Figure 1 : Construction, production and characterization of CASP3*-LENA.

From: Therapeutic potential of HIV protease-activable CASP3

Figure 1

(a) Genetic structure of CASP3 and the genetic modification to produce HIV-1 protease-activable CASP3*. The cellular (gray triangle, digesting D28-S29 and D175-S176 junctions) and HIV-1 (black triangle) protease proteolytic sites are indicated. The CASP3 D28 was replaced with amino acids 127–136 (VSQNYPIVQ) from HIV-1 Gag (according to the HXB2 coordinate). The amino acids 129–134 (QNYPIV) from HIV-1 Gag according to the HXB2 coordinate was inserted after CASP3 D175. HIV-1 protease targets the YP junction. The enzyme is active when the p17 and p12 subunits are dimerized. (b) Production of CASP3*-LENA and its mechanism of action. The CASP3* was placed at the 5’ end of wild type (WT) gag-pol to yield CASP3*-gag-pol. The proteolytic cleavage sites within Gag for HIV-1 protease and the proteolytic products are indicated MA, matrix; CA, capsid; and NC, nucleocapsid. The CASP3*-gag-pol and VSV-G expression vectors were co-transfected into 293T cells. Then, the VSV-G-encapsidated LENA containing activated CASP3* protein was produced in the culture supernatant. CASP3*-LENA enters cells via an endocytotic route, with the content released from endosomes at the site of membrane fusion. The action points of Bafilomycin A1 (BAF) and the CASP3 inhibitor Z-DEVD-FMK are indicated. (c) Expression of CASP3*-gag-pol and CASP3*-LENA production. The cell lysates (Cell) and culture supernatants (Sup) of 293T cells transfected with either wild type (WT) or CASP3*-gag-pol (CASP3*) expression vector were analyzed by Western Blot. The corresponding protein for each band is indicated. (d) Immunofluorescence assay showing the distribution of WT Gag or CASP3*-Gag (CASP3*) in 293T cells transfected with either WT or CASP3*-gag-pol (CASP3*) expression vector. Red and blue represent the anti-p24CA monoclonal antibody-stained signal and the Hoechst 33258-stained nucleus, respectively. Magnification, x630; scale bar, 10 µm. (e) Detection of CASP3 enzyme activity in purified CASP3*-LENA. CASP3*-LENA was produced either in the presence or absence of nelfinavir (NFV) and collected by ultracentrifugation over a 20% sucrose layer. DMSO was used as a control. The amount of CASP3*-LENA and the cleavage pattern of Gag were examined by Western blot (left). CASP3 enzyme activity was detected in the CASP3*-LENA lysate (right).