Characterisation of p12 mutants in a Mo-MLV vector system. (A) Schematic representation of the Mo-MLV Gag-Pol expression plasmid used in this study, showing the amino acid sequence of p12. The blocks of residues changed in each substitution mutant and the mutant names are indicated. (B) LacZ-encoding wild type or mutant Mo-MLV virus-like particles (VLPs) were produced in 293T cells by transient transfection and reverse transcriptase (RT) activity was quantified by a modified ELISA as a measure of virus production. Results show the mean and range of three independent experiments. (C) Equivalent RT units of VLPs from (B) were used to challenge D17 cells. Infectivity was measured by detection of β–galactosidase activity in a chemiluminescent reporter assay. Results are plotted as the percentage of infectivity compared to wild type Mo-MLV and show the mean and range of three independent experiments. (D) Immunoblot analysis of VLPs from (B) showing p12 (bottom panel) and capsid (top panel) protein processing. The middle panel shows that the CRL-1890 monoclonal antibody fails to recognize some mutant p12 proteins. (E) D17 cells were infected with wild type or mutant VLPs and total DNA was isolated at various times post infection as indicated. The relative amounts of minus strand strong stop (left panel) or second strand extension (right panel) were measured by qPCR. Results are representative of at least three independent experiments.