Figure 4
The ability of p12 mutants to saturate TRIM5alpha and Fv1 activity. (A and B) Supernatants from 293T cells containing LacZ-encoding N-tropic MLV (N-MLV) tester viruses, with or without p12 mutations, or B-MLV were serially diluted 2-fold and used to challenge TE671 cells, which express TRIM5alpha (A), or B3T3 cells, that express Fv1b (B). After 4 to 6 hours, the cells were infected a second time with a fixed dose of GFP-encoding N-MLV reporter virus. The percentage of GFP positive cells is plotted for each volume of tester virus. (C) Mo-MLV p12 mutants were converted to N-tropic viruses by changing 3 residues in CA. These mutants (named N/Mo) were used as tester viruses in TE671 cells as described in (A). (D) Mixed particles that contained a mixture of N- or B-tropic CA in addition to mutated and wild type p12 were synthesized as follows: 90% N-MLV mutant 6 with 10% wild type B-MLV (N6/B, orange line), 90% B-MLV mutant 6 with 10% wild type N-MLV (B6/N, brown line) and 90% N-MLV mutant 6 with 10% wild type N-MLV (N6/N, dark red line). These viruses were used to test abrogation of TRIM5alpha restriction in TE671 cells as described in (A). All graphs are representative of at least three independent experiments.