Figure 1.
Incorporation of PB transposase in LPs leads to efficient DNA transposition by lentiviral transduction of target cells. (A) Schematic representations of third-generation lentiviral packaging constructs showing the individual entities of the GagPol polyprotein. In the standard packaging construct (upper), Gag is composed of matrix (MA), capsid (CA), nucleocapsid (NC) and p6, whereas Pol consists of protease (PR), reverse transcriptase (RT) and integrase (IN; here, a defective integrase containing the D64V mutation). The protein reference based on molecular mass is provided above the corresponding entity. For production of protein-transducing viruses, the protein of interest (POI) is inserted between a Lyn-derived myristoylation signal (Myrlyn) and a codon-optimized GagPol-coding sequence (lower). A HIV-1 protease cleavage site SQNY/PIVQ is inserted between POI and GagPol. In this study, POIs include GFP, NanoLuc and hyPBase transposase protein. (B) Schematic representation of the production of a LP containing a specific protein of interest (LP-POI). LPs were generated by co-transfecting 293T cells with pPOI-gagpol-D64V and pMD.2G encoding the viral surface protein VSV-G. Black dots indicate units of hyPBase protein. (C) Lentiviral transduction of GFP protein to HEK293 cells. Flow cytometric analysis was performed 24 h post-transduction. Histogram shows the relative level of GFP fluorescence. The black curve represents cells that were transduced with LP-GFP pseudotyped with VSV-G (152 ng p24), whereas the gray curve is indicative of cells that were treated with LP-GFP without VSV-G (174 ng p24). (D) Lentiviral transduction of NanoLuc to HeLa cells. Luminescence was measured 48 h post-transduction and normalized to the p24 content of LP-NanoLuc particles with and without VSV-G. (E) Evidence of incorporation of hyPBase into LPs. Western blot analysis was performed on LP lysates (three right lanes) using antibodies for the HA tag (top blot) and p24 (bottom blot). IDLV that did not incorporate hyPBase was used as negative control, and lysate from 293T cells transfected with pCMV-HAhyPBase served as positive control. (F) Protease-directed cleavage of the GagPol-fused hyPBase after virus release and maturation. To restrict viral protein processing, LP-producing 293T cells were treated with the protease-inhibitor saquinavir (SQV), or DMSO as a control. Blotting membrane of LP was initially incubated with anti-p24 antibody (middle blot) and reused for anti-HA antibody after stripping (right blot). (G) DNA transposition catalyzed by lentivirally delivered hyPBase protein. HeLa cells were transfected with 1.95-µg pPBT-PGK-puro (or co-transfected with 50-ng pCMV-hyPBase as a positive control, left column) 1 day before transduction with LP-hyPBase, LP-hyPBmut or LP-hyPBase(-VSV-G) (in all cases 250 ng p24) and selected for puromycin resistance.