Protein transduction from retroviral Gag precursors

Abstract

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

Protein transduction by pseudotyped lentivirus-like nanoparticles

“…The protein of interest Protein transduction by LENA T Aoki et al can be fused to the C-terminus of Gag or inserted into the middle of the Gag protein (for example, between the MA and CA). 11,12 Foreign protein fusion to the C-terminus of Gag would destroy the frameshift signal required to produce Gag-pol. Thus, Pol would no longer be produced.…”

“…These data show that a protein transduction system based on LENA would have an extra level of safety compared with the protein transduction system based on a retroviral vector system. 11 Protein transduction in a broad sense, referring to the transport of protein across the cell membrane, is a useful technique in experimental molecular biology. It does not require de novo transcription and translation, and the transferred protein functions immediately after the transduction.…”

“…This is noteworthy because modification of Gag often results in reduction of viral productivity and infectivity. 11,12 It has been reported recently that a protein transduction using murine leukemia virus is achievable by embedding a foreign gene in gag. However, the viral productivity and infectivity need to be improved by WT Gag-pol provided in trans upon viral production.…”

A bispecific transmembrane antibody simultaneously targeting intra‐ and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

“…Gibbon ape leukemia virus (GALV) pseudotyped retroviral vector particles were produced by co-transfecting HEK 293T cells with the respective retroviral transfer plasmid together with packaging and envelope plasmids M634 (pcDNA3.MLVgp) 17 and M620 18 using polyethylenimine transfection as described elsewhere. 19 Vesicular stomatitis virus protein G (VSV-G) pseudotyped lentiviral vector particles were produced by cotransfecting HEK 293T cells with the respective lentiviral transfer plasmid together with packaging and envelope plasmids pCMVDR8.91 and pMD2.G.…”

Protein delivery using engineered virus-like particles

“…In addition, there are a few reports where VLPs generated using nonstructural HIV proteins (e.g., Nef and Vpr) were used to deliver foreign proteins (25)(26)(27). Recently, Voelkel et al (28) reported that they were able to deliver proteins (GFP and functional Flp recombinase) to cells using murine retroviral particles. However, in all these cases, Gag-Pol was used to generate the VLPs.…”