FIG. 3.

Reconstitution of integration activity with HMG I(Y) deletion derivatives and HMG I-C. (A) Diagram of HMG I illustrating the locations of breakpoints studied. (B) Reconstitution of salt-stripped MoMLV PICs with deletion derivatives of HMG I(Y) proteins. Integration was carried out in vitro and analyzed on Southern blots probed with a labeled MoMLV LTR. HMG I(Y) derivatives used for reconstitution are indicated above the autoradiograms. II, integration intermediate produced by integration in vitro; circle, autointegration product; cDNA, unreacted viral DNA; aa, amino acids. Triangles above the lane indicate 1 or 0.2 μg was added. Lanes: 1, no added protein; 2 to 3, reconstitution with full-length HMG I; 4 and 5, reconstitution with an HMG I(Y) derivative lacking the acidic C-terminal domain; 6 to 9, reconstitution with HMG I(Y) derivatives containing two A/T hooks; 10 to l5, reconstitution with single A/T hooks. Molarities for 1 μg in 100-μl reaction mixtures were as follows: full-length HMG I(Y), 0.85 μM; 1–90, 1 μM; 9–75, 1.25 μM; 50–91, 2 μM; AT-1, 4.7 μM; AT-2, 4.7μM; and AT-3, 5 μM. (C) Reconstitution of salt-stripped HIV-1 PICs with deletion derivatives of HMG I(Y). Assay and labeling are as in panel B, except that the triangles indicate 5 or 1 μg was added. The assays of single A/T hooks contained 5 μg. (D and E) Reconstitution of MoMLV PICs (D) and HIV-1 PICs (E) with HMG I-C. The amounts added were 5, 1, or 0.2 μg for MoMLV or 5 μg for HIV-1.