We have recently demonstrated an essential role of the domain of 145-150 amino acid in the E2 glycoprotein of Sindbis virus in the interaction with cellular heparan sulfate (HS) and in the infection of mouse embryonic fibroblasts (MEF) cells. In this study, we constructed and characterized the mutants of Sindbis-like virus XJ-160 in which Tyr-146 and/or Asn-149 in the E2 glycoprotein had been substituted with His and Arg, respectively. Unlike parental virus XJ-160, mutants with either or both substitutions were able to infect wild-type mouse embryonic fibroblasts (MEF-wt) or MEF-Epi-/- cells which produce mutant HS. Significantly more infectious particles were released from MEF-wt than from MEF-Epi-/- cells. The mutant virus with both substitutions release was inhibited by pre-incubation of virus with heparin or pre-treatment of BHK-21 cells with HS-degrading enzyme. Both XJ-160 and the mutant viruses retained substantial neurovirulence in suckling mice. Our findings provide further support to the importance of positively charged residues in the HS-binding site of E2 in mediating Sindbis virus infection of MEF cells.