Identification of host proteins associated with retroviral vector particles by proteomic analysis of highly purified vector preparations.
The Moloney murine leukemia virus (MMLV) 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 MMLV 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 one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. 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 MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin beta1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is 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 retrovirus-mediated gene therapy are discussed.
Segura
María Mercedes
MM
Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Quebec, Canada.
Garnier
Alain
A
Di Falco
Marcos Rafael
MR
Whissell
Gavin
G
Meneses-Acosta
Angélica
A
Arcand
Normand
N
Kamen
Amine
A
D002460:Cell Line; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification*; D016253:Microscopy, Immunoelectron; D008979:Moloney murine leukemia virus / Q000737:chemistry* / Q000302:isolation & purification*; D014461:Ultracentrifugation
D008565:Membrane Proteins