Effect of p51↓RNH ± T477A mutations on viral particle protein composition. Western blots of wild-type (WT) and p51↓RNH ± T477A mutant viruses (1 μg viral p24) generated by transfection of 293T cells and probed with (A) anti-PR, (B) anti-RT, (C) anti-IN, and (D) anti-p24 antibodies. The positions of molecular size markers are shown to the left of each panel. Arrows to the right of each panel indicate the positions and molecular masses of immunoreactive viral proteins. The relative mean proportion of p66 RT to p51 RT (p66:p51) and the total viral content of RT, IN and CA were determined from multiple experiments (n = 3) by densitometric scanning analysis of ECL-exposed blots under subsaturating conditions. Statistical significance of the T477A compensatory effect was determined for each individual mutant virus relative to its non-substituted counterpart using a one-tailed Student's t-test assuming equal variance. Asterisks indicate the degree of statistical significance in relation to the size of the type I error: (*)p < 0.10, *p < 0.05, **p < 0.01. In Figure 2A and 2B, WT and WT+T477A samples (two leftmost lanes) are from a different gel than the rest of the samples, as the number of wells in the electrophoresis apparatus was unable to accommodate all samples simultaneously. However, all electrophoresed samples had the same amount of p24 (see Methods) and were processed simultaneously (using two identical electrophoresis apparatus). Both resultant gels were imaged simultaneously by chemiluminescence as described in Materials and Methods.