Quantitative proteomic analysis of lentiviral vectors using 2-DE

Proteomics. 2009 Jul;9(14):3666-76. doi: 10.1002/pmic.200800747.

Abstract

Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Electrophoresis, Gel, Two-Dimensional / methods*
  • Genetic Vectors / metabolism*
  • Humans
  • Lentivirus / metabolism*
  • Proteins / metabolism
  • Proteomics / methods
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Viral Proteins / metabolism

Substances

  • Proteins
  • Viral Proteins