Figure 8
Two-plasmid genetic complementation assay to test the ability of MPMV virus like particles (VLPs) to package transfer vector viral RNA following Pr78Gag-His6-tag expression in eukaryotic cells. (a) Graphical representation of MPMV full-length Gag expression plasmids with and without His6-tag (FN7 and FN9 respectively) and MPMV transfer vector, SJ238 which provides the substrate for packageable RNA. (b) Design and rationale of the MPMV 2-plasmid genetic complementation assay. VLPs produced by the eukaryotic Gag-expression plasmids (FN7 and FN9) should allow packaging of the transfer vector RNA expressed from SJ2 due to the presence of the packaging signal (Ψ) on its RNA. The 293T cells were co-transfected with the either of the two Gag-expression plasmids along with SJ2 and fractionated into nuclear and cytoplasmic fractions. The cytoplasmic fractions were analyzed for transfer vector (SJ2) RNA expression, while the virus particles were tested for their ability to package SJ2 RNA using RT- PCR. (c) MPMV full-length Gag expression plasmids with (FN7) and without His6-tag (FN9) were transfected into 293T cells along with SJ2 and western blots were performed on cell lysates to detect Gag proteins using anti-Pr78Gag polyserum (panel I) and β-actin proteins using a monoclonal antibody as a control (panel II). Western blots on ultracentrifuged virus particles using anti-Pr78Gag polyserum (panel III). RT-PCR using MPMV transfer vector (SJ2)-specific primers (OTR1161 and OTR1163) amplifying a 530 bp fragment from the cDNAs prepared from the cytoplasmic (panel IV) and virion RNAs amplified for 30 (panel V) and 50 (panel VI) cycles, respectively. Experiments were conducted multiple times with reproducible results and representative blots are shown. Blots/gels have been cropped to show the relevant parts only and full images are provided in Supplementary Data.
