Factors defining these cleavage events in vivo remain poorly defined. One
Factors defining these cleavage events in vivo remain poorly defined. One of the most intriguing polyVelpatasvir web protein proteolytic cleavage events is the RT p51RNH cleavage needed to form the obligate p66/p51 RT heterodimer. While all three pol-derived enzymes (PR, RT, IN) are active only as oligomers, only RT is a heterodimer. We previously showed that mutations introduced into the RT p51RNH protease recognition and cleavage site, whichAbram et al. Retrovirology 2010, 7:6 http://www.retrovirology.com/content/7/1/Page 4 ofFigure 2 Effect of p51RNH ?T477A mutations on viral particle protein composition. Western blots of wild-type (WT) and p51RNH ?T477A mutant viruses (1 g viral p24) generated by transfection of 293T cells and probed with (A) anti-PR, (B) anti-RT, (C) anti-IN, and (D) antip24 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 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27607577 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.Abram et al. Retrovirology 2010, 7:6 http://www.retrovirology.com/content/7/1/Page 5 ofFigure 3 Effect of p51RNH ?T477A mutations on ordered intravirion processing of Gag and Gag-Pol polyproteins. Virus-containing culture supernatants derived from the transfection of COS-7 cells in the presence of various concentrations of ritonavir were subjected to SDS10 PAGE resolution and Western blotting analysis. (A) Pr160Gag-Pol and (B) Pr55gag polyprotein processing intermediates were visualized with anti-RT and anti-p24 monoclonal antibodies respectively, followed by ECL exposure. Analyses of p51RNH mutant viruses containing the wildtype 477T or the mutant 477A are in the left and right panels, respectively. The positions of molecular size markers are shown to the left of each panel. Lines to the right of each panel indicate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27735993 the positions and estimated molecular masses of predicted polyprotein processing intermediates [24,27].we predicted would result in accumulation of unprocessed RT p66, instead resulted in severe attenuations of HIV-1 infectivity due to inappropriate intravirion degradation of RT by the viral protease [22]. Based on these findings, we suggested that the proteolytic cleavage at the RT p51RNH junction to form the RT p66/p51 heterodimer was essential to stabilize RT and to prevent extensive intravirion HIV PR-mediated degradation of RT. HIV has an extraordinary adaptive capacity, and.