In order to dissect the relationship between the virus receptor binding and the mediation of virus entry by the retroviral envelope protein (Env), it was first tried to genetically modify the receptor binding specificity of the Env protein of the ecotropic Moloney murine leukemia virus (MLV). A specific site (Pro-79) was found in the variable region A of the Env protein, where the insertion of peptide ligands, epidermal growth factor (EGF) and stromal-derived factor-1α (SDF-1α), was possible without abolishing viral incorporation of the Env protein and its ecotropic entry function. The MLV vector containing the EGF-Env protein did not show the EGF receptor-dependent transductkon of cells. The MLV vector containing the SDF-1α-Env protein (S3), on the other hand, transduced specifically human cells expressing CXCR4, the receptor for SDF-1α, at titers of 10[?]-10[?] CFU/ml. Further experiments showed that the CXCR4-dependent transduction was based on the specific intertaction berween the SDF-1α moiety of the SDF-1α-Env protein and the CXCR4 and independent of the ecotropic entry function. The S3 Env protein was further modified so that it may lose the tropism towards the ecotropic MLV receptor (mCAT-1) but retain that towards the CXCR4. The MLV vector containing the modified SDF-1α-Env protein (S3-D84K) lost the ecotropic entry function, but could transduce the CXCR4-expressing human cells at titers higher than 10[?] CFU/ml; namely this vector is taken for a tropism-redirected vector that should be applicable to in vivo studies in mice. Introducing the His-8-to-Arg substitution abolished the ability of the S3-D84K Env protein to mediate the transduction through CXCR4 but not that to bind to CXCR4, indicating that the His-8 residue of the S3-D84K Env protein is likely to function as a key residue in regulatign a series of postbinding membranefusion events as was proposed for the same residue of the wild-type Env protein. Further experiments showed that the His-8 residue of the S3-D84K Env protein might be fully functional. This study suggests that the receptor specificity of the Moloney MLV Env protein can be varied while maintaining the ability to activate the envelope-cell membrane fusion function. This provides the basis for constructing the tropism-redirected retroviral cectors.Thesis (Ph. D. in Biological Science)--University of Tsukuba, (A), no. 3334, 2004.3.25Includes bibliographical reference
