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Caveolae as an additional route for influenza virus endocytosis in MDCK cells
Nunes-Correia, Isabel; Eulálio, Ana; Nir, Shlomo; Lima, Maria C. Pedroso deClathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is inter...
Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation...
Nunes-Correia, Isabel; Eulálio, Ana; Nir, Shlomo; Düzgünes, Nejat; Ramalho-Santos, João; Lima, Maria C. Pedroso deFluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidyl...
Interaction of cationic liposomes and their DNA complexes with monocytic leukem...
Pires, Pedro; Simões, Sérgio; Nir, Shlomo; Gaspar, Rogério; Düzgünes, Nejat; Pedroso de Lima, Maria C.Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and [zeta] potential of cationic liposomes (...
Interactions of Influenza Virus with Cultured Cells: Detailed Kinetic Modeling...
Nunes-Correia, Isabel; Ramalho-Santos, João; Nir, Shlomo; Lima, Maria C. Pedroso deWe performed a detailed kinetic analysis of the uptake of influenza virus (A/PR8/34) by Madin Darby canine kidney (MDCK) cells in culture. Experimental procedures were based on the relief of fluorescence self-quenching of the fluorescent probe octadecylrhodamine B chloride (R18) incorporated in the viral envelope. Equilibrium for binding of influenza virus to MDCK cells (2.5 × 106/mL) was reached quicker with t...
Partial fusion activity of influenza virus toward liposomes and erythrocyte gho...
Ramalho-Santos, João; Lima, Maria C. Pedroso de; Nir, ShlomoFinal extents of fusion of influenza virus (A/PR/8/34 strain) with neutral and partially acidic liposomes were monitored with (i) a fluorescence resonance energy-transfer assay in which the liposomes were labeled and (ii) by the dequenching of octadecylrhodamine, initially incorporated in the viral membrane. The latter assay was also employed in the fusion of influenza virus and Sendai virus with erythrocyte gh...
A common mechanism for influenza virus fusion activity and inactivation
Ramalho-Santos, João; Nir, Shlomo; Düzgünes, Nejat; Carvalho, Arsélio Pato de; Lima, Maria da Conceição Pedroso deThe fusion of influenza virus (A/PR/8/34 strain) with PC- 12 cells was monitored by a fluorescence assay, and the results were analyzed with a mass-action model which could explain and predict the kinetics of fusion. The model accounted explicitly for the reduction in the fusion rate constant upon exposure of the virus to low pH, either for the virus alone in suspension or for the virus bound to the cells. When...
Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temp...
Lima, Maria da Conceição Pedroso de; Ramalho-Santos, João; Martins, Maria de Fátima; Carvalho, Arsélio Pato de; Bairos, Vasco; Nir, ShlomoWe have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecylrhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus2013cell fusion. When the temperature was lowered from 37°C to 2...
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