There is increasing evidence that soluble glycosaminoglycans such as heparin can interfere with the infectivity of various viruses, including ecotropic murine leukemia viruses (MLVs). The ecotropic MLV, Friend MLV (F-MLV) and the neuropathogenic variants A8 MLV and PVC-211 MLV, were susceptible to heparin-mediated inhibition of infection of NIH 3T3 cells. To investigate the interaction between the envelope glycoprotein (Env) of MLV and heparin, we prepared vesicular stomatitis virus-based pseudotyped viruses carrying the Env of F-, A8, or PVC-211 MLVs. Surface plasmon resonance analyses indicated that the Env of AS and PVC-211 MLVs had a higher binding activity to heparin than that of F-MLV. We examined the influence of N- or O-sulfation of heparin on binding activity to Env and on the inhibition of the infectivity of MLV and pseudotyped viruses carrying Env. This analysis indicated that the O-sulfate groups of heparin play a major role in determining Env-dependent inhibitory effects. (C) 2011 Elsevier Inc. All rights reserved.