FIG. 4.
Effect of anti-integrin function-blocking MAbs on viral replication in transfected COS-1 cells. Cells were cotransfected with αvβ3-encoding plasmids as described in the text. Thirty minutes prior to infection, paired transfected cell cultures were incubated at room temperature with the following function-blocking anti-integrin MAbs at a concentration of 25 μg/ml (all antibodies were from Chemicon International Inc): anti-αvβ3 (clone LM609; MAB1976), anti-αvβ5 (clone P1F6; MAB1961), anti-αvβ6 (clone 10D5; MAB2077Z), anti-α5 (clone CLB-705; MAB1986), and anti-β1 (clone 6S6; MAB2253). Transfected and nontransfected cultures were infected in pairs with type A12 at an MOI of 1 PFU/cell in the presence of the antibodies. One nontransfected culture pair was infected with the HS-binding O1 Campos variant vCRM4 (43) at an MOI of 1 PFU/cell. After an adsorption period of 45 min at 37°C, all cultures were washed with a low-pH buffer (25 mM MES [morpholineethanesulfonic acid, pH 5.5], 140 mM NaCl) to inactivate any nonadsorbed or noninternalized virus. After the addition of medium, one of the infected pairs was immediately frozen at −70°C to determine viral infectivity remaining at the end of the adsorption period (shaded bars). The other pair was incubated for 24 h at 37°C (solid bars) and then placed at −70°C. After thawing, cell debris was removed by centrifugation, and plaque titer was determined on BHK-21 cells.