Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus assembly phenotype.
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.
Guibinga
Ghiabe H
GH
Department of Pediatrics, Center for Molecular Genetics, University of California at San Diego School of Medicine, La Jolla, CA 92093-0634, USA.
Hall
Frederick L
FL
Gordon
Erlinda M
EM
Ruoslahti
Erkki
E
Friedmann
Theodore
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000502:physiology; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000502:physiology; D016254:Mutagenesis, Insertional; D010641:Phenotype; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012190:Retroviridae / Q000235:genetics*; D013696:Temperature; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*; D019065:Virus Assembly; D014841:von Willebrand Factor / Q000737:chemistry
C028391:G protein, vesicular stomatitis virus; D008024:Ligands; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014841:von Willebrand Factor; D003094:Collagen