Quantitative proteomic analysis of lentiviral vectors using 2-DE

Area of Science:

• Gene Therapy
• Virology
• Proteomics

Background:

• Lentiviral vectors (LV), derived from HIV-1, are promising gene therapy tools due to their ability to infect diverse cell types.
• The cellular protein composition of LV particles is not well-characterized, impacting vector safety and efficacy.
• Understanding LV protein content is crucial for optimizing clinical gene therapy applications.

Purpose of the Study:

• To quantify and identify cellular proteins associated with purified lentiviral vectors (LV).
• To distinguish between incorporated viral proteins and co-purified contaminants.
• To define the protein stoichiometry of infectious HIV-1-derived LV particles.

Main Methods:

• Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry.
• Proteinase K treatment to differentiate incorporated (I-proteins) from co-purified (C-proteins) cellular proteins.
• Analysis of vesicular stomatitis virus G-pseudotyped LV purified for clinical gene therapy.

Main Results:

• Identified 10 co-purified (C-proteins) and 18 incorporated (I-proteins) cellular proteins associated with LV.
• Quantified copy numbers for core I-proteins, ranging from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per particle.
• Discovered three novel incorporated proteins: guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain, and hnRNP core protein A1.

Conclusions:

• This study provides the first comprehensive definition of protein stoichiometry in infectious HIV-1-derived LV.
• The findings enhance our understanding of LV composition, crucial for improving gene therapy vector design and safety.
• Detailed protein characterization of LV is essential for advancing their clinical application.