Identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations

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DOIResolve DOI: http://doi.org/10.1128/JVI.01909-07
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TypeArticle
Journal titleJournal of Virology
Volume82
Issue3
Pages11071117; # of pages: 11
Subjectanalysis; bio; Biotechnology; Cell Adhesion; Cell Line; Cells; chemistry; Chromatography,Liquid; Electrophoresis; Electrophoresis,Polyacrylamide Gel; Gene Therapy; Genetic Vectors; Humans; isolation & purification; Mass Spectrometry; Membrane Proteins; Microscopy,Immunoelectron; Moloney murine leukemia virus; Protein; Proteins; Retroviridae; Sodium; Tags; therapy; Ultracentrifugation; Viral Proteins; Virion
AbstractThe Moloney murine leukemia virus (MoMLV) belongs to the retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MoMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate zonal ultracentrifugation. Viral proteins were fractionated by 1D-SDS-PAGE, in-gel tryptic digested and subjected to liquid chromatography/tandem mass spectrometry analysis (LC-MS/MS). Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MoMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild type MoMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of 8 host membrane proteins were identified including cell adhesion proteins integrin {beta}1 (fibronectin receptor subunit beta) and MFG-E8, tetraspanins CD81 and CD9 and late endosomal markers CD63 and Lamp-2. Membrane proteins are particularly attractive since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retroviral-mediated gene therapy are discussed.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Biotechnology Research Institute
Peer reviewedNo
NRC number47822
NPARC number12401004
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Record identifier96350d6e-3593-4013-9b81-f48344755755
Record created2009-10-26
Record modified2016-05-09
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