Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Figure 5

RT accumulation in HeLa cells treated with inhibitors of proteasomal and lysosomal proteolysis. Western blotting of HeLa cells transfected with pVaxRT1.14opt-in and pVaxRT1.14oil after 18-hour incubation with MG132 (5 μМ), epoxomicin (0,1 μМ), E64 (10 μМ), leupeptin (10 μg/ml), aprotinin (10 μg/ml), and pepstatin А (7,5 μМ) or without inhibitors. The blots were stained with monoclonal anti-RT antibodies. To control equal loading of the samples on the gel, the membranes were stripped and restained with anti-β-actin antibodies (a). Relative content of RТ1.14opt-in and RТ1.14oil in the samples treated with proteasomal inhibitors (b). Relative content of RТ1.14opt-in and RТ1.14oil in the samples treated with lysosomal inhibitors; no difference in response to lysosomal inhibitors between RT1.14opt-in- and RT1.14oil-expressing cells was detected (all values > 0,05) (c). In (b) and (c), protein content in the untreated samples is taken for 1. Graphs in (b) and (c) represent the results of three independent experiments, +SD. (Mann–Whitney test).

(a)

(b)

(c)