The production of potentially targetable VSV-G-pseudotyped retrovirus vectors has been hampered by inadequate understanding of the structure-function relationships of the VSV-G protein. In these studies we demonstrate assembly and production of MLV-based and HIV-1-based vector particles using VSV-G proteins modified by the insertion of a peptide ligand into a site corresponding to amino acid position 24 of the native VSV-G molecule. The inserted ligand represents the decapeptide encoding the collagen-binding domain of von Willebrand factor. We have used deconvolution microscopy to demonstrate that the modified VSV-G molecules sequester in perinuclear structures and are unavailable for assembly of infectious virus particles at the cell surface under standard tissue culture conditions at 37 degrees C. In contrast, at a lower permissive temperature of 30 degrees C, the modified VSV-G protein traffics appropriately to the cell surface and participates in useful titers. Furthermore, VSV-G-pseudotyped MLV-based and HIV-1-based vectors displaying the collagen-binding domain demonstrate a statistically significant increased attachment to a collagen matrix as indicated by an ELISA-like cell binding assay and by a focus transduction assay.