An essential role for the His-8 residue of the SDF-1alpha-chimeric, tropism-redirected Env protein of the Moloney murine leukemia virus in regulating postbinding fusion events.

Abstract

To use retroviral vectors for the cell-specific delivery of genes, it is necessary to redirect their receptor tropism to cell-specific receptors. Previously, we reported that a Moloney murine leukemia virus (MLV) retroviral vector containing a human stromal-derived factor-1alpha (SDF-1alpha)-chimeric envelope protein (Env) (S3) acquired the ability to transduce human cells via CXCR4, the cognate receptor for SDF-1alpha, while retaining the ability to transduce mouse cells via mCAT1. We constructed expression plasmids for derivatives of the S3 Env protein; S3-D84K containing an Asp-84-to-Lys (D84K) substitution, S3-H8R-D84K containing D84K and an additional His-8-to-Arg substitution, and S3-D84K-RY containing D84K and additional Gln-227-to-Arg plus Asp-243-to-Tyr substitutions which have been suggested to suppress the loss of function of His-8. Cellular expression, virion incorporation, and entry functions of these derivatives were investigated. All three derivatives were incorporated into virions. The S3-D84K vector lost its ecotropism, but could transduce CXCR4-expressing human and mouse cells at titers of 10(3) to 10(4) colony-forming units (cfu)/ml. The S3-H8R-D84K vector did not show transduction, although its Env protein could bind to CXCR4. The transduction titer of the S3-D84K-RY vector via CXCR4 was slightly lower than that of the S3-D84K vector. These results indicate that the His-8 residue of the S3-D84K Env protein is indispensable and may be fully functional in postbinding membrane fusion. Insertion of a ligand at Pro-79 of the Moloney MLV Env protein has proved to be a valuable strategy for constructing direct targeting retroviral vectors, since it permits the formation of a redirected Env protein without ecotropism, and it does not disrupt the function of the essential His-8 residue.