (NC) was detected in several European countries where AS03-adjuvanted Pandemrix vaccine had been used. NC is a chronic neurological disorder characterized by excessive daytime sleepiness and cataplexy. In human NC, the hypocretin-producing neurons in the hypothalamus or the hypocretin signaling pathway are destroyed by an autoimmune reaction. Both genetic (e.g. HLA-DQB1*0602) and environmental risk factors (e.g. Pandemrix) contribute to the disease development, but the underlying and the mediating immunological mechanisms are largely unknown. Influenza virus hemagglutinin is known to bind gangliosides, which serve as host cell virus receptors. Anti-ganglioside antibodies have previously been linked to various neurological disorders, like the Guillain-Barré syndrome which may develop after infection or vaccination. Because of these links we screened sera of NC patients and controls for IgG anti-ganglioside antibodies against 11 human brain gangliosides (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b) and a sulfatide by using a line blot assay. Samples from 173 children and adolescents were analyzed: 48 with Pandemrix-associated NC, 20 with NC without Pandemrix association, 57 Pandemrix-vaccinated and 48 unvaccinated healthy children. We found that patients with Pandemrix-associated NC had more frequently (14.6%) anti-GM3 antibodies than vaccinated healthy controls (3.5%) (P = 0.047). Anti-GM3 antibodies were significantly associated with HLA-DQB1*0602 (P = 0.016) both in vaccinated NC patients and controls. In general, anti-ganglioside antibodies were more frequent in vaccinated (18.1%) than in unvaccinated (7.3%) individuals (P = 0.035). Our data suggest that autoimmunity against GM3 is a feature of Pandemrix-associated NC and that autoantibodies against gangliosides were induced by Pandemrix vaccination.

Topics: Adolescent; Autoantibodies; Child; Child, Preschool; Europe; G(M3) Ganglioside; HLA-DQ beta-Chains; Humans; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Influenza, Human; Mass Vaccination; Narcolepsy

To elucidate the molecular mechanisms of transmission of influenza viruses between different host species, such as human and birds, binding properties of sialic acid-containing carbohydrates that are recognized by human and/or avian influenza viruses were characterized by the surface plasmon resonance (SPR) method. Differences in the binding of influenza viruses to three gangliosides were monitored in real-time and correlated with receptor specificity between avian and human viruses. SPR analysis with ganglioside-containing lipid bilayers demonstrated the recognition profile of influenza viruses to not only sialic acid linkages, but also core carbohydrate structures on the basis of equilibrated rate constants. Kinetic analysis showed different binding preferences to gangliosides between avian and human strains. An avian strain bound to Neu5Acalpha2-3nLc4Cer with much slower dissociation rate than its sialyl-linkage analog, Neu5Acalpha2-6nLc4Cer, on the lipid bilayer. In contrast, a human strain bound equally to both gangliosides. An avian strain, but not a human strain, also interacted with GM3 carrying a shorter carbohydrate chain. Our findings demonstrated the remarkable distinction in the binding kinetics of sialic acid-containing carbohydrates between avian and human influenza viruses on the lipid bilayer.

Topics: Carbohydrates; G(M3) Ganglioside; Humans; Influenza, Human; Kinetics; Ligands; Lipid Bilayers; Models, Biological; Models, Chemical; N-Acetylneuraminic Acid; Orthomyxoviridae; Surface Plasmon Resonance; Time Factors