HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner
Figure 2
Differences in DNA binding between NCp15 and NCp7 and the effect of NCp15 proteolysis.
(A,B) EMSA using G4-DNA (5 nM) and increasing amounts of NCp7 or NCp15. Complexes were formed for 15 min. at 37°C before electrophoresis. Note that in the case of NCp15 there is only one shifted G4-DNA species (A), whereas quantification of the DNA bound fraction as a function of the total NCp concentration (B) shows a 6-fold drop in affinity for G4-DNA with NCp15 (solid line) compared to NCp7 (dashed line). (C,D,E) TEM visualization of ssDNA (5 nM) without protein (C) or with saturating amounts (3.4 µM) of NCp7 (D) or NCp15 (E). (F,G,H) TEM visualization of NCp15 proteolysis in the presence of ssDNA after 15 min. (F and G) and after 40 min. (H) incubation with saturating amounts of NCp bound to ssDNA and a NCp15-to-PR ratio of 30 corresponding to [NCp15] = 3.4 µM, [PR] = 100 nM and [ssDNA] = 5 nM. The nascent aggregates revealed in (F) are shown with a higher magnification in (G). Scale bars in each panel correspond to 150 nm.