FIG. 6.
Incorporation of LysRS, ProRS, and GagPol into HIV-1 made in murine cells. (A) Viral incorporation of human or mouse LysRS. GM11686-cycT1 cells were cotransfected with BH10 DNA and DNA coding for either human LysRS (hLysRS) or murine LysRS (mLysRS), both of which were tagged with V5. Forty-eight hours posttransfection, viruses were collected from the supernatant, and cells and viruses were lysed. Western blots of cell and viral lysates were probed with anti-V5 and anti-β-actin or with anti-V5 and anti-CAp24, respectively. (B) Fluorescence anisotropy experiment, showing binding of mouse LysRS (mLysRS) to FITC-CAp24. Only a representative data set is shown, but measurements were carried out at least three times. The equilibrium dissociation constant (Kd) is shown. (C) Incorporation of ProRS. GM11686-cycT1 cells were transfected with BH10, and 48 h posttransfection, viruses were collected from the supernatant, and cells and viruses were lysed. Western blots of cell lysate were probed with anti-ProRS, and Western blots of viral lysates were probed with anti-ProRS and anti-CAp24. (D) Incorporation of GagPol. GM11686-cycT1 cells and 293T cells were transfected with BH10. Forty-eight hours posttransfection, viruses were collected from the supernatant and lysed. Western blots of viral lysates were probed with anti-CAp24 and anti-RT.