Quantitative proteomic analysis of lentiviral vectors using 2-DE.
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.
Denard
Jérôme
J
GENETHON, 91002 Evry, France.
Rundwasser
Stéphanie
S
Laroudie
Nicolas
N
Gonnet
Florence
F
Naldini
Luigi
L
Radrizzani
Marina
M
Galy
Anne
A
Merten
Otto-Wilhelm
OW
Danos
Olivier
O
Svinartchouk
Fedor
F
D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000378:metabolism
D011506:Proteins; D014764:Viral Proteins